摘要:

Communication Influence of functional and cross-linking monomers and the amount of template on the performance of molecularly imprinted polymers in binding assays Ecevit Yilmaz, Klaus Mosbach* and Karsten Haupt*† Lund University, Department of Pure and Applied Biochemistry, Chemical Center, PO Box 124, S-22100 Lund, Sweden. E-mail: klaus.mosbach@tbiokem.lth.se Received 17th February 1999, Accepted 16th March 1999 An evaluation of molecularly imprinted polymers has been performed using radioligand binding assays.The bronchodilator theophylline was chosen as a model analyte and template molecule. The effect of type and amount of functional and cross-linking monomers, as well as of different combinations of functional and cross-linking monomers has been studied with respect to the yield of highaffinity binding sites and their dissociation constants. Copolymers of trifluoromethyl acrylic acid with divinylbenzene yielded the highest, and the combination of trifluoromethyl acrylic acid with ethylene glycol dimethacrylate the lowest binding capacities.Polymers can be imprinted with very small amounts of template molecule without jeopardising their affinity and specificity. Although binding isotherms revealed a slight decrease in the absolute number of high-affinity sites in polymers prepared with low template concentrations, the relative yield in high-affinity sites increased. This extends the potential applicability of non-covalent molecular imprinting to template molecules which are expensive, not available in large quantities, or poorly soluble.The degree of cross-linking also seems to play a less important role for the recognition properties of the polymer in binding assay applications. Even only 19% crosslinked polymers were still specific, which could for instance facilitate the preparation of thin polymer films or membranes. Antibodies are routinely utilised as analytical reagents in clinical and research laboratories,1 and one of their most common applications is in immunoassays.2 Molecularly imprinted polymers (MIPs) which are artificial, tailor-made receptors, share the most important feature of antibodies: the capability to specifically bind a target molecule.3,4 It has been shown that MIPs can be substituted for antibodies in applications such as immunoassays, immunoextraction or immunosensors. 5 Since the first introduction of an assay system based on molecularly imprinted polymers,6 a competitive radioligand binding assay for the drugs theophylline and diazepam, there have been numerous reports describing immunoassay-type applications of MIPs.7–10 The protocols for the preparation of the MIPs used in these applications are mostly identical with protocols developed earlier for (chiral) chromatography.11 From a practical point of view, these protocols often suffer from certain limitations.These may be the poor solubility of the analyte (template) in the imprinting mixture, or the limited availability of the template, of which a considerable amount is needed in order to prepare the MIP in the first place.However, the requirements for MIPs intended for binding assays differ in many points from those used in separation. For example, a MIP used as a chiral stationary phase in chromatography should have the highest possible number of binding sites combined with fast on–off kinetics of analyte binding, whereas the binding strength is somewhat less important because the enantiomers are separated through multiple association–dissociation phenomena with the imprinted stationary phase during their travel through the column. MIPs used in immunoassay-type applications, on the other hand, should contain sites of the highest possible quality and thus affinity, to obtain a high specificity and sensitivity of the assay.Binding capacity and kinetics are somewhat less important, because only a very small quantity of analyte is applied to the polymer, and because the system is allowed to reach equilibrium before the unbound or bound analyte is quantified.5,6 The present paper describes how imprinting recipes can be adapted specifically for binding assays and sensors, using a polymer imprinted with the bronchodilating drug theophylline as a model system.Experimental Ethylene glycol dimethacrylate (EDMA), methacrylic acid (MAA) and 2-(trifluoromethyl) acrylic acid (TFMAA) were from Merck (Darmstadt, Germany).Acrylamide (AAm) was from Bio-Rad (Richmond, CA, USA). 2,2A-Azobis(2,4-dimethyl valeronitrile) (ABDV) was from Wako (Osaka, Japan). Theophylline, caffeine, theobromine and 3H-theophylline (specific activity 0.8 mCi mol21) were from Sigma (St. Louis, MO, USA). Technical grade divinylbenzene (DVB) containing 80% DVB and 20% ethylvinylbenzene, and styrene were purchased from Aldrich (Steinheim, Germany) and treated with basic alumina immediately prior to use, to remove polymerisation inhibitors.Anhydrous quality toluene and tetrahydrofuran (THF) were from Labscan (Dublin, Ireland). All other chemicals were of analytical grade and used as obtained. Preparation of polymers Appropriate amounts of cross-linker (CL), functional monomer (M), porogenic solvent (1.65 ml ml21 monomers), ABDV (radical initiator; 0.7 mol% of the polymerisable units) and the template (T) theophylline were added into a screw-capped borosilicate test tube, sonicated until a clear solution was obtained, and cooled on ice.After sparging with nitrogen for 2 min the mixture was polymerised at 45 °C for 12 h. The resultant hard polymer monolith was ground in a mechanical mortar. The polymer particles were suspended in acetone, sonicated for 5 min and then allowed to settle. Particles sedimenting within 5 min (coarse) and particles not sedimenting after 10 min centrifugation at 5000g (fine) were discarded. The remaining particles were washed by incubation in methanol– acetic acid (8 : 2, v/v) (33) and methanol (33) for 2 h each time on a rocking table, followed by centrifugation.Finally, the solvent was removed by centrifugation and the particles were † Present address: Institut National de la Transfusion Sanguine, INSERM U76, 6, rue Alexandre Cabanel, 75739 Paris Cedex 15, France. E-mail: karsten.haupt@tbiokem.lth.se Anal. Commun., 1999, 36, 167–170 167dried in vacuo.In this way, particles with an average diameter of 5–30 mm were obtained. Control polymers were prepared in the same way but without the addition of the template. Radioligand binding assays The polymer particles were suspended in the incubation solvent toluene–THF (9 : 1, v/v) and appropriate volumes were added into 1.5 ml polypropylene test tubes, followed by the radioligand 3H-theophylline (0.4 pmol, if not otherwise stated), varying amounts of a solution of a competing ligand if appropriate, and solvent to a total volume of 1 ml.The samples were incubated on a rocking table for 12 h. After centrifugation, 500 ml of supernatant was withdrawn and added to 10 ml Ecoscint O scintillation liquid (National Diagnostics, Atlanta, GA, USA). Radioactivity was measured by liquid scintillation counting using a Rackbeta 1219 counter (LKB Wallac, Turku, Finland). Results and discussion Choice of functional monomers and cross-linker A range of imprinted polymers have been prepared containing different combinations of cross-linker (EDMA or DVB) and functional monomer (MAA, TFMAA or AAm).All polymers were analysed for binding of 3H-theophylline in equilibrium binding experiments. The different combinations as well as the binding characteristics of the imprinted polymers compared to the corresponding non-imprinted control polymers are shown in Table 1. Looking at the cross-linker, it can be concluded that polymers containing DVB show better binding of the analyte and lower non-specific binding (with the exception of polymers containing AAm).This can probably be attributed to the less polar nature of DVB as compared to EDMA, which helps to avoid interaction of T with CL and promotes complex formation between T and M. For the functional monomer, the situation is somewhat more complex. Acrylamide-containing polymers perform less well than the other monomers regardless of which CL was used. Even though the binding capacities of the AAm-polymers are high, 40% of non-specific binding to the control polymers as compared to the imprinted polymers makes them less suitable for equilibrium assays.However, AAm-polymers have been demonstrated to be very useful for the preparation of chiral stationary phases for chromatography.12 Comparing MAA and TFMAA, in combination with EDMA, the MAA-polymer has a 9 times higher capacity than the TFMAA-polymer, whereas in combination with DVB, the TFMAA-polymer is slightly better.The TFMAA/DVB polymer has a 40 times higher capacity than the TFMAA/EDMA polymer. Apparently, TFMAA which is more acidic and a better hydrogen bond donor than MAA due to the negative inductive effect of the three fluoro substituents, forms a more stable complex with T.13 However, for the same reason, it is to a larger extent able to interact with the ester functionalities of EDMA, which results in a lower binding capacity for the analyte, and in a five times higher non-specific binding.Thus, at least in our model system, a combination of TFMAA with EDMA would not be a very good combination. Ratio functional monomer : template A current criticism of imprinted polymers is the significant initial amount of template needed to prepare the polymer. To prepare a five gram batch of MIP, with the currently used ratio M: T of 4 : 1 to 10 : 1,6–13 depending on the molecular weight of T, up to several hundred mg of the template are needed.For MIPs used as stationary phases in chromatography, there might be no circumvention to this problem, since high load capacities are required for this application and as much T as possible has to be used to imprint the polymer. However, this is not the case for equilibrium binding applications. In a typical competitive assay protocol, depending on the label, only about 1 pmol or less of labelled analyte is applied per mg polymer. Aiming at reducing the costs of imprint preparation, we had found in preliminary experiments that it was possible to reduce the amount of T in the polymer recipe.5 We therefore investigated how much T is actually needed to create a sufficient number of binding sites.Fig. 1a shows a series of titration experiments of a fixed amount of 3H-theophylline with TFMAA/DVB copolymers that were imprinted with different amounts of theophylline, M : T ranging from 4 : 1 to 5000 : 1. Surprisingly, at M : T as high as 500 : 1, the imprinted polymer still bound significantly more of the analyte than the non-imprinted control at the same polymer concentration.Even at a ratio of 5000 : 1(!), the imprinting effect is clearly visible. Fig. 1b shows a plot of the amount of polymer required to bind 50% of the radiolabelled Table 1 Binding of 3H-theophylline to polymers containing different combinations of functional monomer and cross-linker. Amount of imprinted polymer necessary to adsorb 50% of the 3H-theophylline added to the assay, and binding of 3H-theophylline to the same amount of a nonimprinted control polymer relative to the imprinted polymer MAA AAm TFMAA EDMAa Imprinted polymer needed/mg 0.9 0.6 8 Control polymer binds (%) 2 40 10 DVBa Imprinted polymer needed/mg 0.3 1.1 0.2 Control polymer binds (%) 0 40 0 a EDMA-polymers contained 83% cross-linker; DVB-polymers were 69% cross-linked due to the presence of 20% ethylvinylbenzene in the commercial DVB.Fig. 1 (a) Binding of 3H-theophylline to imprinted polymers prepared at different M : T ratios, and to a non-imprinted control polymer as a function of polymer concentration. (b) Amount of polymer (P50) required to adsorb 50% of the added 3H-theophylline as a function of M : T.The polymers consisted of DVB (20 molar equivalents) and TFMAA (4 molar equivalents) and were prepared with toluene as the porogenic solvent. 168 Anal. Commun., 1999, 36, 167–170theophylline as a function of the M : T ratio.At ratios between 4 : 1 and 100 : 1, the required amount of polymer was � 1 mg ml21 and varied only slightly, the 12 : 1 polymer showing the highest binding. Only at the considerably higher ratio of 5000 : 1, the necessary amount of polymer increased substantially to 13 mg ml21. It seems to be logical that at a low ratio ( = more T), the total number of imprinted sites created is higher. However, due to the situation in non-covalent imprinting where association and dissociation processes of the prepolymerisation complex occur simultaneously at equilibrium, the number of good (high affinity) sites is smaller.On the other hand, at higher ratios ( = less T), the overall number of sites created is smaller, but due to the large excess of M the equilibrium is shifted toward complex formation, and the yield in good sites is higher. One advantage of using such high M :T ratios in the imprinting protocol is that solubility problems can be avoided in most cases, and even very polar compounds can be imprinted using non-polar solvents.Quantification of the affinity In an attempt to quantify the affinity of the polymers for theophylline, binding isotherms of 3H-theophylline binding to the MIPs were recorded. Non-covalently imprinted polymers normally contain a heterogeneous population of binding sites, and two- or three-site binding models have been used to determine approximates for the dissociation constants and number of binding sites.6 We have chosen to use an even further approximation by varying the labelled analyte concentration in a rather narrow concentration range (0.3–8 nM), and to determine apparent dissociation constants only for the high affinity sites, using a one-site Langmuir-type binding isotherm QB = QMappS/(KDapp + S) where QB is the amount of theophylline bound to the polymer at equilibrium, QMapp the apparent number of high affinity binding sites, S the free analyte concentration at equilibrium and KDapp the apparent dissociation constant.Although this does not reflect the real situation in the polymer, it allows a comparison of the different polymers with each other. Fig. 2 shows the binding isotherms obtained for polymers prepared with M : T of 4 : 1, 12 : 1, 100 : 1 and 500 : 1. The dissociation constants and the number of binding sites calculated from the isotherms are represented in Table 2. As can be seen, the high-affinity sites of all three polymers have dissociation constants in the same order of magnitude, the 100 : 1 polymer having the lowest KDapp and the 12 : 1 polymer the highest number of high affinity sites.This is consistent with the results from the titration experiments (Fig. 1). However, the apparent yield of high affinity binding sites (relative to the amount of template added) increases considerably with M : T (Table 2), which is logical since the high excess of functional monomer shifts the equilibrium toward complex formation.It has to be added here that when the original imprinting solvent (toluene) was used in the binding assays, the apparent yields obtained were at least one order of magnitude higher. However, for practical reasons (solubility of theophylline) a mixture of toluene and tetrahydrofuran (9 : 1, v/v) was used. Binding specificity of the polymers In order to verify that the polymers are specific for theophylline, we have determined the binding of theophylline (1,3-dimethylxanthine) and the related compounds theobromine (3,7-dimethylxanthine) and caffeine (1,3,7-trimethylxanthine) in competitive radioligand binding assays using 3H-theophylline. For this experiment, imprinted polymers with M : T of 4 : 1, 12 : 1, 100 : 1 and 500 : 1 were used.For all four polymers, the crossreaction with theobromine and caffeine was less than 0.1% as compared to theophylline. In a control experiment, a polymer was imprinted with caffeine at M : T of 100 : 1, and analysed in the same way as the theophylline polymers, but using 14Ccaffeine as the radioligand.The cross-reaction of this polymer with theophylline and theobromine was 10% and 3%, respectively, relative to caffeine. Although this confirms that the polymers are imprinted, it also indicates that care should be taken when interpreting cross-reactivities. These may in part reflect differences in the physiochemical properties of the mpounds (polarity, basicity etc.), in addition to the templatecomplementarity of the imprinted sites.Degree of cross-linking For chromatography applications, it has been shown that a high degree of cross-linking of the imprinted polymer was necessary to obtain good separations. Although a sufficient mechanical stability of the polymer is certainly required for applications such as HPLC, this might also indicate that a certain rigidity of the polymer matrix is necessary to preserve the imprinted memory.For example, the bifunctional cross-linker EDMA has mostly been used at approximately 80%11 and trifunctional CLs such as trimethylolpropane trimethacrylate at 50%14 of the total molar amount of M+CL in the polymer. Polymers used in equilibrium assays and as recognition layers in sensors are subjected to much less mechanical stress than in HPLC. We therefore wanted to know whether the degree of cross-linking can be reduced without abolishing the recognition properties of the polymer.This would make the polymer more flexible which could be an advantage for certain applications requiring thin imprinted films or membranes. At the same time it might be possible to incorporate more functional monomer into the polymer and to use more template, thus resulting in a higher binding capacity. We have prepared MAA/DVB-polymers having a M : T ratio of 12 : 1, which contained varying amounts of CL (19, 36 and 69%). In order to keep the overall monomer concentration Fig. 2 Binding isotherms of 3H-theophylline binding to imprinted polymers prepared with M : T of 4 : 1, 12 : 1, 100 : 1 and 500 : 1. Polymer composition: see Fig. 1. Table 2 Apparent dissociation constants (KDapp) and apparent number of high affinity binding sites (QMapp) for polymers prepared with different M: T ratios, and relative yield of high affinity binding sites. Relative Ratio KDapp/nMa QMapp/nmol g21a yield (%) 4 : 1 10.0 ± 0.5 22.8 ± 0.8 0.007 12 : 1 9.4 ± 0.7 40.9 ± 2.2 0.039 100 : 1 8.1 ± 0.9 8.2 ± 0.6 0.063 500 : 1 14.7 ± 2.4 2.9 ± 0.4 0.120 aKDapp and QMapp were calculated from the binding isotherms in Fig. 2 by non-linear least squares parameter estimation. Anal. Commun., 1999, 36, 167–170 169constant, appropriate quantities of styrene were added. Fig. 3 shows the binding of 3H-theophylline to the different polymers as a function of polymer concentration. Surprisingly, the lower cross-linked polymers had recognition properties comparable to the polymer with 69% cross-linking, although the apparent number of high quality sites decreased with the degree of crosslinking.The specificity of the sites was the same for all polymers. Less than 0.1% crossreactivity was observed with both caffeine and theobromine as compared to theophylline. With the corresponding control polymers, the same low amount of non-specific binding was observed independent of the degree of cross-linking. This demonstrates that a molecular memory can be introduced by molecular imprinting even in lowcrosslinked polymers.Conclusions The results described in the present paper show that the recipes for imprinted polymers to be used in applications such as equilibrium binding assays or sensors are considerably more flexible than previously anticipated, thus allowing the polymer recipes to be optimised specifically for these applications, regarding the choice of functional monomer and cross-linker with respect to the template of interest.It should be possible to use high functional monomer : template ratios in many cases. This may result in an increased relative yield of high-affinity binding sites, and the consumption of template is lowered. Moreover, solubility problems will be less prevalent in such systems, and less polar solvents can be used for imprinting which stabilise the prepolymerisation complex and yield better quality sites. It seems possible, if required, to reduce the degree of cross-linking of the polymer, which makes it more flexible and which may allow more functional monomer and template to be used to prepare the polymer.It is of particular note that, owing to its non-polar nature, DVB appears to be a good choice as the cross-linker, yielding polymers not only with higher analyte binding, but also with reduced non-specific binding. These findings extend the potential applicability of noncovalent molecular imprinting to template molecules which are expensive, not available in large quantities or which are very polar and thus only poorly soluble in organic solvents.Addendum During the reviewing process of the present manuscript it came to our knowledge that a related study on the aspect of template concentration in morphine-imprinted polymers has recently been published by others.15 They found that up to an M : T of 150 : 1, the imprinted polymer had a higher binding capacity than the non-imprinted control, although the overall capacity decreased with increasing M : T.Comparing these data with our data on theophylline-imprinted polymers, the lowest template concentration that still yields imprints is higher for morphine than for theophylline. Apart from the different monomers and cross-linkers used, this is probably also due to a higher degree of non-specific binding. The stronger basicity of morphine compared to theophylline causes it to bind more strongly to carboxyl groups in the polymer which are not situated in an imprinted site.These findings also show that imprinting with low template concentrations is possible and, although depending on the nature of the template, might be broadly applicable to polymers for binding assays. Acknowledgements K. H. acknowledges financial support by the EU Human Capital and Mobility program. References 1 D. J. Anderson, B. Guo, Y. Xu, L. M. Ng, L, J. Kricka, K. J. Sokgerboe, D. S.Hage, L. Schoeff, J. Wang, L. J. Sokoll, D. W. Chan, K. M. Ward and K. A. Davis, Anal. Chem., 1997, 69, 165R. 2 D. S. Hage, Anal. Chem., 1995, 67, 455R. 3 K. Mosbach and O. Ramström, Bio/Technology, 1996, 14, 163. 4 G. Wulff, Angew. Chem., Int. Ed. Engl., 1995, 34, 1812. 5 K. Haupt and K. Mosbach, Trends Biotechnol., 1998, 16, 468. 6 G. Vlatakis, L. I. Andersson, R. Müller and K. Mosbach, Nature (London), 1993, 361, 645. 7 M. Muldoon and L. Stanker, J. Agric. Food Chem., 1995, 43, 1424. 8 L. I. Andersson, Anal. Chem., 1996, 68, 111. 9 O. Ramström, L. Ye and K. Mosbach, Chem. Biol. 1996, 3, 471. 10 K. Haupt, A. G. Mayes and K. Mosbach, Anal. Chem., 1998, 70, 3936. 11 B. Sellergren, Enantiomer separation using tailor-made phases prepared by molecular imprinting, in A practical approach to chiral separations by liquid chromatography, ed. G. Subramanian, VCH, Weinheim, Germany, 1994, pp. 69–93. 12 Y. Cong and K. Mosbach, J. Org. Chem., 1997, 62, 4057. 13 J. Matsui, O. Doblhoff-Dier and T. Takeuchi, Anal. Chim. Acta, 1996, 343, 1. 14 M. Kempe, Anal. Chem., 1996, 68, 1948. 15 A. G. Mayes and C. R. Lowe in Drug Development Assay Approaches, Including Molecular Imprinting and Biomarkers, ed. E. Reid, H. M. Hill and I. D. Wilson, Guilford Academic Associates, Guilford, UK, 1998, pp. 28–36. Paper 9/01339C Fig. 3 Binding of 3H-theophylline to imprinted and control polymers prepared with different degrees of cross-linking, as a function of polymer concentration. The polymers were DVB/MAA-polymers containing 17% MAA, with a M : T of 12 : 1, prepared with toluene as the porogenic solvent. To vary the degree of cross-linking, styrene was partially substituted for DVB. 170 Anal. Commun., 1999, 36, 167–170

ISSN:1359-7337    

DOI:10.1039/a901339c

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Maximising microdialysis sampling by optimising the internal probe geometry Nelson Torto,*a† Ekaterina Mikeladze,b Lo Gorton,a Elisabeth Cs�oregic and Thomas Laurelld a Department of Analytical Chemistry, University of Lund, PO Box 124 SE 221 00 Lund, Sweden. E-mail: Nelson.Torto@analykem.lu.se; Fax: +46 46 222 4544; Tel: +46 46 222 0103 b Group of Biochemical Neuropharmacology I, Beritashvili Institute of Physiology, Georgian Academy of Science, Tbilisi, Georgia c Department of Biotechnology, University of Lund, Lund, Sweden d Department of Electrical Measurements, University of Lund, Lund, Sweden Received 11th February 1999, Accepted 17th March 1999 An in-house microdialysis probe equipped with 3 different inner cannulae exhibiting different inner and outer radii was used to sample glucose, maltotriose, maltopentaose and maltoheptaose as model analytes in order to evaluate its performance by means of optimised internal probe geometry.The results were correlated to the inner cannula ratio (Rir), defined as the inner cannula’s inner radius divided by the inner cannula’s outer radius.Extraction fraction (EF) or relative recovery (RR) showed a dependency on Rir for all the investigated analytes at all the perfusion rates. The EF for glucose improved up to 10% for a nominal increase of 0.27 in Rir. The sensitivity of the EF of saccharides to Rir decreased with the size of molecule. Therefore the Rir represents a parameter that should be further investigated in order to maximise the performances of concentric type microdialysis probes.Introduction To date, the only parameters available to optimise the extraction fraction (EF) or relative recovery (RR) for microdialysis probes for in vivo and in vitro experiments are: membrane cut-off, membrane length, perfusion rate as well as the membrane material. This is despite the extensive theoretical and experimental treatment of the microdialysis experiment in areas of neuroscience, pharmacology and biotechnology.1–5 The choice of microdialysis probes is based on their size and membrane cut-off.In biotechnological applications, the microdialysis probe’s size is not a limiting factor. However in neurochemistry applications, small probe dimensions are very important. Renno et al. have recently described a short microdialysis probe (1 mm) used to measure precisely the concentrations of amino acid neurotransmitters in delineated and pre-measured areas of the central nervous system.6 Monitoring of processes which exhibit fast kinetics where the uptake of neurotransmitters, decomposition, reaction cascades or dilution is experienced presents a problem when larger ( > 1 mm) probe designs with lower EF are used.However the probe’s size relative to the nerve terminals is very important as neurotransmitters could be measured at release sites if accessed by a small probe design. Very often measurements are carried out in secondary release areas where diffusion considerations for neurotransmitters into these areas and the microdialysis membrane have to be addressed.7 Similarily, hydrolytic characteristics of enzymes are best studied by sampling at the site of their action.Microdialysis has recently been coupled successfully to many analytical techniques enabling sampling and subsequent sensitive measurement of neurotransmitters, metabolites, administered drugs or hydrolysates.6,8–12 However, microdialysis achieves high RR only at ultra-slow perfusion rates.13 The disadvantages of working at low perfusion rates are obvious as these include flow fluctuation as well as longer periods to generate samples as this consequently limits timely monitoring. As a sampling technique, microdialysis offers time averaged rather than time point data.The latter is preferred as the time resolution of microdialysis sampling could be improved if the performance of microdialysis probes (large or small) is maximised.This could be realised if probes exhibiting high EF at high perfusion rates are used to acquire analytes at very short sampling times. Microdialysis literature does not indicate efforts to maximise the performance of microdialysis probes by utilising the dimensions of the internal probe geometry. The inner cannula dimension has not been a parameter accessible to the research community. This is due to the fact that most of the microdialysis research is carried out using commercially available probes.Their cost as well as design inflexibility limits the user’s ability to change any parts of the probe without damaging it. Further, it has come to our attention that the in situ tunable probe14 achieves higher EF compared to commercially available probes equipped with membranes exhibiting the same characteristics, cut-off and effective dialysis length.15,16 This is attributed to the probe’s flexible design and its internal hydrodynamics.Therefore, in this work the performance of an in situ tunable probe equipped with inner cannulae exhibiting different inner and outer diameters is reported by evaluating it for the sampling of glucose, maltotetraose, maltopentaose and maltoheptaose. The preliminary data show a dependency of EF on Rir (defined as the inner cannula’s inner radius divided by the inner cannula’s outer radius) for a microdialysis membrane with a constant inner radius.The results indicate a possibility to enhance the performance of microdialysis probes by utilising inner cannula dimensions. Experimental Reagents Glucose, maltotriose, maltopentaose, and maltoheptaose were obtained from Sigma (St. Louis, MO, USA). The 50% w/w NaOH was obtained from J. T. Baker, (Deventer, Holland) and NaOAc was obtained from Merck (Darmstadt, Germany). All solutions were prepared using HPLC grade water obtained from a Millipore system (Millipore, Bedford, USA).† On leave from: Department of Chemistry, University of Botswana, P/Bag 0022, Gaborone, Botswana. Anal. Commun., 1999, 36, 171–174 171Microdialysis experiment An in-house microdialysis probe14 was equipped with inner cannulae exhibiting different inner and outer diameters respectively, see Table 1. The probe was fitted with a freshly cut polysulfone membrane from Fresenius A/G (St. Wendel, Germany) with a 10 mm effective dialysis length and a 30 kDa cut-off.Microdialysis experiments were carried out by inserting the membrane fitted microdialysis probe into a 5 ml vial housed in a Pierce heating and stirring module no. 18971 (Pierce, Rockford, USA). The probe was perfused at 9, 7, 5, 3 and 1 ml min21 to evaluate the EF of saccharides. The perfusion water was delivered by a CMA/100 syringe pump and 20 ml of dialysate were injected by a CMA/160 on-line injector, all from CMA Microdialysis AB (Stockholm, Sweden). All experiments were carried out at 40 °C.Chromatographic system A Dionex 500 chromatographic system, controlled by PeakNet software, Dionex (Sunnyvale, California, USA) was used to separate and detect the carbohydrates during the investigations. Chromatographic and detection conditions have been reported elsewhere.17 Results and discussion The concentric type of microdialysis probe has been most widely used in neurochemistry, pharmacokinetics and bioprocess monitoring compared to the loop as well as the linear types of microdialysis probes.This preference indicates an advantage characteristic of a well designed probe. However, although the application of microdialysis and its coupling to analytical techniques are increasing,1 there are a few parallel efforts to maximise the performance of the popular concentric design. By utilising the various models presented to describe the mass transport processes during microdialysis experiments, 3,5,18 the analysis and data presented herein demonstrates the significance of the inner cannula dimensions as an additional parameter to increase the performance of a concentric type microdialysis probe.Bungay et al.5 have shown dialysate EF to be dependent on the perfusion rate (Qd), resistances to dialysate (Rd), membrane (Rm) and tissue or external solution (Rext) respectively as in eqn. (1). EF = 1 – exp 1 d m d ext – ( ) Q R R + + é ë ê ù û ú (1) For a concentric probe with components and geometries described in Fig. 1, the inner cannula role can be demonstrated. The resistances to diffusion can be expressed independently as shown in eqn. (2), R r D S = D eff f (2) where Dr ( = rb 2 ra) is the thickness of the cylindrical flow path along which the dialysates travel, Deff is the effective diffusion coefficient, S is the membrane surface area and f is the volume fraction available for the dialysates.5 However, for these investigations only the resistance to dialysates needs to be considered as it is the only resistance affected by the inner cannula dimensions; R r r L r D d edl eff = é ë êê ù û úú 13 70 b a b p – (3) where Deff is the dialysate effective diffusion coeffecient and the fraction 13/70 is based on an approximation for uniform flux for a given geometry of the probe channel and the ratio of the annulus residence time.5 From Fig. 1, eqn. (1), (2), and (3) it is obvious that if the EF has to be maximised, the Ledl and ra should have their maximum values.Deff could be affected by Rext or the interaction of the membrane with the analyte and/or biomatrix. Ledl is often maximised using a long membrane which is normally 3 and 10 mm in length for in vivo and in vitro work respectively. As long as the membrane size is maintained for a given microdialysis probe, the value of rb will always be constant. However, the value of ra depends on the geometry of the inner cannula. From eqn. (1), as EF increases the value of Rd is decreased.This is achieved by increasing the outer radius of the inner cannula. Increasing ro can be carried out independently or simultaneously with ra. In this study, the potential to increase the performance of microdialysis probes was investigated using glucose and higher oligosaccharides as model analytes. Glucose has been monitored extensively in peripheral tissue, skeletal muscles and organs.3 Maltotetraose, maltopentaose and maltoheptaose were used to evaluate their performance when sampling larger analytes.The effect of the inner cannula ratio (Rir) as expressed in eqn. (4), was evaluated. R r r ir o = a (4) Other than a recent report by Jolly and Vezina,19 to the best of our knowledge, there has been no publication that has detailed the dimensions of the inner cannula for the concentric type of microdialysis probes. The commercially available probe systems lack the desired flexibility that enable adaptable use in a diverse research environment which requires design and optimisation of experiments.Of course, flexible probe designs can only be realised when research is carried out with in-house constructed probes as this approach reduces the long term costs of acquiring probes. In-house constructed probes also allow the user to maximise the performance of the probe by changing the membrane type, membrane cut-off, membrane effective dialysis length as well as the inner cannula dimensions and the overall probe size.However, for applications using in-house designs where silica or other tubing has been used to fabricate a microdialysis probe, the dimensions of the tubing (inner cannula) are sometimes given. For example O’Shea et al.12 used PE tubing with an equivalent Rir = 0.46, Schneiderheinze and Hogan20 used a fused silica capillary of Rir = 0.5, Renno et al.6 a fused Table 1 Dimensions of the inner cannulae used in the investigationsa Cannula ro/mm ra/mm Rir A 0.15 0.10 0.67 B 0.135 0.08 0.59 C 0.125 0.05 0.40 a The value for rb was 0.25 mm for all the investigations.Fig. 1 A cross-section of a microdialysis probe showing the inner cannula, membrane, glued tip, outer cannula and the effective dialysis length (Ledl). The symbols ro, ra and rb represent the outer radius of the inner cannula, the inner radius of the inner cannula and the inner radius of the microdialysis membrane respectively. 172 Anal. Commun., 1999, 36, 171–174capillary Rir = 0.69 and Kaptein et al.13 also used a fused silica capillary of Rir = 0.83.In these examples the inner radius or surface area of the microdialysis membrane were not given. Therefore a direct comparison of the probe’s performances is not possible, although disparity in Rir is obvious as also reflected by commercially available probes. The CMA 10, 12, and 20 type microdialysis probes have Rir = 0.4. Agtho Tho’s AB microdialysis probes have an Rir = 0.8 and 0.66.Effect of inner cannula ratio on extraction fraction for saccharides Fig. 2a shows the EF profile for glucose, maltotriose, maltopentaose and maltoheptaose using a probe with Rir = 0.67. The EF of saccharides increases with a decrease in perfusion rate and is dependent upon the size (diffusion coefficient) of the analyte molecules. Fig. 2b shows the effect of Rir on glucose EF for microdialysis probes equipped with inner cannulae exhibiting Rir = 0.4, 0.59 and 0.67. It is evident that EF increases as Rir is increased.The EF values evaluated for glucose at lower perfusion rates ( < 5 ml min21) were improved by at least 5% as the Rir was increased from 0.4 to 0.67. However, as shown in Fig. 2c, the performance of inner cannulae A and B is indistinguishable for glucose. The EF also shows a dependency on Rir for all investigated inner cannulae especially at lower perfusion rates. Effect of inner cannula ratio on the extraction fraction for maltoheptaose EF values evaluated for maltoheptaose at all the investigated perfusion rates showed similar trends to that of glucose.The EF exhibited a dependency on Rir at lower perfusion rates. However, the EF of maltoheptaose did not show a comparable increase to that of glucose or maltotriose for the different inner cannulae as shown in Fig. 3a. This change in EF as a result of an increased analyte size has almost always been linked to diffusion coefficients, but it could also be associated with the inner cannulae dimensions.Fig. 3b shows an EF profile for maltopentaose, which is comparable to that of glucose even though their EFs (see Fig. 2a) are significantly different. Conclusions Given that samples acquired by microdialysis are compositionally unique, as they cannot be carried out in duplicates or higher, these preliminary results sufficiently indicate a possibility to increase the performance of microdialysis probes by utilising the internal probe geometry.These studies demonstrated the feasibility to efficiently utilise Rir to maximise the performance of a tunable microdialysis probe for in vivo and in vitro work especially if inner cannula (A) is used instead of (C). It has also been emphasized that the inner cannula dimensions should be chosen based on performance considerations for other probe designs. The impact of these data in fabricating smaller or larger probes depending on application can only be realised if more Fig. 2 (a) EF profile for saccharides when utilising a probe with an inner cannula exhibiting an Rir = 0.67. (b) EF profile for glucose when utilising a probe with inner cannulae exhibiting an Rir = 0.40, 0.59 and 0.67. The shaded bars represent the given perfusion rates in ml min21. (c) EF for glucose at all perfusion rates with inner cannulae exhibiting Rir = 0.40 (C), 0.59 (B) and 0.67 (A). Fig. 3 (a) EF for maltoheptaose at all investigated perfusion rates with inner cannulae exhibiting an Rir = 0.40 (C), 0.59 (B) and 0.67 (A).Compare with 2(c) and 3(b). (b) EF for maltopentaose at all investigated perfusion rates with inner cannulae exhibiting an Rir = 0. 40 (C), 0.59 (B) and 0.67 (A). Anal. Commun., 1999, 36, 171–174 173extensive investigations are carried out that would systematically vary the dimensions of the inner cannula in relation to the microdialysis membrane’s surface area, membrane type as well as inner diameter. The EF for glucose has been demonstrated to increase up to 10% for a 0.2 increase in Rir.Although the investigations were carried out for n @ 3, the present changes are significant. The results also show an increase in the EF for all investigated saccharides: glucose, maltotriose, maltopentaose and maltoheptaose for all perfusion rates with increasing Rir. Acknowledgements EC/INTAS 96-1432, MFR, SSF (EC & EM), the Swedish Board for Technical and Industrial Development (NUTEK) as well as the Swedish Natural Science Research Council (NFR) are acknowledged for financial support.References 1 N. Torto, T. Laurell, L. Gorton and G. Marko-Varga, Anal. Chim. Acta, 1999, 379, 281. 2 N. Torto, T. Laurell, L. Gorton and G. Marko-Varga, Trends Anal. Chem., 1999, in the press. 3 T. E. Robinson and J. B. Justice Jr, in Microdialysis in the Neurosciences, ed. J. P. Huston, Elsevier, Amsterdam, 1991. 4 J. Kehr, J. Neurosci. Methods, 1993, 48, 251. 5 P. M. Bungay, P.F. Morrison and R. L. Dedrick, Life Sci., 1990, 46, 105. 6 W. M. Renno, M. A. Mullet, F. G. Williams and A. J. Beitz, J. Neurosci. Methods, 1998, 79, 217. 7 G. Di Chiara, Trends Pharm. Sci., 1990, 11, 116. 8 N. Torto, R. Hofte, R. A. van der Hoeven, U. Tjaden, L. Gorton, G. Marko-Varga. and C. van Bruggnik, J. Mass Spectrom., 1998, 33, 334. 9 S. Y. Zhou, H. Zuo, J. F. Stobaugh, C. E. Lunte and S. Lunte, Anal. Chem., 1995, 67, 594. 10 M. I. Davies and C. E. Lunte, Chem. Soc. Rev., 1997, 26, 215. 11 B. L. Hogan, S. M. Lunte, J. F. Stobaugh, and C. E. Lunte, Anal. Chem., 1994, 66, 596. 12 T. J. O’Shea, M. W. Tetling-Diaz, S. M. Lunte, C. E. Lunte and M. L. Smyth, Electroanalysis, 1992, 4, 463. 13 W. A. Kaptein, J. J. Zwaagstra, K. Venema and J. Korf, Anal. Chem., 1998, 70, 4696. 14 T. Laurell and T. Buttler, Anal. Methods Instr., 1995, 2, 197. 15 T. Buttler, C. Nilsson, L. Gorton, G. Marko-Varga and T. Laurell, J. Chromatogr A., 1996, 725, 41. 16 N. Torto, T. Buttler, L. Gorton, G. Marko-Varga, H. Stålbrand and F. Tjerneld, Anal. Chim. Acta, 1995, 313, 15. 17 N. Torto, J. Bång, S. Richardson, T. Laurell, G. Nilsson, L. Gorton and G. Marko-Varga, J. Chromatogr. A., 1998, 806, 265. 18 J. K. Hsiao, B. A. Ball, P. F. Morrison, N. I. Mefford and P. M. Bungay, J. NeuroChem., 1990, 54, 1449. 19 D. Jolly and P. Vezina, J. Neurosci. Methods, 1996, 68, 259. 20 J. Schneiderheinze and B. L. Hogan, Anal. Chem., 1996, 68, 3758. Paper 9/01175G 174 Anal. Commun., 1999, 36, 171–174

ISSN:1359-7337    

DOI:10.1039/a901175g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Fluorescent indicators for inositol 1,4,5-trisphosphate based on bioconjugates of pleckstrin homology domain and fluorescent dyes Kenzo Hirose,*ab Hiroshi Takeshimaab and Masamitsu Iinoab a Department of Pharmacology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8654, Japan. E-mail: hirose@calcium.cmp.m.u-tokyo.ac.jp; Fax: +81 3 3815 9360; Tel: +81 3 3812 2111 ext. 3414 b CREST Japan Science and Technology Corporation, Tokyo 113-8654, Japan Received 17th February 1999, Accepted 16th March 1999 Bioconjugates of fluorescent dyes and the recombinant pleckstrin homology (PH) domain of phospholipase Cd1 were produced with the aim of developing a method to quantify inositol 1,4,5-trisphosphate (IP3) in biological samples. We replaced Cys-96 of the PH domain with Ser while retaining Cys-48 to which thiol-reactive fluorescent dyes can be coupled specifically.Acrylodan- and Dapoxyllabelled C96S PH domain mutants exhibited fluorescence upon UV illumination with an emission peak at wavelengths of 505 and 514 nm, respectively.IP3 induced decreases in the fluorescence intensity with a red shift in the emission spectra. The dissociation constants (Kds) of the acrylodanand Dapoxyl-labelled PH domains for IP3 were 659 and 586 nM, respectively. An additional mutation (C96S/V58K) in the PH domain decreased the Kds by ~ 50%, providing a more sensitive method. The results indicate that these bioconjugates are promising as fluorescent indicators for IP3 quantification.Inositol 1,4,5-trisphosphate (IP3) is an intracellular signal molecule controlling the Ca2+ concentration which, in turn, regulates many cellular functions. Stimulation of G proteincoupled receptors or receptor-tyrosine phosphorylation pathways activates phospholipase C, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate resulting in IP3 generation. 1 IP3 opens the IP3 receptor/channels on the intracellular Ca2+ stores resulting in elevation of cytoplasmic Ca2+ concentration in complex spatio-temporal patterns, and regulates Ca2+ dependent cellular events in many types of cells.1,2 The IP3 signal is deactivated by the degradation of IP3 by IP3 5-phosphatase and 3-kinase, which catalyze the dephosphorylation of phosphate at position 5 (ref. 3) and phosphorylation of the hydroxy group at position 3 (ref. 4), respectively. Rapid and simple methods for the quantification of IP3 are useful techniques to understand the regulation of the intracellular metabolism of inositol phosphates.To measure IP3 concentrations in biological samples, two methods have so far been used. The first is based on HPLC separation.5 Various inositol phosphates are separated on an anion exchange column in an HPLC system. IP3 is separated from its isomers such as inositol 1,3,4-trisphosphate and other inositol phosphates, and is quantified using the radioactivity.The major advantage of this method is that various inositol phosphates can be analysed simultaneously owing to each having a specific retention time. The other method utilizes microsomal IP3-binding proteins.6 This method has a great advantage over the HPLC method because it does not require the steps of separation of the inositol phosphate species. The selectivity of the IP3-binding proteins ensures specificity over other inositol phosphate species in ordinary biological samples.Although these two methods are widely used, both methods generally require a radioisotope, which raises safety issues. Fluorescent bioconjugates which consist of a fluorescent compound and a protein have been developed for non-isotopic quantification of various biomolecules.7–10 Hence we designed and constructed fluorescent IP3 indicators composed of an IP3- binding protein and a fluorescent dye. As the IP3-binding protein, we employed a recombinant protein of the pleckstrin homology (PH) domain from phospholipase Cd1, which has been shown to bind specifically to IP3 in preference to other inositol phosphates.11,12 Experimental An amplified cDNA encoding the PH domain of phospholipase Cd1 (residues 11–140) was obtained by the reverse transcriptase polymerase chain reaction (RT-PCR) with a pair of primers based on the published sequence.13 Site-directed mutagenesis was performed to generate PH domains with altered amino acids using a PCR-based site-directed mutagenesis kit (Mutan- Super Express Km, Takara, Tokyo, Japan).cDNAs encoding the wild-type and mutant PH domains, whose sequences were confirmed by an automated DNA sequencer, were subcloned into the bacterial expression plasmid, pET-23a. The recombinant proteins were expressed as His-tagged fusion proteins in E. Coli., BL-21(DE3) and were purified by Ninitrilotriacetate affinity chromatography and subsequent cation exchange chromatography. Yields were typically ~ 50 mg l21 growth medium.The purified proteins were concentrated and stored at 280 °C until use. The thiol-reactive fluorescent dyes, fluorescein iodoacetamide, acrylodan and Dapoxyl(2-bromoacetamidoethyl) sulfonamide (at final concentration 10–20 mM), were then conjugated to the purified PH domains (2 mg) in a 2.5 ml reaction solution containing 150 mM NaCl, 1 mM EDTA and 0.1 M sodium phosphate (pH 8.0). Following reaction for 2 h at room temperature, the dye-labelled proteins were separated from unreacted dye by gel filtration with a phosphate-buffered solution containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.47 mM KH2PO4 (pH 7.4) as a running solution.For the Dapoxyl labelling, 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) (1%) was included in the reaction solution to minimize the aggregation of the dye and was removed by Extracti-Gel D detergent removing gel (Pierce, IL, USA). The typical molar ratio of dye to protein after labelling was estimated to be 0.3–0.6 based on the protein concentration and absorption spectrum.The yield of the dyelabelled protein has not been extensively optimized in the present study. The fluorescence spectra were obtained in the phosphate-buffered solution containing 0.1% CHAPS on a fluorescence spectrophotometer (FP-750, JASCO, Japan) at room temperature. Anal. Commun., 1999, 36, 175–177 175Results and discussion In order to label the PH domain at specific sites, we used a set of thiol-reactive fluorescent dyes for labelling.The PH domain contains two Cys residues at positions 48 and 96, which are potential targets of the thiol-reactive dyes. To eliminate the complexity arising from multiple labelling sites, we substituted Cys-96 of the PH domain with Ser while preserving Cys-48, using site-directed mutagenesis (C96S PH domain). The C96S PH domain was then labelled with a fluorescent dye. The fluorescein-labelled C96S PH domain had the maximal emission at 519 nm.Addition of IP3 induced an increase in fluorescence intensity up to 13% without any shifts in the emission spectra (not shown). This result indicates that the dyelabelled PH domain binds to IP3 and that this binding can be monitored by changes in the fluorescence intensity. Since Cys- 48 is located sufficiently far from the IP3-binding site according to the tertiary structure,14 the fluorescence augmentation of the dye is unlikely to be due to direct contact with bound IP3.Rather, it is likely that the fluorescein detects changes in the microenvironment around the Cys-48 residue as a result of conformational changes in the protein upon IP3-binding. In an attempt to obtain greater changes in the signal intensity, we used other thiol reactive fluorescent dyes, namely acrylodan15 and Dapoxyl(2-bromoacetamidoethyl)sulfonamide,16 which are known to be environment-sensitive. The acrylodan-labelled C96S PH domain also showed changes in the fluorescence emission spectrum (Fig. 1A) but in a pattern different from that observed for the fluorescein-labelled PH domain. IP3 elicited a decrease in the fluorescence intensity at shorter wavelengths (@ ~ 520 nm), while the fluorescence intensity at longer wavelengths (! ~ 520 nm) changed less significantly. This spectral change is advantageous because ratiometric dual-emission spectrofluorometry would allow us more precise quantification by correcting for any differences in the probe concentration.The Dapoxyl-labelled PH domain showed a relatively more pronounced decrease in fluorescence intensity upon IP3 addition (Fig. 1B). The extent of change was greater at shorter wavelengths than at longer wavelengths similar to that observed for the acrylodan-labelled PH domain (see Fig. 1A and B). Taking the data from the acrylodan-labelled PH domain also into consideration, the results suggest that IP3 induces conformational changes in the PH domain and consequently reduces the polarity of the microenvironment around Cys-48.We determined the dissociation constants (Kds) for the acrylodan- and Dapoxyl-labelled C96S PH domains by analysing the changes in the fluorescence intensities (Fig. 2). The Kds were estimated to be 659 ± 13 nM (mean ± standard error of means (SEM), n = 3) and 586 ± 12 nM (mean ± SEM, n = 4) for the acrylodan- and Dapoxyl-labelled PH domains, respectively. Thus, although the values were higher than the Kd of unmodified PH domain ( ~ 100 nM, unpublished data), the dyelabelled PH domains still showed high affinity for IP3.The maximal changes in fluorescence intensity observed were 74% (at 450 nm) and 88% (at 514 nm) for the acrylodan- and Dapoxyl-labelled PH domains, respectively. These results suggest that these bioconjugates are useful indicators of IP3. The major advantage of recombinant proteins is the feasibility of altering the properties of the proteins by manipulation of the cDNA.We introduced an additional mutation V58K into the C96S PH domain, expecting an increase in the affinity of the PH domain for IP3, because Val-58 is located near the binding site of IP3.14 Therefore a phosphate group from IP3 may come in contact with the e-amine of the substituted Lys. Accordingly, we constructed the mutant PH domain C96S/V58K and labelled the expressed protein with fluorescent dyes. Both the acrylodanand Dapoxyl-labelled C96S/V58K PH domains changed their fluorescence emission spectra upon addition of IP3 in a manner Fig. 1 Fluorescence emission spectra of the (A) acrylodan- and (B) Dapoxyl-labelled C96S PH domains of phospholipase Cd1. The number indicates the concentration of IP3 in mM. Fig. 2 Analysis of the IP3 concentration-dependence of the fluorescence intensity for the determination of Kd. The fluorescence intensity was normalized so that the intensity without IP3 addition is 100%. The data is representative of three and four experiments for (A) acrylodan- and (B) Dapoxyl-labelled PH domains, respectively.The data (circles) are fitted by an equation, 1 2 A 3 {Kd + [IP3] + C 2 ([IP3]2 + 2 [IP3]Kd 2 2C[IP3] + 2CKd + C2 + Kd 2)1/2}/(2C), where A and C are the maximal changes in normalised fluorescence intensity [(Fmax 2 Fmin)/Fmax] and the concentration of the dye-labelled PH domain (60 nM), respectively. The equation was derived from the chemical equilibration in the formation of a 1 : 1 complex of IP3 and the PH domain. 176 Anal.Commun., 1999, 36, 175–177similar to the observations in the C96S PH domain. From an analysis of the dose-dependent changes in fluorescence intensities we determined that the introduction of V58K indeed increased the affinity for IP3: the Kds for the acrylodan- and Dapoxyl-labelled C96S/V58K were 410 ± 14 nM (mean ± SEM, n = 3) and 382 ± 2 nM (mean ± SEM, n = 4). Although this mutation reduced the extent of changes in fluorescence intensity (57% for acrylodan and 72% for Dapoxyl), the improvement in the affinity is advantageous for the sensitive quantification of IP3.In this work, we developed fluorescent indicators for IP3 by conjugating mutant PH domains with fluorescent dyes and found that acrylodan- and Dapoxyl-labelled mutant PH domains (C96S and C96S/V58K) are useful for the quantification of IP3. The proposed method is advantageous over the previously used methods in which the use of radioisotopes makes the assays complicated.In addition, no requirement of separation steps for sample preparation allows for a potential new application. For example, IP3 production and/or degradation reaction may be monitored without interrupting the reaction. If the bioconjugate is introduced into cells, IP3 may be directly imaged within the living cells in the same manner that Ca2+ is visualized by fluorescent indicators such as fura-2 (ref. 17). Further screening for the suitable mutations and conjugation dyes should optimize the properties which are necessary for these specific applications. In conclusion, the method using protein-fluorescent dye conjugates presented here may prove to be a valuable substitute for conventional radioisotopic methods for analysis of the IP3 content of cells. Acknowledgements Supported in part by the Ministry of Education, Science, Sports and Culture of Japan. The authors thank Dr M.Tanabe for her technical assistance and Dr K. Kikuchi for valuable comments on the manuscript. References 1 M. J. Berridge, Nature, 1993, 361, 315. 2 D. E. Clapham, Cell, 1995, 80, 259. 3 C. P. Downes, M. C. Mussat and R. H. Michell, Biochem. J., 1982, 203, 169. 4 R. F. Irvine, A. J. Letcher, J. P. Heslop and M. J. Berridge, Nature, 1986, 320, 631. 5 H. Binder, P. C. Weber and W. Siess, Anal. Biochem., 1985, 148, 220. 6 S. Palmer, K. T. Hughes, D. Y. Lee and M. J. Wakelam, Cell. Signal., 1989, 1, 147. 7 G. Gilardi, L. Q. Zhou, L. Hibbert and A. E. G. Cass, Anal. Chem., 1994, 66, 3840. 8 M. Burne, J. L. Hunter, J. E. T. Corrie and M. R. Webb, Biochemistry, 1994, 33, 8262. 9 V. Schauer-Vukasinovic, L. Cullen and S. Daunert, J. Am. Chem. Soc., 1997, 119, 11 102. 10 J. S. Marvin and H. W. Hellinga, J. Am. Chem. Soc., 1998, 120, 7. 11 M. Yoshida, T. Kanematsu, Y. Watanabe, T. Koga, S. Ozaki, S. Iwanaga and M. Hirata, J. Biochem., 1994, 115, 973. 12 M. A. Lemmon, K. M. R. O. B. Ferguson, P. B. Sigler and J. Schlessinger, Proc. Natl. Acad. Sci. U.S.A., 1995, 92, 10 472. 13 P. G. Suh, S. H. Ryu, K. H. Moon, H. W. Suh and S. G. Rhee, Cell, 1988, 54, 161. 14 K. M. Ferguson, M. A. Lemmon, J. Schlessinger and P. B. Sigler, Cell, 1995, 83, 1037. 15 F. G. Prendergast, M. Meyer, G. L. Carlson, S. Iida and J. D. Potter, J. Biol. Chem., 1983, 258, 7541. 16 Z. Diwu, Y. X. Lu, C. L. Zhang, D. H. Klaubert and R. P. Haugland, Photochem. Photobiol., 1997, 66, 424. 17 D. A. Williams, K. E. Fogarty, R. Y. Tsien and F. S. Fay, Nature, 1985, 318, 558. Paper 9/01274E Anal. Commun., 1999, 36, 175–177 177

ISSN:1359-7337    

DOI:10.1039/a901274e

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Photoluminescent oxygen sensing using palladium tetrakis(4-carboxyphenyl)porphyrin self-assembled membrane on alumina Yutaka Amao,a Keisuke Asaia and Ichiro Okura*b a Aerodynamic Division, National Aerospace Laboratory, Jindaiji-higashi, Chofu, Tokyo 182-8522, Japan. b Department of Bioengineering, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. E-mail: iokura@bio.titech.ac.jp Received 26th January 1999, Accepted 16th March 1999 An optical oxygen sensor based on the photoluminescent quenching of palladium tetrakis(4-carboxyphenyl)porphyrin (PdTCPP) self-assembled membrane on an alumina plate has been developed.The luminescence intensity of PdTCPP membrane decreased with increase of oxygen concentration, indicating that this membrane is a highly sensitive device for oxygen concentration. The signal changes of this membrane are large enough to allow quantification of oxygen with good sensitivity (I0/I100 > 3).The response times of the sensor are 36 s on going from argon to oxygen and 148 s from oxygen to argon. This is a photostable sensing membrane that exhibits minimal decrease (ca 5%) in initial intensity after continuous irradiation for 24 h. A variety of devices and sensors based on photoluminescent quenching of organic dyes have been developed for measuring oxygen pressure. Many optical oxygen sensors are composed of organic dyes, such as polycyclic aromatic hydrocarbons1,2 and transition metal complexes,3–7 immobilized in an oxygen permeable polymer.As organic dyes interact with polymer molecules, the properties of sensing membranes strongly depend on the polymer matrices. To overcome this problem, selfassembled membranes (SAM) have been exploited. SAM techniques have attracted much attention as effective methods to design a solid surface with a well-defined composition, structure and thickness for interfacial optical studies.8–12 Among a number of SAM techniques, the use of compounds with the carboxy functional group is most prevalent in preparing a SAM of an organic compound on a metal oxide surface (Al2O3, Fe2O3, etc.).Compounds with the carboxy functional group form a SAM on a metal oxide surface via a chemisorptive ester bond. As the sensing dyes are arranged on a solid surface without the support of polymer by using this technique, a highly sensitive device for oxygen detection will be established by using SAM. On the other hand, among luminescent dyes, palladium porphyrins reveal strong room-temperature phosphorescence with high quantum yield and a long natural lifetime.13 Therefore, these compounds have frequently been utilized as oxygen sensitive dyes.14,15 Among porphyrins, palladium tetrakis(4- carboxyphenyl)porphyrin (PdTCPP) is suitable as an optical oxygen sensing device using SAM because of the formation of a stable membrane on alumina and its carboxyl group.In this study we hope to describe the fabrication of PdTCPP luminescence compound for oxygen sensing as a self-assembled membrane on an alumina plate and the optical oxygen sensing properties of this sensing membrane.Experimental Materials and preparation of sensing membrane Palladium tetrakis(4-carboxyphenyl)porphyrin (PdTCPP) was synthesized with refluxing tetrakis(4-carboxyphenyl)porphyrin and excess palladium chloride in dimethylformamide (DMF) solution [UV-visible spectrum: lmax/nm (DMF): 418 (Soret), 523, 556 (Q-band); emission spectrum: lemission/nm (DMF): 700 (lexcitation: 418 nm)].The membrane was constructed by dipping the alumina plate (for TLC; obtained from Merck Ltd., Poole, Dorset, UK) into 0.1 mmol dm23 PdTCPP–dimethyl sulfoxide (DMSO) solution for 24 h. After modification, the plate was rinsed with water and ethanol several times. PdTCPP physically adsorbed onto alumina was removed by ultrasonication and then the membrane was dried under vacuum overnight.The excitation spectrum of membrane was the almost same as the absorption spectrum of PdTCPP in DMF solution. As the number of PdTCPP molecules adsorbed on the alumina surface was not determined, the concentration was estimated to be ca 1025 mol dm23 from luminescence intensity. Spectroscopic measurements Steady state luminescence spectra of the membranes were measured using a Hitachi Model F4010 fluorescence spectrometer with a 150 W xenon lamp as a visible excitation light source.Excitation and emission bandpasses were 5.0 nm. The sample membranes were mounted at a 45° angle in the quartz cell, for steady state luminescence experiments, to minimize light scattering from the sample and substrate. Different oxygen concentrations (in the range 0–100%) in a gas stream were produced by controlling the flow rates of oxygen and argon gases entering a mixing chamber. The total pressure was maintained at 760 Torr. All experiments were carried out at room temperature.Results and discussion Luminescence spectrum The membrane showed strong phosphorescence at 700 nm when excited at wavelengths attributed to the Soret and Q bands, as is shown in Fig. 1. The phosphorescence intensity of the membrane depended on the oxygen concentration. The intensity decreased with an increase of oxygen concentration, indicating that this membrane can be used as an optical oxygen sensing device based on phosphorescence quenching by oxygen. The ratio I0/I100 is used as a measure of the sensitivity of the sensing membrane, where I0 and I100 represent the phosphorescence intensities detected from a membrane exposed to 100% argon and 100% oxygen, respectively.The I0/I100 of this membrane is estimated to be 17.7. In the case of the PtTFPP [platinum tetrakis(pentafluorophenylporphyrin)] –polystyrene (PS) system, on the other hand, the I0/I100 value has previously been reported to be 3.0.16 The I0/I100 of the new membrane is greater than that of PtTFPP– PS membrane, indicating that PdTCPP self-assembled membrane is a highly sensitive device for oxygen. Anal.Commun., 1999, 36, 179–180 179Oxygen sensing Fig. 2 shows a plot of the phosphorescence of PdTCPP as a function of oxygen concentration (I0 and I are phosphorescence intensities in the absence and in the presence of oxygen, respectively). The plot for this membrane exhibits considerable linearity at lower oxygen concentrations, although the curvature decreases at higher oxygen concentrations.At lower concentrations, the intensities from the photoexcited PdTCPP are quenched by oxygen according to the Stern–Volmer equation (I0/I = 1 + KSV [O2]; KSV is the Stern–Volmer quenching constant) as well in homogeneous system. At higher concentrations, on the other hand, plots of nearly all sensors based on luminescence quenching are nonlinear, mainly because of the simultaneous presence of static and dynamic quenching.Demas et al. reported a multi-site model with an oxygen-accessible site and an oxygen difficultly-accessible site, respectively.17 In this model, the sensor molecule can exist in two or more sites, each with its own characteristic quenching constant. The Stern– Volmer plot becomes as follows: I0/I = {S[fn/(1 + KSVn[O2])]}21 (1) where fn is the fractional contribution of the oxygen-accessible site or the oxygen difficultly-accessible site; KSVn is the quenching constant for each accessible site.In Fig. 2, the solid line is the best fit using the above equation (n = 2). Thus, there are two oxygen accessible sites for sensing membrane; one is an oxygen-accessible site (KSV1 = 4.56%21, f = 0.945) and the other is an oxygen difficultly-accessible site (KSV2 = 0.00190%21, f = 0.055). The KSV2 is a very low value and makes little contribution (f = 0.055) compared with KSV1, indicating that the oxygen difficultly-accessible site cannot be attributed to PdTCPP (emission from background scattering, etc.).Operational stability, response time and photostability Fig. 3 shows the typical dynamic response of the sensor when switching between fully oxygenated and fully deoxygenated atmospheres. The response times of the sensor are 36 s on going from argon to oxygen and 148 s on going from oxygen to argon (65 s on going from argon to oxygen and 200 s on going from oxygen to argon for Pd coproporphyrin-silicon rubber system15). The signal changes were fully reversible and measurement hysteresis was not observed.This is a photostable sensing membrane that exhibits minimal decrease (ca. 5%) of initial intensity after continuous irradiation for 24 h. Conclusions An optical oxygen sensing technique was prepared using the PdTCPP self-assembled membrane on an alumina plate. This sensing membrane showed good linearity using modified Stern– Volmer plots in total oxygen concentration region and possesses good operational stability and reproducibility.These results show that a PdTCPP self-assembled membrane can be developed as a sensitive oxygen sensing device. The surface structure of the PdTCPP membrane is now being studied using FT-IR. The present work is partially supported by the Grant-in-Aid for Scientific Research on Priory-Area-Research from the Ministry of Education, Science, Sports and Culture of Japan (10145211). References 1 E. D. Lee, T. C. Werner and W.R. Seitz, Anal. Chem., 1987, 59, 279. 2 W. Xu, R. Schmidt, M. Whaley, J. N. Demas, B. A. DeGraff, E. K. Karikari and B. L. Farmer, Anal. Chem., 1995, 67, 3172. 3 P. Hartmann, M. J. P. Leiner and M. E. Lippitsch, Anal. Chem., 1995, 67, 88. 4 M. G. Sasso, F. H. Quina and E. J. H. Bechera, Anal. Biochem., 1986, 156, 239. 5 E. Singer, G. L. Duveneck, M. Ehrat and H. M. Widmer, Sens. Actuators A, 1994, 41, 542. 6 E. R. Carraway, J. N. Demas, B. A. DeGraff and J. R. Bacon, Anal.Chem., 1991, 63, 332. 7 J. R. Bacon and J. N. Demas, Anal. Chem., 1987, 59, 2780. 8 R. G. Nuzzo, F. A. Fusco and D. L. Allara, J. Am. Chem. Soc., 1987, 109, 2358. 9 M. D. Porter, T. B. Bright, D. L. Allara and C. E. D. Chidsey, J. Am. Chem. Soc., 1987, 109, 3559. 10 P. E. Laibinis and G. M. Whitesides, J. Am. Chem. Soc., 1992, 114, 1990. 11 A. Ulman, An Introduction to Ultrathin Organic Films From Langmuir– Blodgett to Self-Assembly, Academic Press, San Diego, CA, USA, 1991. 12 J. D. Burgess, M. C. Rhoten and F. M. Hawkridge, J. Am. Chem. Soc., 1998, 120, 4488. 13 K. Kalyanasundaram, Photochemistry of Polypyridine and Porphyrin Complexes, Academic Press, New York, NY, USA, 1992. 14 D. B. Papkovsky, Sens. Actuators B, 1995, 29, 213. 15 P. M. Gewehr and D. T. Delpy, Med. Biol. Eng. Comput., 1993, 31, 11. 16 S.-K. Lee and I. Okura, Anal. Commun., 1997, 34, 185. 17 J. N. Demas, B. A. DeGraff and W. Xu, Anal. Chem., 1995, 67, 1377. Paper 9/00721K Fig. 1 Luminescence spectra of PdTCPP sensing membrane. 1, 100% argon; 2, 20% oxygen; and 3, 100% oxygen. Excitation wavelength was 523 nm. Fig. 2 A plot of the phosphorescence of PdTCPP sensing membrane as a function of oxygen concentration. The solid line is the best fit using equation (1) (n = 2). Fig. 3 Response time and relative intensity change for PdTCPP sensing membrane on switching between 100% argon (a) and 100% oxygen (b). Excitation and monitoring wavelengths were 523 and 700 nm, respectively. 180 Anal. Commun., 1999, 36, 179–180

ISSN:1359-7337    

DOI:10.1039/a900721k

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Luminol immobilized anion-exchange resin as an indicator phase for a chemiluminescence oxygen gas sensor Takuya Okamoto,a Ken-ichiro Tanaka,a Hajime Goto,a Jin-Ming Lin,a Masaaki Yamada*a and Yasukazu Asanob a Department of Applied Chemistry, Graduate School of Engineering, Tokyo Metropolitan University, Hachi-ohji, Tokyo 192-0397, Japan. E-mail: yamada-masaaki@c.metro-u.ac.jp b Department of Chemical Science and Engineering, Ariake National College of Technology, Higashi-Hagio, Ohmuta, Fukuoka 836-0097, Japan Received 22nd February 1999, Accepted 15th March 1999 An indicator phase, luminol immobilized on a strongly basic anion-exchange resin, was evaluated for the chemiluminescence sensing of O2 in N2 gas.The base catalyzed luminol chemiluminescence was monitored with a silicon photodiode installed in a flow cell, into which the luminol-loaded resin was packed. The signal was dependent significantly on resins and solvents used for the immobilization.Oxygen gas concentrations at ppm levels in a N2 gas and a city gas were determined by means of 1 ml sample injection. Chemical sensors are of increasing interest for the continuous and real-time monitoring of analytes, and have been reported on the basis of various sensing principles.1,2 One of the most intensively explored types is optical sensors,3 which are roughly classified into absorbance and luminescence sensors, depending on the origin of the optical signal.Recently, considerable efforts have been devoted to the development of luminescence sensors,4,5 because of their intrinsic advantages over other sensor types. Until several years ago, only a few sensors based on chemiluminescence (CL) have been reported,6–8 despite their advantages over fluorescence sensors in that they do not require an excitation light source and spectral separation of exciting and emitted light. Very recently, however, CL sensors have been developed upon the recognition of their usefulness. 9–11 This paper describes the indicator phase for a flow-through CL sensor capable of monitoring O2 in a N2 gas by use of a simple photodiode detection system. The indicator phase is a strongly basic anion-exchange resin on which luminol was immobilized. The anion-exchange resin may function as a base catalyst and thus is suitable for an indicator phase matrix12,13 because most of CL reactions proceed at basic conditions. The present O2 gas sensor is based on the measurement of the CL generated by the heterogeneous reaction of luminol adsorbed on the base catalyst with O2.Experimental Apparatus A flow system consists of a sensor, a sample injector and a standard gas generator. The sensor is composed of a flow cell (10 mm id 3 5 mm depth), made by hollowing out a stainless steel block and a silicon photodiode with a 10 3 10 mm sensitive zone (S1227-1010, Hamamatsu Photonics, Japan), mounted on a lid of the flow cell (Fig. 1). The photodiode was operated at 15 V from a DC voltage supplier and followed by a photosensor amplifier (C2719, Hamamatsu Photonics, Japan). A stainless steel gas sampler with a sample loop of 1 ml was mainly used for the introduction of sample gas into a N2 carrier gas. The standard O2 sample gas was prepared by means of a standard gas generator (Reinetsu RGD-2, Tokyo Gas Chemicals, Japan). Stainless steel tubes (1/16 in id) were used for flow lines. The peak height of the signal recorded was mainly measured as a CL signal.Absorption spectra of luminol were measured with a spectrophotometer (UV-2200, Shimadzu, Japan). Gas chromatographic determinations of O2 in real samples (a regular N2 gas and a city gas) were conducted on a gas chromatograph (GC-7A, Shimadzu, Japan) using a molecular sieve 13X(60/80) column (2 m 3 3 mm id, 50 °C), a thermal conductivity detector (125 mA, 60 °C) and a He carrier gas (20 ml min21). Reagents All chemicals were of analytical-reagent grade and used as received.The water was prepared using MILLI-XQ equipment. For the immobilization of luminol, the following strongly basic anion-exchange resins (size 0.4–0.6 mm) were used as supporting materials: Amberlite (IRA-400, -410, -458, -900, -910 and -958) and Amberlyst series (A-26 and -27). An O2 gas sample was prepared by passing a 99.6% O2 gas or a low concentration standard O2 gas (99.7 ppm) and a high purity N2 gas (99.9999%, < 0.2 ppm O2) through the standard gas generator.The N2 gas was also used for a carrier gas. Another low concentration standard O2 gas (5.06 ppm) was used without further dilution. Immobilization Five grams of each resin (OH-form) was treated two times with a 20 ml portion of a solvent (water or organic solvent) by sonicating for 5 min. After decantation, a 2 ml portion of the resin was immersed in the solvent (20 ml) in which 20 mg of luminol was dispersed; the resin was equilibrated with luminol by shaking for 30 min.The luminol loading was confirmed by means of the measurement of the absorbance of luminol (lmax Fig. 1 Schematic illustration of the flow cell. A, silicon photodiode; B, anion-exchange resins; C, carrier gas. Anal. Commun., 1999, 36, 181–183 181= 350 nm). After immobilization, the resin was washed well with about 30 ml of the solvent by decantation and stored in the solvent. About 0.4 ml of the luminol-loaded resin was packed into the flow cell and the carrier gas was flowed through to remove the solvent. Results and discussion Selection of anion-exchange resin Eight kinds of luminol-loaded resins were evaluated with respect to the signal for 5.06 ppm O2.The results are shown in Fig. 2, showing the relationships between the signal and the time elapsed, t, after the carrier gas was flowed to the cell. It is found from the figure that the Amberlyst resins are different from the Amberlite resins in the time dependences of the signals.With the amberlyst resins, the signals gradually increase and the times required for the signals to reach a maximum, tmax are long (ca. 5 h). After tmax, the signals are likely to be constant. The A-26 resin gives the highest maximum signal, ca. 10 times higher than that for the A-27 resin. On the other hand, the Amberlite resins provide no (IRA- 400, -410 and -458) or low (IRA-900, -910 and -958) signals and short lifetimes, although their tmax are shorter than those in the Amberlyst resins. The length of tmax seems to be associated with the extent of the removal of the solvent (in this case, ethanol) used for the immobilization as stated later.Although any clear elucidation can not be made for the time dependences of the signals, the different features between both resins might be correlated with their chemical properties, i.e. the Amberlyst resins are prepared for use in organic solvents. For further experiments, the Amberlyst resins were selected by virtue of higher sensitivity and longer lifetime.Effect of solvent used for the immobilization Initially, the resin was treated with the luminol-dispersed water and allowed to dry overnight under reduced pressure before being packed into the cell. This procedure resulted in low sensitivity and long tmax (ca. 10 h). It seems likely that the low sensitivity is due to the marked consumption of luminol in air through the packing of the luminol-loaded resin and the long tmax due to the difficulty of the removal of water by the N2 carrier gas.Thus, in place of water, volatile solvents such as acetone, acetonitrile, methanol and ethanol were examined using Amberlyst A-27, and each indicator phase prepared was packed into the cell without any procedure for removing solvent in advance. The results are shown in Fig. 3. Compared to the treatment with water, the sensitivity was improved 16 times by use of ethanol and tmax was shortened by one-third by use of acetonitrile.Judging from the fact that the indicator phase wetted with solvent is inactive against O2 (see the signals at zero time in Fig. 3), the increase in the sensitivity is explained by the suppressed deterioration of the indicator phase on exposure to air. For subsequent experiments, methanol was chosen in view of its tmax and sensitivity, and the signals were taken with tmax = 5 h. Optimization For the determination of the operating conditions, the amount of immobilized luminol and the flow rate of the carrier gas were examined using the Amberlyst resins with respect to the signal for 5.06 ppm O2.For both resins, they were determined to be 5.6 3 1025 mol (ml resin)21 and 20 ml min21, respectively. Calibration graphs Under the above optimized conditions, the CL sensors were investigated from the analytical point of view. Calibration graphs showed linear correlations represented by the following equations of Amberlyst A-26 and A-27 in the concentration range 0–100 ppm, respectively.I = 26C + 7.5 (r2 = 0.999) I = 8.0C + 0.87 (r2 = 0.999) where I (10213 A) is the CL signal and C is the concentration of O2 (ppm). Blank experiments (i.e. C = 0) gave signals corresponding to 0.2 ppm O2, indicating that air entered the flow line through the gas sampler used. The detection limits (S/ N = 3) were calculated to be 0.04 ppm for the A-26 resin and 0.2 ppm for the A-27 resin.The signal profiles for 5–30 ppm O2 are depicted in Fig. 4. The relative standard deviations (n = 10) for 2 ppm O2 are 1.0% for the A-26 resin and 1.4% for the A-27 resin. Lifetime In order to check the lifetime of the sensors, the stability of the indicator phases was explored. On the freshly prepared indicator phase, the 5.06 ppm O2 sample gas was continuously flowed at the flow rate of 20 ml min21 in place of the N2 carrier gas. The results are shown in Fig. 5. After tmax (ca. 5 h), the Fig. 2 Effect of anion-exchange resins on the CL signal for O2. 2, IRA- 900 and -910; 8, IRA-958; -, A-26; 5, A-27. Conditions: 5.6 3 1025 mol luminol (ml resin)21; O2, 5.06 ppm; solvent used for immobilization, ethanol; carrier gas flow rate, 20 ml min21. Fig. 3 Effect of organic solvents used for the immobilization of luminol on the CL signal for O2. -, acetone; 8, acetonitrile; 5, methanol; 2, ethanol. Conditions as in Fig. 2 except for resin (Amberlyst A-27) and solvent. 182 Anal. Commun., 1999, 36, 181–183signals with the A-26 and A-27 resins remain constant for 9 and 5 h, respectively, and then gradually decrease, 95, 60, 46% and 70, 38, 24% of each maximum signal at t = 20, 40, 60 h, respectively. With A-26, the signal at t = 230 h was almost the same as that at t = 85 h with A-27. The relative standard deviations of the signals (n = 10) at every 1 h in the vicinity of t = 20, 40, 60 h were 0.4, 0.3, 0.3% for A-26 and 1.2, 0.8, 0.9% for A-27, respectively.This means that the present sensors are usable for flow-through sensing of O2, although not for continuous monitoring for a long time. Simple calculations, i.e. assuming 100% reaction efficiency, show that in 20 h luminol (L22) changes into 3-aminophthalate (AP22) by about 24% of the initial amount. L22 + O2 ? AP22 + N2 This suggests that the gradual decrease in signal is due to the decrease in the effective concentration of luminol caused by the deposition of AP22 on the surface of the indicator phase.Although the durability of both indicator phases against the continuous sample supply is not enough, long-running of the system will be accomplished through an intermittent sample supply using a gas sampler. Determinations of O2 in real samples The Amberlyst A-27 CL sensor was used for the determinations of O2 in a regular N2 gas and a city gas prepared from natural gas. According to the gas suppliers, the N2 gas and the city gas contain less than 2 and 100 ppm O2, respectively, as impurities.One ml of each sample gas was injected into the gas chromatography column and after gas chromatographic detection each effluent was subjected to on-line CL sensing. The O2 concentrations were directly evaluated with calibration graphs and determined to be 5.0 ± 0.1 ppm (n = 5) in the N2 gas and 2.0 ± 0.9 ppm (n = 5) in the city gas, which were in agreement with the results (4.7 ± 0.5 ppm and 2.0 ± 0.8 ppm, respectively) of the gas chromatographic method.Conclusions A simple method for O2 gas sensing was developed by using an inexpensive CL detection device. The indicator phase may easily be prepared and its sensitivity is high enough to detect O2 at sub-ppm levels. The monitoring of O2 at ppb levels, the ultimate goal of this work, will be established by both replacing the present photodiode by a photomultiplier tube and preventing the permeation of air into the flow line. For practical use, however, the time required for a signal to reach a maximum must be reduced markedly.A study is now on going with regard to the on-line removal of the solvent used for immobilization under reduced pressure. The present indicator phase can also be applied to the detection of other oxidative gases such as NO2, SO2 and Cl2. Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research (No. 09650894) from the Ministry of Education, Science and Culture of Japan.We are thankful to Tokyo Gas Chemicals for use of the standard gas generator. References 1 K. Cammann, U. Lemke, A. Rohen, J. Sander, H. Wilken and B. Winter, Angew. Chem., Int. Ed. Engl., 1991, 30, 516. 2 M. Valcarcel and M. D. Luque de Castro, Flow-through (Bio)- Chemical Sensors, Elsevier, Amsterdam, 1994. 3 O. S. Wolfbeis, Fiber Optic Chemical Sensors and Biosensors, CRC Press, Boca Raton, FL, vol. I and II, 1991. 4 O. S. Wolfbeis, Anal. Proc., 1991, 28, 357. 5 M. J. P. Leiner, Anal. Chim. Acta, 1991, 255, 209. 6 O. S. Wolfbeis, Fiber Optic Chemical Sensors and Biosensors, CRC Press, Boca Raton, FL, 1991, vol. I, p. 37. 7 M. Ishii and M. Yamada, J. Flow Injection Anal., 1994, 11, 154. 8 L. J. Blum, Bio- and Chemi-Luminescent Sensors, World Scientific, Singapore, 1997. 9 G. E. Collins and S. L. Rose-Pehrsson, Anal. Chem., 1995, 67, 2224. 10 A. Kuniyoshi, K. Hatta, T. Suzuki, A. Masuda and M. Yamada, Anal. Lett., 1996, 29, 673. 11 W. Qin, Z. Zhang and C. Zhang, Anal. Chim. Acta, 1998, 361, 201. 12 T. Nakagama, M. Yamada and T. Hobo, Anal. Chim. Acta, 1990, 231, 7. 13 F. Yoshimura, T. Suzuki, M. Yamada and T. Hobo, Bunseki Kagaku, 1992, 41, 191. Paper 9/01414D Fig. 4 CL signal profiles. Conditions as in Fig. 2 except for resin (Amberlyst A-27), solvent (methanol) and O2 concentration. Fig. 5 Durability of the indicator phases. 2, A-26; 5, A-27. Conditions as in Fig. 2 except resin and solvent (methanol). Anal. Commun., 1999, 36, 181–183 183

ISSN:1359-7337    

DOI:10.1039/a901414d

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Novel sorbent extraction technique using a chelating agent impregnated porous PTFE filter tube: preconcentration of In(iii) with a bis(2-ethylhexyl) hydrogen phosphate (HDEHP) loaded porous PTFE filter tube Masahiko Murakami* and Takeo Takada Department of Chemistry, College of Science, Rikkyo (St. Paul’s) University 3-34-1 Nishi-ikebukuro, Toshima-Ku, Tokyo 171, Japan. E-mail: mmura@rikkyo.ac.jp or GHD01412@niftyserve.or.jp; Fax: +81 3 5992 3434 Received 18th March 1999, Accepted 6th April 1999 The sorption and elution of In(iii) on a porous PTFE filter tube impregnated with bis(2-ethylhexyl) hydrogen phosphate (HDEHP) was studied.A 1 mg portion of In(iii) in 1000 ml of sample solution was quantitatively complexed with HDEHP adsorbed onto a porous PTFE filter tube by passing the solution through the micropores of the filter tube. Preconcentrated In(iii) was then quantitatively recovered provided that the elution, which consisted of cyclical filtering of 0.1 ml of 4 mol l21 hydrochloric acid through the filter tube for 1 min, was repeated 3 times.The total volume of the eluent was only 0.3 ml in this case; therefore, over 3300-fold of enrichment was attained within 90 min of total preconcentration time. This method was applicable to the preconcentration of In(iii) in highly saline samples. A 2.5 mg portion of In(iii) could be quantitatively extracted from 500 ml of synthetic seawater and be recovered into 0.5 ml of 4 mol l21 hydrochloric acid, with subsequent determination by flame AAS.The average recovery and the RSD of the results were 100% and of 1.4%, respectively (n = 5). Introduction Recently, a sorbent extraction (or liquid–solid separation) technique has been applied to preconcentrate the analyte and to eliminate matrix interference prior to AAS, ICP-AES, and ICPMS analysis of trace elements, as an alternative to the liquid– liquid extraction technique.1 Sorbent extraction has several advantages over traditional liquid–liquid extraction.The degree of preconcentration is determined by the volume of sample from which the analyte is preconcentrated and by the volume of eluent required for the removal of the analyte from the solid sobent, so that significantly high enrichment factors may be achieved, provided that the volume of the eluent is reduced. Sorbent extraction is suited to on-line applications which offer several benefits compared with traditional batch approaches, e.g., minimum sample handling, reduced amounts of reagents and man-hours, and hence reduced sample contamination.In this technique, several types of mini-columns have been used, packed with commercially available or laboratory made ionexchange or chelating resins. However, preparing columns which are fit for use in ultra trace analysis is difficult and laborious, as there are contamination problems. The use of conventional columns is also limited by the amount of solution which can be handled.Because of the large dead volume of the column system, it is difficult to reduce the volume of the final eluent. Therefore, it is not possible to achieve the high enrichment factor (thousand fold) required for the analysis of rare earth elements (REEs) in seawater samples. From the standpoint of the dead volume, the use of a membranetype sorbent is more desirable than that of a sorbent column if sufficient capacity is obtained; the long layer of sorbent present in the conventional column is not needed, because the chromatographic behavior is not required in the preconcentration.Hence, in the present report, we have proposed a new sorbent extraction system involving a porous PTFE filter tube (pore size 1 mm, id 1 mm, and od 2 mm) which is impregnated with a hydrophobic chelating agent. In this work, we have tried two new approaches; one of them is the use of PTFE as a substrate for the sorbent. It is a simple technique to introduce a chelating function to a sorbent without any chemical process.In previous work, various porous substrates (silicagel,2,3 Amberlite XAD- 2,4,5 XAD-4 and XAD-7,6 Seralite SRA-400,7 Dowex 1 38 and Lewaitit MP5080,8 ODS (C18),9 activated carbon,10 and so on) have been impregnated with and/or adsorbed with chelating agent. In these studies the porous PTFE has not been applied, however, we think it has some suitable properties for this purpose.PTFE has strong hydrophobicity and it strongly adsorbs hydrophobic species, so it is expected to firmly immobilize the water insoluble reagent layer thereby allowing it to be used as a solid sorbent. Additionally, in terms of contamination, the PTFE based sorbent is advantageous as it is chemically stable and is a very clean material. The other approach in the present work is the application of a ‘tube-shaped’ filter as the substrate for the sorbent; it has generally been used as an aeration filter. In comparison with the preconcentration system based on the use of a membrane disk with metal chelating properties,11 our approach has several advantages. The filter tube itself not only has a small dead volume but also needs no apparatus to hold it, as it can be held by plugging it into pump tubing.In the conventional membrane disk system, a membrane filter holder is necessary to hold the sorbent membrane disk. This apparatus gives rise to significant dead volume and sample contamination.Our new technique would ensure a high enrichment factor by preconcentrating metal ions from a large volume of sample, and then eluting it to a small volume of eluent. In the present work, the preconcentration of indium(iii) with a bis(2-ethylhexyl) hydrogen phosphate (HDEHP) impregnated porous PTFE filter tube has been studied, as a preliminary study to the application of this system for the preconcentration of rare earth elements in seawater and their subsequent determination.The reason why we selected HDEHP as a chelating agent and In(iii) as an analyte is that HDEHP is a very strong complexing agent for REEs and has successfully been used for their extraction,12 and that In(iii) is extractable by HDEHP as are to REEs, although it can be analysed by flame AAS in contrast to REEs. Hence, in this paper, the preconcentration of In(iii) from matrix free sample or synthetic seawater with the HDEHP impregnated PTFE filter tube has been studied to validate our approaches. Anal.Commun., 1999, 36, 185–188 185Experimental Reagents Porous PTFE filter tube (pore size 1 mm) was obtained from UNIVERSAL Co., Ltd. (Toshima-ku, Tokyo, Japan). Bis (2-ethylhexyl) hydrogen phosphate was purchased from Tokyo Kasei Co., Ltd. (Kita-ku, Tokyo, Japan). All chemicals were reagent grade and were used as received. Water was redistilled from all-glass apparatus. Procedures Impregnation of HDEHP into porous PTFE filter tube.A porous PTFE filter tube (25 mm 3 1 mm id, 2 mm od) was sealed at one end with a heat-shrinkable plastic tube. The tube and 25 ml of 25% HDEHP in hexane solution were added to a 50 ml conical flask. To obtain sufficient impregnation of the tube with the solution, an ultrasonic wave was applied to the flask. Then the tube was dried by air at room temperature. Preconcentration. The preconcentration system consisted of a MasterFlex model 7524-10 peristaltic pump with a Tygon pump tube connected to the HDEHP loaded PTFE filter tube, with PTFE tubing (Fig. 1(a)). Then 100–1000 ml of solution containing 2.5 mg of In(iii), which had been acidified to pH 2 with nitric acid, was filtered through the filter tube at a flow rate of 6 ml min21. Preconcentration capacity. To estimate the sorption capacity of In(iii) on the 25 mm tube, the amount of In(iii) sorbed on to the tube was determined as follows. A 100 ml portion of solution containing 100 mg of In(iii) was filtered through the tube at a flow rate of 6 ml min21, and then residual In(iii) in the solution was measured.The amount of HDEHP loaded onto the tube was measured by weighing the tube before and after the process of the reagent impregnation. Elution. The preconcentrated In(iii) was eluted with 0.1–1 ml of 4 mol l21 hydrochloric acid at a flow rate of 3 ml min21. For circulating elution, the eluting acid was circulatively filtered through to the tube as shown in Fig. 1(b), for a period of 1–3 min. This process was repeated 3 times with fresh acid each time, to obtain a quantitative elution recovery. Recovery for spiked synthetic seawater sample. Synthetic seawater was prepared as described by Riley and Skirrows.13 A 500 ml portion of the synthetic seawater was spiked with 2.5 mg of In(iii), and filtered through the PTFE filter tube following the procedure mentioned above, at a flow rate of 6 ml min21. The preconcentrated In(iii) was recovered by successive circulating elution with 0.2 ml, followed by 0.2 ml, and then 0.1 ml of 4 mol l21 hydrochloric acid; at a flow rate of 3 ml min21 and elution period of 1 min.The eluents (0.5 ml in total) were mixed together in a single vial and analysed by AAS. Measurements. The concentrations of In(iii) in the eluent and the residues were determined by AAS on a Jarrel-Ash model AA782 (Nippon Jarrel-Ash Co., Ltd., Kyoto, Japan) instrument equipped with an air–acetylene flame atomizer.Results and discussion Sorption Effect of pH. The effect of the pH of the sample solution on the sorption of In(iii) was studied for samples of matrix free solution and highly saline synthetic seawater. For the matrix free samples, In(iii) was quantitatively extracted in the pH range 0.6 to 3.0 (Fig. 2(a)). In the case of the highly saline sample, quantitative extraction was not observed under pH 2; the range of the quantitative extraction was shifted to pH 2 to 5.8 (Fig. 2(b)). Such an effect of the salinity on the optimum pH range for the extraction may be complicated and is not explainable now, though it is assumed that it is affected by the dissociation of the acidic proton in HDEHP, dissolution of the reagent into the sample solution, the salting out effect on extraction, and so on. From the above results, the optimum pH range was estimated to about pH 2 to 3, because In(iii) can be quantitatively extracted regardless of the salinity of the sample. Effect of tube length.The effect of the filter tube length on the extraction was studied using the 25, 50, and 100 mm lengths of tube. Quantitative preconcentration recovery of 25 mg of In(iii) was obtained at all the lengths studied. Thus, to decrease the dead volume of the system as much as possible, the 25 mm filter tube was selected and used in our later studies. Effect of the flow rate. The effect of flow rate of sample solution on the extraction recovery of In(iii) was studied over the range of 1 to 6 ml min21.The results are shown in Table 1. It is notable that quantitative recovery is obtained except in the case of 1 ml min21; this indicates that at too low a flow rate the sorption efficiency is rather lowered in this system. It suggests Fig. 1 Schematic diagram of (a) the preconcentration set-up and (b) the circulating elution apparatus; 1, PTFE filter tube; 2, peristaltic pump; 3, sample solution; 4, PTFE filter tube; 5, heat-shrinkable plastic tube for end cap; 6, PTFE tubing to pump; 7, eluting acid.Fig. 2 Effect of pH on sorption recovery of 2.5 mg of In(iii) from 100 ml of (a) matrix free solution and (b) synthetic seawater sample at a flow rate of 3 ml min21; tube length 2.5 cm. Table 1 Effect of flow rate on preconcentration recovery Preconcentration recovery (%) 1 ml min21 2 ml min21 3 ml min21 4 ml min21 6 ml min21 77 103 103 100 100 186 Anal. Commun., 1999, 36, 185–188that a flow rate as low as 1 ml min21 causes insufficient liquidpressure to filter the solution through all the pores of the filter tube, resulting in a decrease in sorption capacity.Therefore, 6 ml min21 (the maximum possible flow rate of the present pump system) was chosen as the optimum flow rate, taking into account the time required for the preconcentration procedure. Preconcentration capacity and composition of preconcentrated species. To estimate sorption capacity and composition of the preconcentrated species, the amount of HDEHP loaded on the tube and the limiting amount of preconcentrated In(iii) were measured.It was observed that under the present conditions, about 13 mg (4.1 3 1025 mol) of HDEHP was loaded on the 25 mm tube and about 630 mg of In(iii) (5.5 3 1026 mol) could be sorbed on the tube. The results indicate that the tube system gives a large sorption capacity; and it is quite enough for the present purpose. Additionally, the composition of the preconcentrated species of In(iii) can be inferred from the results.The molar ratio of the preconcentrated In(iii) to the initially loaded HDEHP is calculated to be about 1 : 6, considering that the effective amount of HDEHP must be estimated at about 80% of the above value; about 20% of the total length of the tube was used as the margin to connect the tube to the tubing from the pump, and this connecting section does not work as the sorbent. The above molar ratio compares well with that obtained using liquid–liquid extraction of some trivalent metals with HDEHP.Peppard et al.14 reported that it can be assumed that the molar ratio of the species extracted into HDEHP solution in toluene, M3+ to HDEHP, is about 1 : 6, when the extraction is carried out in the low hydrogen-ion concentration range at more than pH 0. The above fact suggests that both of the extracted species and the reaction process for extraction are similar to those observed in liquid–liquid extraction.Elution Effect of types and concentrations of acids on elution recovery. It is known that HDEHP works as a very strong complexing agent for In, REEs, and Y, and the elution or back extraction of these elements with various mineral acids is very difficult.12 Thus, elution of preconcentrated In(iii) was first studied by using 1 ml of 1–6 mol l21 of hydrochloric and of nitric acid, at a constant flow rate of 3 ml min21. It was observed that the highest elution recovery was attained at 4 mol l21 hydrochloric acid, however, quantitative recovery was not obtained under all the conditions studied.It was also found that the preconcentrated In(iii) was not satisfactorily recovered when the eluting acid passed through the tube at any flow rate, and that it could not be improved by changing the volume of eluting acid; a recovery of about 65% was obtained under all the conditions studied. Thus, circulating elution was tried; the eluting acid which had passed through the tube was returned back to the tube again and again as shown in Fig. 1(b). As the circulating time was increased, the recovery slightly increased. It remained almost constant at about 80% with a 1–3 min circulation period and could not be improved by a further increase in the circulation period. Effect of acid volume and repeating of elution process. The above results suggest that the single elution process is insufficient to recover preconcentrated In(iii).Quantitative recovery would be obtained provided the elution is repeated sequentially with fresh acid at each elution step. However, an increase in total volume of eluent is inevitable in this case, thus to reduce the total volume of the eluent as much as possible and hence to obtain a higher enrichment factor, the volume of the eluting acid used in each step must be reduced. Therefore, the effect of the volume of 4 mol l21 hydrochloric acid solution on the elution recovery was studied at a circulation period of 1 min.Table 2 shows that there is no decrease in the elution recovery for a volume of range 1.0 to 0.18 ml; it is worth noting that even when the volume decreased to 0.1 ml, a recovery of 67% was obtained. Hence elution with 0.1 or 0.2 ml of fresh acid at each elution step was repeated sequentially several times, and the recovery at each elution step was determined. The results are shown in Table 3. It was found that the total elution recovery increased step-by-step when the elution was repeated sequentially, and quantitative total recovery could be obtained by three sequential steps of elution with a total volume of eluent of only 0.3 ml.Noticeably the use of the porous PTFE filter tube as the sorbent enables the use of as low as 0.1 ml of eluent, without considerable loss in its volume. This is one of the important advantages of this system, such a decrease in the volume of the final eluent directly contributes to the improvement of the enrichment factor. Enrichment factor.To estimate the total enrichment factor obtained by the present system, 1000 ml of 1 ng ml21 In(iii) solution was used as the sample, and the total recovery was studied at a flow rate of 6 ml min21. In this case, two of the 25 mm tubes and pumps were simultaneously operated to shorten the preconcentration time. Under the present conditions 1000 ml of sample solution could be treated within 90 min.Although the concentration of In(iii) decreased to 1 ng ml21, quantitative sorption recovery was obtained, and no effect in the increase in sample volume on the total recovery was observed. In the elution step, preconcentrated In(iii) on both filter tubes could quantitatively be recovered by three-times sequential elution with 0.1 ml 4 mol l21 hydrochloric acid; recovery was first 64%, then 30%, and finally 10%, and the total recovery was 104%. The analyte was extracted from 1000 ml of sample solution and was then eluted to 0.3 ml of the eluent in total; thus, over 3300 fold of enrichment was achieved.The present results suggest that higher enrichment will be easily achieved provided that the initial sample volume is increased. Recovery for spiked synthetic seawater sample. To verify the applicability of the present system to the preconcentration of trace In(iii) from seawater, the extraction and elution of 2.5 mg of In(iii) from 500 ml of synthetic seawater was studied. In this study, the preconcentrated In(iii) was recovered with 0.5 ml of eluting acid in total, although quantitative elution was attainable by using 0.3 ml of acid, as mentioned in above section.This is because the minimum volume of solution required for flame AAS measurement is estimated to be about 0.5 ml; therefore the enrichment factor is about 1000-fold in this case. Table 4 shows the results of five independent determinations of 2.5 mg of In(iii) in synthetic seawater.Quantitative recoveries were obtained from the highly saline sample with good reproducibility, even though 1000-fold enrichment was applied. The results indicate that the sorption and elution of In(iii) is little Table 2 Effect of volume of 4 mol l21 HCl solution (refluxing for 1 min) Elution recovery (%) 0.10 ml 0.18 ml 0.25 ml 0.40 ml 1.00 ml 68 78 79 81 79 Table 3 Effect of repeating of refluxing elution Volume of 4 mol l21 HCl Recovery (%) of each elution for each elution/ml 1st 2nd 3rd total (1 + 2 + 3) 0.2 68 26 8 102 0.1 67 26 8 101 Anal.Commun., 1999, 36, 185–188 187affected by the coexistence of salt matrices, and suggests that the present method is applicable to real natural water samples such as seawater. Conclusions Use of the chelating agent impregnated porous PTFE filter tube provides a rapid, simple and clean technique for concentration of trace elements in aqueous samples. The filter tube was repeatedly useable provided that the used tube was soaked with hexane or chloroform to remove the adsorbed reagent, and then the tube was dried and fresh reagent loaded again.The most attractive feature of this system is that it permits the handling and recovery of a very small volume of eluent, and hence it yields a very high enrichment factor of over thousand-fold. The present method does fully execute its own ability when it is used as the preconcentration system for analytical methods requiring a relatively low sample throughput, i.e.GFAAS, HPLC, ICPMS with direct injection nebulization, and so on. By combining with such techniques, its very high enrichment factor can directly be attained. Hence, the application of our approach to the preconcentration of REEs in seawater followed by ICP-MS determination is now in progress. References 1 R. A. Nickson, S. J. Hill and P. J. Worsfold, Anal. Proc., 1995, 32, 387. 2 A. Haruta, K. Matsumoto and K. Terada, Anal. Sci., 1989, 5, 319. 3 C. Samara and Th. A. Kouimtzis, Fresenius’ Z. Anal. Chem., 1987, 327, 509. 4 K. Brajter, E. O. � Sleszy�nska and M. Staskiewicz, Talanta, 1988, 35, 65. 5 P. B. Barrera, G. G. Campos, M. F. Novais and A. B. Barrera, Talanta, 1998, 46, 1479. 6 S. Blain, P. Appriou and H. Handel, Analyst, 1991, 116, 815. 7 L. Joseph and V. N. S. Pillani, Analyst, 1989, 114, 439. 8 P. Burba, Fresenius’ J. Anal. Chem., 1991, 431, 709. 9 M. B. Shabani, T. Akagi and A. Masuda, Anal. Chem., 1992, 64, 737. 10 X. Peng, Z. Jiang and Y. Zen, Anal. Chim. Acta., 1993, 283, 887. 11 X. Wang, Z. Zuang, C. Yang and F. Zhyu, Spectrochim. Acta, Part B, 1998, 53, 1437. 12 M. B. Shabani, T. Akagi, H. Shimizu and A. Masuda, Anal. Chem., 1990, 62, 2709. 13 J. P. Riley and G. Skirrows, Chemical Oceanography 1, Academic Press, New York, 1965, p. 648. 14 D. F. Peppard, G. W. Mason, J. L. Maier and W. J. Driscol, J. Inorg. Nucl. Chem., 1957, 4, 334. Paper 9/02146I Table 4 Total recovery from synthetic seawater samplea (n = 5) Added/mg l21 Found/mg l21 Total recovery (%) RSD (%) 5.0 5.0 100 1.4 a Volume of sample: 500 ml (pH 2); total volume of eluent: 0.5 ml. 188 Anal. Commun., 1999, 36, 185&ndash

ISSN:1359-7337    

DOI:10.1039/a902146i

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Rapid determination of enzyme purity by a microdialysis-based assay Sara Richardson,*a Gunilla S. Nilsson,a Nelson Torto,†a Thomas Laurellb and Lo Gortona a Department of Analytical Chemistry, Center for Chemistry and Chemical Engineering, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden. E-mail: Sara.Richardson@analykem.lu.se; Fax: +46 46 2224544; Tel: +46 46 2228164 b Department of Electrical Measurements, University of Lund, Lund, Sweden Received 10th March 1999, Accepted 12th April 1999 Microdialysis was shown to be useful as a fast on-line sampling method for determining the purity of starch hydrolysing enzymes.The enzymes were characterised using their hydrolytic properties. b-Amylases and pullulanases from different sources and/or manufacturers were investigated, with maltose, maltoheptaose, pullulan, and potato amylopectin starch (PAP) as substrates. The hydrolysis products were sampled via an on-line microdialysis probe and determined in a high-performance anion-exchange chromatographic (HPAEC) system.Comparison between the expected (theoretical) hydrolysis products with those obtained in the experiments made it possible to determine impurities in the enzymes. Two of the b-amylases and one pullulanase released unwanted hydrolysis products, indicating trace impurities in the enzyme preparation. Microdialysis sampling allows on-line sampling and eliminates separate sample preparation and clean-up steps.On-line microdialysis coupled to a HPAEC system is therefore a fast and simple technique for analysing enzyme hydrolysates. Introduction When using hydrolytic enzymes for analytical purposes, the absence of contamination from other hydrolases in the enzyme preparation is essential to obtain accurate results. Although highly purified enzyme products are available, the possibility of the presence of small amounts of unwanted enzymes should not be discounted.1 Development of fast methods for determining the purity of enzymes is therefore of interest.Enzymes with different specificities are commonly used for structural analyses of starch, that is used in large quantities in food, paper, and pulp industries. Since the starch structure and the amylose/amylopectin ratio affect the properties of starch, it is important to understand the relationships that exist between starch structure and its properties. Analysis of the products obtained from enzymic hydrolysis of starch provides structural information such as chain length distribution, inner and outer chain length, b-limit value, A/B-chain ratio, and amylose content.2–8 Enzymic degradation is also useful when studying the positions of substituents in modified starch and cellulose. 9–11 Determination of enzyme purity may be performed either by analysing the protein homogeneity, or by studying the substrate specificity of the enzyme preparations. The most widely used analytical technique for determination of the number of protein components in an enzyme preparation is electrophoresis.12,13 Gel filtration, affinity chromatography, and analytical ultracentrifugation are other analytical techniques for investigating protein homogeneity.14 Enzyme activity and substrate specificity can be determined by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD).15–18 HPAEC separates and PAD detects mono-, di-, oligo- and polysaccharides19,20 with chain lengths (CL) of up to 80.5,7 Microdialysis is a reliable technique for continuous sampling from medical environments21 and has also found applications in bioprocess monitoring.17,22 Microdialysis sampling is based on the perfusion of a microdialysis probe fitted with a semipermeable membrane, which acts as a barrier to large molecules, such as enzymes, dextrins, and starch, but is permeable to smaller molecules, such as starch hydrolysis products.By choosing a dialysis membrane with an appropriate molecular weight cut-off (MWCO), a selective step is introduced, as the molecular mass of the analyte affects the recovery. Advantages of using microdialysis in the sampling of enzymic hydrolysis products are: on-line sampling and sample clean-up, continuous sampling, and automated injection. Thus, the need for separate sample preparation and clean-up steps is eliminated, which in turn leads to a reduced over-all analysis time.In addition, microdialysis probes can be tailor-made for a particular bioprocess by choosing a membrane with the desired length, MWCO, and chemical properties. On-line microdialysis sampling coupled to HPAEC-PAD has proved to be a very suitable combination of techniques for analysis of the hydrolysis products from enzymic degradation of polysaccharides, and for characterisation of the hydrolytic and catalytic properties of enzymes.18,22,23 As analytical chromatographic columns and reference electrodes are sensitive to proteins and other sample matrices associated with enzymic hydrolysates, introduction of on-line microdialysis results in simultaneous sample clean-up, thereby enhancing the performance of this technique.This work presents a method for determination of the purity of starch hydrolysing enzymes by on-line microdialysis sampling coupled to HPAEC-PAD for characterisation of the hydrolysis products. b-Amylase and pullulanase purchased from several manufacturers were investigated.By identification of the hydrolysis products and subsequent comparison with the theoretically expected products, it was possible to detect activities from impurities (contaminating hydrolases) in the enzyme preparations. Experimental Reagents/Chemicals Glucose, maltose, maltotriose, maltopentaose, maltoheptaose (degree of polymerisation, DP, 1–3, 5, and 7), and pullulan (cat. no. P-4516, lot. no. 34H0051), were from Sigma Chemical Co.(St. Louis, MO, USA). Potato amylopectin starch (PAP) was a † On leave from: Department of Chemistry, University of Botswana, P/Bag 0022 Gaborone, Botswana. Anal. Commun., 1999, 36, 189–193 189gift from Lyckeby Stärkelsen (Kristianstad, Sweden) and Svalöf Weibull AB (Svalöv, Sweden). All substrates were prepared in 25 mM citrate buffer, pH 6.0. The water used was Millipore water from a Milli-Q system, Millipore (Bedford, MA, USA). Chromatographic system A chromatographic system, HPAEC-PAD, from Dionex Corp. (Sunnyvale, CA, USA) was used for separation and detection of the hydrolysis products.It consisted of CarboPac PA-100 preand analytical columns, a GP40 gradient pump, and an ED40 electrochemical detector with a gold working electrode with the following waveform: E1 = 0.10 V (td = 0.20 s, t1 = 0.20 s), E2 = 0.70 V (t2 = 0.19 s), and E3 = 20.75 V (t3 = 0.39 s) vs. a Ag/AgCl(sat) reference electrode (Antec, Amsterdam, The Netherlands).The system was controlled by PeakNetTM software from Dionex. Elution of debranched starch was performed using a gradient programme with 150 mM NaOH (eluent A) and 500 mM NaOAc prepared in 150 mM NaOH (eluent B). Eluent A decreased linearly and was 70% at 0 min, 60% at 2 min, 40% at 10 min, 20% at 30 min, and 0% between 30 and 31 min. At the end of the run, eluent A was readjusted to 70% for equilibration of the column (B = 100% 2 A). The oligosaccharides (DP 1–7) liberated from hydrolysis of maltoheptaose and maltose were separated isocratically with 70% A and 30% B. The flow rate was 1.0 ml min21 in all experiments.Identification of the peaks was performed by comparison with the retention times of commercial standards. Injecting various concentrations of the standards provided corrections for detector response for different oligomers. The corrected detector responses were then used when calculating the ratios of hydrolysis products.Microdialysis sampling Microdialysis sampling was performed using an in-laboratory fabricated microdialysis probe with in situ tuneable extraction fraction and an effective dialysis length of 10 mm,24 fitted with a 30 kDa MWCO polysulfone membrane from Fresenius A/G (St. Wendel, Germany). The membrane-fitted probe was inserted into the test solution, which was kept in a 5 ml vial housed in a heating and stirring module (No. 18971, Pierce, Rockford, IL, USA) at 37 °C. The probe was perfused with water at 7 ml min21 using a CMA/100 microinjection pump from CMA/Microdialysis (Solna, Sweden).The substrate solution was sampled via the probe and 20 ml of the dialysate were injected onto the chromatographic system by a CMA/160 on-line injector. The experimental set-up of the microdialysis- HPAEC-PAD system is shown in Fig. 1. Enzymes purity Four preparations of b-amylase (1,4-a-d-glucan maltohydrolase, EC 3.2.1.2) from different sources purchased from ICN Biomedical, Inc.(Aurora, OH, USA), Megazyme International (Bray, County Wicklow, Ireland), and Sigma, were used in this investigation (Table 1). One unit (U)‡ of b-amylase was defined as the amount of enzyme that liberates 1 mmol of maltose per minute at pH 4.8 at 25 °C, according to the manufacturers. Pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) preparations were from Sigma, ICN, and Megazyme (Table 1). One unit of pullulanase liberates 1 mmol of maltotriose from pullulan at pH 5.0, 25 °C, according to the manufacturers. ‡ 1 U = 16.67 nkat.Fig. 1 Schematic presentation of the analyte pathway, the microdialysis probe, and the experimental set-up of the microdialysis-HPAEC-PAD system. Table 1 Enzymes used in the investigation Enzyme Origin Manufacturer Cat. no. Lot. no. b-Amylase Barley ICN 160058 21631 b-Amylase Sweet potato ICN 159863 88366 b-Amylase Barley Megazyme E-BARBP 50302 b-Amylase Sweet potato Sigma A-7005 24H7075 Pullulanase Enterobacter aerogenes Sigma P-5420 64H4056 Pullulanase Aerobacter aerogenes ICN 321721 88637 Pullulanase Klebsiella pneumoniae Megazyme E-PULKP 30701 190 Anal.Commun., 1999, 36, 189–193The purity of the different b-amylases was determined by monitoring hydrolysis products of maltose or maltoheptaose substrates. Three millilitres of 50 mM of the substrate solution were incubated with 10 U of b-amylase for 2 h at 37 °C with continuous stirring, prior microdialysis sampling and injection onto the chromatographic system.Investigation of the purity of pullulanase was performed with maltoheptaose, pullulan, or PAP substrates. Three millilitres of 50 mM maltoheptaose or 3 ml of 100 mM pullulan were incubated with 5 U of pullulanase for 2 h at 37 °C with continuous stirring. PAP was boiled (100 °C) in water for 15 min, then diluted with citrate buffer, pH 6.0, to a final ion strength of 0.025 M and a final substrate concentration of 4 mg ml21 ( ~1 mM when debranched), and subsequently incubated with 5 U of pullulanase for 4 h at 37 °C. The hydrolysis products were then sampled via the microdialysis probe and injected onto the column.Substrates were analysed before enzymic hydrolysis by microdialysis sampling of blank solutions (no enzyme, only substrate). The presence of mono- and/or oligosaccharide contaminants in the enzyme preparations was determined by microdialysis sampling of blank solutions containing only the enzyme but not the substrate.Maltase activity in the b-amylases was investigated by measuring the amount of reducing sugars liberated after incubation with maltose, using the Somogyi–Nelson copper sulfate method.25,26 Four millilitres of 1 mM maltose were incubated with 200 U b-amylase in 50 mM citrate buffer, pH 4.8, at 37 °C for 1 h. One millimolar maltose was used as blank, and the method was calibrated against glucose (0.125–2.0 mM). One unit of maltase activity hydrolyses 1 mmol of maltose per minute at 37 °C, pH 4.8.27 Results and discussion The purity of different b-amylases and pullulanases was investigated by hydrolysis of different substrates and subsequent monitoring of the products. The determinations were performed with on-line microdialysis sampling in combination with HPAEC-PAD.Starch hydrolysing enzymes can be characterised by their hydrolytic action (Fig. 2). a-Amylase is an endo-enzyme that randomly catalyses the hydrolysis of 1,4-a- glucosidic linkages, resulting in a production of maltooligosaccharides.b-Amylase and a-glucosidase are exo-enzymes that catalyse the hydrolysis of 1,4-a-glucosidic linkages from the non-reducing ends of the chains. a-Glucosidase is also called maltase, since it readily hydrolyses maltose to a-glucose. Pullulanase is a debranching enzyme that catalyses the hydrolysis of 1,6-a-glucosidic linkages in starch.28 Starch is comprised of the mainly linear polymer amylose and the highly branched amylopectin.The most commonly used starches, i.e., from maize, wheat, and potato, contain approximately 20–25% amylose.29 However, there are natural mutants of e.g. maize (waxy), that are essentially absent of amylose, as well as those with high amylose content. New potato varieties have been developed by genetic engineering,30 which produce amylose deficient amylopectin starch. In this investigation a PAP sample was used as the substrate. b-Amylase b-Amylase is used in structural studies of amylopectin to determine the b-amylolysis limit and the outer chain length.3 Hydrolysis of starch liberates b-maltose; however, hydrolysis of linear chains with an odd number of glucose residues gives, in addition, maltotriose residues from the reducing end.31 b- Amylase from sweet potato has been reported to contain a- glucosidase impurity.27 Four b-amylase preparations were investigated in this work, see Table 1.Maltose and maltotriose were used as substrates for studying unwanted or unexpected side reactions.Maltose is not a substrate for b-amylase and should thus not be hydrolysed. Maltoheptaose should theoretically be hydrolysed to maltose and maltotriose in a ratio of 2 : 1. Neither b-amylase from Megazyme (barley) (Fig. 3A) nor ICN (barley malt) contained any detectable impurities when maltose was used as the substrate. b-Amylase (sweet potato) from Sigma released a significant amount of glucose when incubated with maltose (Fig. 3B), as did b-amylase (sweet potato) from ICN (Fig. 3C). For comparison, the increase in reducing power after hydrolysis of maltose was measured with the Somogyi–Nelson copper sulfate method,25,26 see Table 2. These results confirm that there are contaminating hydrolases in sweet potato b-amylase from ICN and Sigma. Hydrolysis of maltoheptaose by b-amylase from Megazyme and ICN (barley malt) released maltose and maltotriose respectively in a ratio of 2 : 1 (Fig. 4A), which is the expected result for pure enzyme preparations. Megazyme reports the contamination with a-amylase to be 1/3 million of b-amylase on an activity basis, but this impurity, if present, was far too low to be detected in this investigation, and should thus not cause misleading results if used in structural studies of saccharides. The detection limit for glucose under prevailing experimental conditions was 0.15 mM. ICN, on the other hand, does not report any contaminating activities in this product.However, in Figs. 4B and C, which show the chromatograms from the hydrolysis of maltoheptaose by b-amylase from Sigma (sweet potato) and ICN (sweet potato), respectively, the release of glucose, in addition to the expected maltose and maltotriose, is inconsistent with a pure enzyme without any impurity of a-glucosidase and/ or a-amylase. According to the manufacturer, the content of contaminating a-amylase is 2% (w/w) in the b-amylase from Sigma, while ICN does not report any contaminating activities in this product.Fig. 2 Schematic model of the action of starch hydrolysing enzymes. Fig. 3 Products released from hydrolysis of maltose by b-amylase from (A) Megazyme (barley), (B) Sigma (sweet potato), and (C) ICN (sweet potato). Anal. Commun., 1999, 36, 189–193 191The total time for every analysis using on-line microdialysis sampling was about 15 min, 10 min for sampling and 5 min for separation and detection.The software used allowed for autoinjections every seventh minute when using isocratic elution. Pullulanase Pullulanase debranches amylopectin and liberates linear chains. The enzyme is used when studying the structure of branched polysaccharides.3 Three pullulanases were tested in this investigation (Table 1) with pullulan, maltoheptaose, and PAP respectively, as substrates. Pullulanase without contaminating hydrolases hydrolyses pullulan to maltotriose; maltoheptaose is not hydrolysed at all, while hydrolysis of PAP results in release of linear chains. The three pullulanases hydrolysed pullulan to a single product, maltotriose, which is the expected result.However, when maltoheptaose was used as substrate, hydrolysis by pullulanase from Sigma released maltose, maltotriose, and maltopentaose (Fig. 5). Obviously there are contaminating activities in this enzyme preparation. The first peak in the chromatogram corresponds to an unknown substance that is also present in the blank enzyme suspension.According to the manufacturer the enzyme preparation was tested for contaminating activities from a-amylase, b-amylase, and dextranase, but no such activities were reported. Pullulanase from Megazyme and ICN did not hydrolyse maltoheptaose. Megazyme reports contaminations with a-glucosidase and a-amylase of less than 0.001% on an activity basis. These contaminants, if present, could not be detected in these experiments.In Fig. 6 the chain length distribution patterns of PAP hydrolysed by pullulanases from Megazyme and Sigma are shown. The patterns are similar; the only difference is the additional peak at 3 min in the chromatogram from hydrolysis by pullulanase from Sigma. This small peak corresponds to maltotriose and is an indication of an impurity in the enzyme, since the shortest chain in amylopectin is CL 6.5,7 Hydrolysis of PAP by pullulanase purchased from ICN resulted in the same debranching pattern (not shown) as the hydrolysis by the enzyme from Megazyme.In all experiments, on-line microdialysis sampling allowed injection of the hydrolysate without the need for separate cleanup steps (e.g. precipitation and centrifugation) for removal of the enzyme. Conclusions Microdialysis sampling in combination with HPAEC-PAD is a fast and automated technique for determining the purity of starch hydrolysing enzymes. Hydrolysis products were sampled and analysed without the need for time-consuming sample preparation and clean-up.Results obtained showed that pullulanase and b-amylase (barley) preparations from ICN and Megazyme were pure, since no unwanted hydrolysis products were detected. Acknowledgements This work was supported by Lyckeby Stärkelsen, Kristianstad, Sweden, The Swedish Research Council for the Engineering Sciences (TFR), Centre for Amphiphilic Polymers (CAP), Lund, Sweden, and the Swedish Board for Technical and Industrial Development (NUTEK). Potato amylopectin starch Fig. 4 Products released from hydrolysis of maltoheptaose by b-amylase from (A) Megazyme (barley), (B) Sigma (sweet potato), and (C) ICN (sweet potato). Table 2 Maltase activity of different b-amylases determined by the Somogyi–Nelson method (n = 3, s < 0.001), and products obtained from DP 2 or DP 7 after hydrolysis by different b-amylases Manufacturer Origin Specific maltase activity/U mg21 protein Products from hydrolysis of DP 2 Products from hydrolysis of DP 7 Sigma Sweet potato 0.009 DP 1 DP 1, DP 2, DP 3 Megazyme Barley 0.003 — DP 2, DP 3 ICN Sweet potato 0.007 DP 1 DP 1, DP 2, DP 3 ICN Barley 0.002 — DP 2, DP 3 Fig. 5 Products released from hydrolysis of maltoheptaose by pullulanase from Sigma. Fig. 6 Hydrolysis products obtained from debranching of potato amylopectin starch by pullulanase from (A) Megazyme, and (B) Sigma. 192 Anal. Commun., 1999, 36, 189–193(PAP) was a gift from Svalöf–Weibull AB, Svalöv, Sweden and Lyckeby Stärkelsen.References 1 D. J. Holme and H. Peck, Analytical Biochemistry, Longman Scientific and Technical, 2nd edn., 1993, p. 303. 2 S. Hizukuri and Y. Maehara, Carbohydr. Res., 1990, 206, 145. 3 D. J. Manners, Carbohydr. Polym., 1989, 11, 87. 4 K. S. Wong and J. Jane, J. Liq. Chrom. Rel. Technol., 1997, 20, 297. 5 I. Hanashiro, J.-I. Abe and S. Hizukuri, Carbohydr. Res., 1996, 283, 151. 6 Q. Zhu, R. Sjöholm, K. Nurmi and E. Bertoft, Carbohydr.Res., 1998, 309, 213. 7 K. Koch, R. Andersson and P. Åman, J. Chromatogr. A, 1998, 800, 199. 8 H. Fredriksson, J. Silvero, R. Andersson, A.-C. Eliasson and P. Åman, Carbohydr. Polym., 1998, 35, 119. 9 Q. Zhu and E. Bertoft, Int. J. Biol. Macromol., 1996, 21, 131. 10 P. A. M. Steeneken, A. C. Tas, A. J. J. Woortman and P. Sanders, Spec. Publ.-R. Soc. Chem., 1997, 205, 153. 11 M. Gohdes and P. Mischnick, Carbohydr. Res., 1998, 309, 109. 12 U. K. Laemmli, Nature, 1970, 227, 680. 13 D. Wu and F. E. Regnier, J. Chromatogr., 1992, 608, 349. 14 T. Palmer, Understanding Enzymes, Prentice Hall/Ellis Horwood, 4th edn., 1995, p. 300. 15 F. W. Willenbrock, D. C. A. Neville, G. S. Jacob and P. Scudder, Glycobiology, 1991, 1, 223. 16 K. Tyagarajan, J. G. Forte and R. R. Townsend, Glycobiology, 1996, 6, 83. 17 N. Torto, T. Buttler, L. Gorton, G. Marko-Varga, H. Stålbrand and F. Tjerneld, Anal. Chim. Acta, 1995, 313, 15. 18 C. M. Zook and W. R. LaCourse, Anal. Chem., 1998, 70, 801. 19 T. Lu, W. R. LaCourse and J. Jane, Starch/Stärke, 1997, 49, 505. 20 K. Koizumi, Y. Kubota, T. Tanimoto and Y. Okada, J. Chromatogr., 1989, 464, 365. 21 C. E. Lunte, D. Scott and P. Kissinger, Anal. Chem., 1991, 63, 773A. 22 N. Torto, T. Laurell, L. Gorton and G. Marko-Varga, Anal. Chim. Acta, 1999, 379, 281. 23 N. Torto, G. Marko-Varga, L. Gorton, H. Stålbrand and F. Tjerneld, J. Chromatogr. A, 1996, 725, 165. 24 T. Laurell and T. Buttler, Anal. Methods Instrum., 1995, 2, 197. 25 N. Nelson, J. Biol. Chem., 1944, 153, 375. 26 M. Somogyi, J. Biol. Chem., 1952, 195, 19. 27 J. J. Marshall and W. J. Whelan, J., Anal. Biochem., 1973, 52, 642. 28 M. Florkin and E. Stotz, Comprehensive Biochemistry, Elsevier, 3rd edn., 1973, vol. 13, p. 212. 29 H. F. Zobel, Starch/Stärke, 1988, 40, 44. 30 P. Hofvander, P. T. Persson, A. Tallberg and O. Wikström, Amylogene HB, Swedish Pat. 9004096-5, Sweden, 1992. 31 A. Guilbot and C. Mercier, The Polysaccharides, ed. G. Aspinall, Academic Press, 1985, vol. 3, p. 232. Paper 9/01895F Anal. Commun., 1999, 36, 189–193 193

ISSN:1359-7337    

DOI:10.1039/a901895f

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Sol–gel horseradish peroxidase biosensor for the chemiluminescent flow determination of hydrogen peroxide Jun Li, Ke-Min Wang,* Xiao-hai Yang and Dan Xiao College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Received 11th March 1999, Accepted 29th March 1999 A chemiluminescent (CL) H2O2 sensor based on horseradish peroxidase (HRP) immobilized by the sol–gel method has been proposed in this paper. A new process of fabricating a non-cracking HRP encapsulated sol–gel membrane has been suggested. Flow injection analysis (FIA) was used to give reproducible results.Under optimum conditions, the sensor showed a linear response toward hydrogen peroxide in the range 0.01–2 mM with a detection limit of 8 mM. The linear relative coefficient r = 0.998. The sensor showed rapid response, good reproducibility (relative standard deviation = 2.4%) and a long lifetime of at least two months. The proposed sensor was used to determine a 30% H2O2 solution that has been stored for a long time.The results were in good agreement with the standard volumetric method. The fabrication of a sensitive H2O2 sensor exhibiting long-term stability is beneficial since many biomolecules can be oxidised by oxidases to produce H2O2. The immobilization of enzymes is one of the most important steps in the research of optical H2O2 chemical sensors because biomolecules are easy to denature.1 In the past few years, the introduction of the sol–gel method to immobilize enzymes has become a new trend for preparing chemical sensors.2–6.The conventional process of sol–gel preparation includes four steps: (1) the hydrolysis of low molecular alkoxysilanes; (2) the condensation of silanol–silanol to produce sol–gel stock solution; (3) the gelation of sol by polymerization of silanol groups; (4) the drying and aging of the gel.7 Presently, the sol–gel method has become a convenient way to prepare a host matrix for heat-sensitive molecules such as proteins and microbes.8,9.The key advantage of sol–gel is that there is little or no structural alteration of the encapsulated species and it is suitable for optical sensors due to its optical transparency and chemical stability. Some optical sensors5,6 are designed to determine inorganic species such as nitrate, carbon monoxide and nitrogen monoxide, etc. The cracking of sol–gel during the drying and aging step, however, restricts its application in chemical sensors.Methods such as the spinning method,2 surface coated method5 or addition of surface-active reagent method,10 have been suggested for preparing a noncracking sol–gel membrane, but they are complex or difficult to reproduce. The chemiluminescence (CL) reaction based on the luminol– H2O2–horseradish peroxidase (HRP) system can be expressed by the following equation.11–13 HRP luminol + 2H2O2 ––? 3-aminophthalate + N2 + 3H2O + hn The amount of generated light is proportional to the concentration of H2O2. Therefore, the CL intensity has often been used for H2O2 assays.Flow injection analysis (FIA) has the features of high precision and quick-rate analysis.14 The combination of CL and FIA has become a useful technique for microanalysis of H2O2. In this paper, the sol–gel method is introduced to fabricate a H2O2 optical sensor by immobilizing HRP as a catalyst of the luminol–H2O2 CL system. A new fabrication and store process is suggested to prepare an enzyme encapsulated sol–gel membrane.The sensitive membrane is optically transparent and a non-cracking gel. Compared to the existing membrane preparation method,2,5,10 the suggested method is easy to control. Moreover, Diaz et al.15 recently proposed a H2O2 biosensor based on HRP immobilized by the sol–gel method. Unfortunately, the precision and response time were not very satisfactory (relative standard deviation = 7.1% and the response time was 300 s).For this sensor, an FIA system is involved to construct a more precise and rapid response sensor. The relative standard deviation is 2.4% and the response time less than 60 s. The HRP encapsulated sol–gel membrane also possessed a longer lifetime than other immobilizing methods.16 The proposed sensor is used to determine a 30% H2O2 solution that has been stored for a long time and gives a satisfactory result. Experimental Reagents Luminol was synthesized according to the literature17 and characterised by IR spectroscopy.A 0.01 M luminol stock solution was prepared by dissolving 0.0885 g dried solid in the smallest amount of 0.1 M NaOH solution, the final volume was made up to 50 ml with buffer solution (5 mM NH4Cl–NH3H2O solution, pH = 9.1). HRP (250 U mg21)† was purchased from Shanghai Biochemical Institute (Shanghai, China). A 0.1 M H2O2 stock solution was prepared by diluting 30% H2O2 and standardized with 0.02 M KMnO4.The H2O2 standard solutions were diluted with buffer from stock solution before use. All other chemicals were of analytical grade and used as received without further purification. Distilled water was used to prepare all aqueous solutions. The HRP encapsulated sol–gel membrane preparation The preparation procedure of the sol–gel stock solution was similar to that proposed by Parang et al.2 2.2 ml tetraethylorthosilicate (TEOS), 0.7 ml H2O and 50 ml 0.1 M HCl were mixed in a glass vial.The mixture was stirred for 3 h and then a clear sol–gel stock solution was obtained. The HRP solution was prepared by dissolved 1 mg HRP in 0.5 ml phosphate buffer, pH = 6.0. Then 0.5 ml sol–gel stock solution and 0.5 ml HRP solution were mixed. 0.2 ml of the mixture was placed on a 25 mm 3 25 mm clean glass plate. Then the glass plate was enclosed in a glass vessel to let the gel age at room temperature for a week. During this period, HRP was encapsulated in the gel’s network formed by the polymerization of silanol groups in the sol. To avoid the membrane losing most of the water and causing cracking, the membrane was immersed in water for 1 min everyday and then enclosed in the glass vessel to keep the gel in a wet state.† 1 U = 16.67 nkat. Anal. Commun., 1999, 36, 195–197 195Instruments and procedures The measuring set-up of the CL hydrogen peroxide sensor was similar to the literature.18 Standard luminol solution and carrier solution were pumped to a flow cell by a two-channel peristaltic pump with the same flow rate of 0.5 ml min21.The sample solution was injected to a flow cell until a stable baseline on the computer was recorded. The total injection volume was only 80 ml. The concentration of hydrogen peroxide was quantified by the peak height of the CL intensity. Results and discussion Features of sol–gel entrapment of HRP Many methods of fabricating a non-cracking enzyme encapsulated sol–gel membrane, such as the spinning method,2 surface coated method5 and addition of surface-active method,14 have been recommended in the literature.However, when the spinning method was used, a poor response was obtained because the amount of catalyst was very small in the thin film. The surface coated method was subsequently adopted to fabricate a sol–gel entrapment enzyme membrane. It was found that the gel was easy to lose from the glass plate when the parafilm was removed from the gel surface.The method of addition of surface-active reagent was not used because the surface-active reagent may affect the catalytic activity of the enzyme. Therefore, an enclosed aging method described in the Experimental section was used to prepare the non-cracking enzyme membrane. Moreover, to avoid HRP leaching from the gel during the aging process and to let the gel contain a certain amount of water, the gel was immersed in water for 1 min every day instead of immersing the gel in buffer solution throughout.The aged sol-gel membrane did not need to be stored in a refrigerator and was just enclosed in a glass vessel at room temperature. Characteristics of the CL hydrogen peroxide sensor Linear response curve of the sensor. Under the optimum experimental conditions ([luminol] = 0.5 mM, flow rate = 0.5 ml min21), series of hydrogen peroxide standard solutions, the concentration ranging from 0.001 to 2 mM, were injected into the CL-FIA system. Fig. 1 demonstrates the typical dynamic response of the sensor. The height of each peak reached its maximum 20 s after the injection of H2O2, and the peak declined to the base line rapidly 40 s after reaching the maximum. The total response time for one sample was 60 s. A linear calibration response curve can be described by the following equation in the range 0.01–2 mM: log Y = 1.7918log C + 3.2556 (1) where Y is the peak height in mV, C is the concentration of hydrogen peroxide (mM) , the linear relative coefficient r = 0.998.The detection limit was approximately 0.008 mM. Reproducibility and lifetime of the sensor. The reproducibility of the CL hydrogen peroxide sensor was studied by injecting 0.1 mM H2O2 5 times. Fig. 2 gives the results. The mean height intensity ± standard deviation was 29.25 ± 0.72 mV The relative standard deviation (RSD) was 2.4%. This result indicates that the sensor has a better reproducibility than the literature.15 The lifetime of the CL hydrogen peroxide sensor depended on the leaching rate of HRP from the sol–gel membrane, which led to a decrease in CL response.We compared the CL intensity of the newly prepared membrane with that of the same membrane after determining the hydrogen peroxide at least one thousand times over 2 months. No obvious decrease of the signal was observed. Compared with other HRP immobilization methods such as adsorption by cellulose membrane, entrapment in polyacrylamide gel and cross-linking by bovine albumin via glutaraldehyde,16 the sol–gel method for immobilizing HRP allows the sensor a long lifetime Application It is known that H2O2 decomposes easily and the real concentration should be determined before use.In order to evaluate the effectiveness of the proposed sensor, a 30% H2O2 solution that had been stored for a long time was determined with the sensor and the results obtained compared with those of the volumetric method.After a suitable dilution to the concentration of the sample within the linear range, the sample was injected into the sensor. Fig. 1 Typical dynamic response of the sensor. The determination was under optimum conditions: [luminol] = 0.5 mM, pH = 9.1, [buffer] = 5 mM, flow rate = 0.5 ml min21; A: [H2O2] = 0.08 mM; B: [H2O2] = 0.2 mM; C: [H2O2] = 0.6 mM. Fig. 2 Reproducibility of the CL hydrogen peroxide sensor. The response signals were obtained under optimum experimental condition as mentioned in Fig. 1. The concentration of hydrogen peroxide used was 0.1 mM. Table 1 Determination of a 30% H2O2 solution that had been stored for a long time using the conventional volumetric method and the proposed sensor Volumetric method (n = 2) Proposed method (n = 4) Concentration/mM Concentration/mM RSD (%) 583.5 ± 7.0 588.3 ± 5.3 0.9 116.7 ± 1.4 114.8 ± 3.8 3.3 196 Anal. Commun., 1999, 36, 195–197The mV value was obtained from the computer and the concentration was calculated according to eqn.(1). The results are listed in Table 1 indicating that the sensor is suitable for determinating H2O2 solution. Acknowledgements This work was supported by the National Natural Science Foundation and National Outstanding Youth Foundation of P.R. China. Reference 1 K. M. Wang, Theories and Methods of Optical Chemical Sensor, Hunan Education Press, Changsha, 1994, ch. 8. 2 U. Parang, P. N. Prasad, F. V. Bright, K. Ramanathan, N. D. Kumar, B.D. Malhotra, M. N. Kamalasanan and S. Chandra, Anal. Chem., 1994, 66, 3139. 3 J. Li, S. N. Tan and H. Ge, Anal. Chim. Acta, 1996, 335, 137. 4 J. Wang, P. V. A. Pamidi and D. S. Park, Anal. Chem., 1996, 68, 2705. 5 J. W. Aylott, D J. Richardson and D. A. Russell, Analyst, 1997, 122, 77. 6 D. J. Blyth, J. W. Aylott, D. J. Richardson and D. A. Russell, Analyst, 1995, 120, 2750. 7 O. Lev, M. Tsionsky, L. Rabinovich, V. Glezer, S. Sampath, I. Pankratov and J. Gun, Anal. Chem., 1995, 67, 22A. 8 B. C. Dave, B. Dunn, J. S. Valentine and J. I. Zink, Anal. Chem., 1994, 66, 1120A. 9 J. Lin and C. W. Brown, Trends Anal. Chem., 1997, 16, 200 10 S. C. Kraus, R. Czolk, J. Reichert and H. J. Ache, Sens. Actuators B, 1993, 15–16, 199. 11 R. W. Marshall and T. D. Gibbson, Anal. Chim. Acta, 1992, 266, 309. 12 F. Preuschoff, U. Spohn, G. Blankenstein, K. H. Mohr and M. R. Kula, Fresenius’ J. Anal. Chem., 1993, 346, 924. 13 A. Berger and L. J. Blum, Enzyme Microb. Technol., 1994, 16, 979. 14 Y. X. Zhou, and G. Y. Zhu, Chin. J. Anal. Chem., 1997, 25, 222. 15 A. N. Diaz, M. C. R. Peinado and M. C. T. Minguez, Anal. Chim. Acta, 1998, 363, 221. 16 J. Z. Li, Z. Z. Zhang and L. Li, Talanta, 1994, 41, 1999. 17 E. C. Horning, Organic Synthesis collective Vol. III (20–29), John Wiley, 1935; Chinese translation by Organ. Chem. Group of Nanjing University, Science Press, Beijing, 1981, pp. 42–43. 18 K. M. Wang, J. Li, X. H. Yang, F. L. Shen and X. Wang, Technical Digest of the Seventh International Meeting on Chemical Sensors, Beijing, 1998, p. 586. Paper 9/01946D Anal. Commun., 1999, 36, 195–197 197

ISSN:1359-7337    

DOI:10.1039/a901946d

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Anion-exchange ability of neutral hydrophobic hypercrosslinked polystyrene N. A. Penner and P. N. Nesterenko* Analytical Chemistry Division, M.V. Lomonosov Moscow State University, Lenin Hills GSP-3, Moscow 119899, Russian Federation. E-mail: PavelN@gsm.chem.msu.su Received 26th March 1999, Accepted 20th April 1999 The characterization of novel neutral hydrophobic hypercrosslinked polystyrene MN-200 was performed. This resin was shown to exhibit anion-exchange properties in the pH range from 2.6 to 4.3 that can be attributed to the presence of protonated carbonyl groups in the framework of the polymer as no other heteroatoms are present, except for oxygen, responsible for the occurrence of positive charge at the surface.The role of high hydrophobicity of MN-200 in retention of anions is discussed. The anion-exchange selectivity of this sorbent to inorganic anions with diluted perchloric acid solutions as eluent was found to be different from that observed for the common anion-exchangers. The main features of MN-200 are weak retention of sulfate and comparatively strong retention of nitrite that can be useful in practical ion chromatography (IC).A new approach to the synthesis of polystyrene polymer sorbents having a crosslinking degree of 100% and higher was invented by Davankov et al.1 These sorbents represent a new generation of polystyrene networks, which follows the traditional gel-type and macroreticular copolymers and offers new possibilities for developing various adsorbing materials.The hypercrosslinked polystyrene (HC-PS) polymers have a highly developed inner surface ( > 1000 m2 g21) and display increased affinity to all kinds of organic solutes.1,2 When swollen, these polymers are distinguished by an exceptionally rigid openwork structure of high permeability. Starting in the mid-1980s, HC-PSs have become frequently used in analytical practice, in general in solid phase extraction of different organic compounds, such as phenols,3 long-chain aliphatic amines,4 synthetic dyes,1 lipids1 and pesticides.5,6 Now, a number of different HC-PC sorbents including neutral MN-200 are produced by Purolite International Ltd. Due to high rigidity and pressure resistance, microparticles of hypercrosslinked polystyrene can be considered as a promising stationary phase for HPLC.MN-200 has been successfully applied to the high-performance separation of phenols,3 alkaline earth and transition metal ions,7 and polyaromatic hydrocarbons. 2 However, as has been pointed out in many studies,2,8 adsorption properties and the retention mechanism on MN-200 are unclear and are different from those of other types of polystyrene-based polymer sorbents.The retention mechanism may involve not only hydrophobic, but also p–p interactions. It is worthwhile underlining the increased affinity of hypercrosslinked polystyrene to aromatic molecules, attributed to the impact of p–p interactions which in the case of MN-200 is much higher than for other related sorbents.2 Because of the highly developed microporous structure of hypercrosslinked polystyrene, size exclusion may also affect retention of substances.One of the surprising properties of neutral non-modified hypercrosslinked polystyrene is wettability by water, taking into account the hydrophobicity of this material. Recently, complete physical and chemical characterization of the neutral hypercrosslinked polystyrene MN-200 was performed5 and the presence of significant positive zeta potential on its surface at pH less than 4.3 was shown (Fig. 1). However, according to elemental analysis data, as well as carbon and hydrogen, MN- 200 contains only traces of chlorine and oxygen in a concentration of between 5 and 6 mass% for the polymer. The exact nature of the functionality on this polymer was determined using spectroscopic techniques.This suggests that the major functional groups are ketones, ethers and alcohols; however, the complex mixture of groups prevented their quantification.5 Therefore, the occurrence of significant positive charge at the surface can be attributed only to the presence of oxygen in the framework of this resin. These positively charged groups are probably protonated carbonyl groups. Therefore, one can propose that the anion-exchange properties for this ‘so called’ neutral resin are attributed to the following possibilities: ï ïdelocalised charge in the whole C OH + C OH + polymer framework. Therefore, the aim of this paper was to study the ionexchange selectivity of neutral hydrophobic hypercrosslinked polystyrene MN-200 to inorganic anions. Experimental Instrumentation The HPLC system consisted of a Model 114 Beckman (Berkeley, CA) high-pressure pump, spectrophotometric detector Micro-UVIS 20 (Carlo Erba Instruments, Milan, Italy) and conductometric detector Conductolyser 5300B (LKB, Bromma, Sweden); Rheodyne (Cotati, CA) Model 7125 injection valve equipped with 100 ml loop.A ZIP (Gomel, Russia) pH-340 pH-meter with glass electrode was used for pH control of eluents. Fig. 1 Zeta potential of MN-150 and MN-200 according to ref. 5. The suspension of microparticles was titrated with either 0.1 mol dm23 hydrochloric acid or 0.1 mol dm23 sodium hydroxide. Anal. Commun., 1999, 36, 199–201 199Materials Perchloric acid (analytical grade, Reakhim, Moscow, Russia) solutions in distilled water were used for the preparation of eluents. 10 mmol dm23 water solutions of sodium and potassium salts were used as solutes. Columns and sorbents The hypercrosslinked polystyrene resin chosen for investigation was MN-200 (Purosep-200, Purolite Int., Pontyclun, Wales). The high rigidity and moderate fragility of this polymer allows fine fractions of microparticles of 10 to 20 mm in size to be obtained by simple crushing of resin particles of original size 40 to 160 mm with a pestle and mortar.A combination of sieving through sieves and sedimentation of suspended particles in water or acetone was used for obtaining the final narrow fraction of microparticles of 12 to 15 mm in size. The stainless steel column 150 33 mm was slurry packed from acetone under constant pressure. The macroreticular porous PS-DVB resin PRLP-S-300, average particle diameter 8 mm, was obtained from Polymer Laboratories (Church Stretton, Shropshire, UK) and used as a reference polystyrene type material.Results and discussion According to the zeta potential curve (Fig. 1), MN-200 may exhibit anion-exchange properties at pH lower than 4.3 while maximum positive charge is observed at pH 2.5–2.7. It should be pointed out that the maximum zeta potential of MN-200 is only 1.5 times lower than for hypercrosslinked polystyrene MN-150 containing ternary amino groups and having an anionexchange capacity of 1.06 mequiv.g21. 5 The retention of inorganic anions was investigated at pH 2.6–4.3 using 0.1–4 mmol dm23 perchloric acid as an eluent (Fig. 2). The retention of all anions is affected by both concentration of the perchloric acid and surface charge. The resulting curves present the sum of these factors and have a maximum around pH 3.5 except for nitrite with a retention maximum at pH 3.0. The obtained retention order, SO4 22 < Cl2 < IO3 – < Br2 < NO32 < I– < < SCN2 ~ NO22 < < IO42, is different from that generally observed for the common anion-exchangers.9 There are two useful features of this retention order.One feature is the very weak retention of sulfate that can shorten the analysis time of water samples usually containing sulfate, which is strongly retained on traditional anion-exchangers. The second difference is the comparatively high retention of nitrite. This allows separation of the nitrite peak relative to others on the chromatogram.These results can be explained from the point of view of dual mechanism of retention of anions. In our opinion, the combination of high hydrophobicity and anion-exchange properties due to a positively charged surface results in the unusual elution order. As it is in the neutral form due to protonation (pKa HNO2 = 3.4), nitrite must be more strongly retained at low pH of the eluent due to hydrophobic interactions. However it is more difficult to find a reasonable explanation for the strong retention of this anion on MN-200 at pH 4.3.At this pH, nitrite is still more strongly retained than thiocyanate. This probably means the existence of a special affinity of polystyrene based resin to nitrite. The relatively strong retention of nitrite was also found in additional experiments performed on a PLRP-S column. Only a few anions were retained by neutral PLRP-S resin in acid eluent (pH 2.85) while nitrite was retained much more strongly (kA = 0.92) than for hydrophobic anions such as thiocyanate (kA = 0.08) and periodate (kA = 0.10).The hydrophobic constituent in the retention of divalent sulfate is less evident than for monocharged anions which could be the possible explanation for the weak retention of sulfate. In spite of good anion-exchange selectivity of MN-200, the efficiency of the chromatographic column used in these initial experiments was not very high and allowed the separation of 2–3 anions (Fig. 3). However, further improvement of efficiency and resolution ability of the column can be achieved with Fig. 2 Plot of capacity factors of some inorganic anions versus pH of the mobile phase. Eluent: perchloric acid solution. UV detection, 210 nm; and conductometric flow rate, 0.8 ml min21. Fig. 3 Chromatogram of some inorganic anions on 15 mm MN-200, column 150 3 3 mm, eluent: 1 mmol dm23 perchloric acid solution. UV detection, 210 nm; and flow rate, 0.8 ml min21. 200 Anal. Commun., 1999, 36, 199–201the application of specially prepared narrow fine particles of MN-200 of spherical shape. Conclusions A new type of anion-exchange selectivity associated with the positive charge localized in the framework of the neutral hypercrosslinked resin MN-200 and with the high hydrophobicity of this resin was found. The usefulness of this resin for separation and determination of inorganic anions were demonstrated. Acknowledgements We thank the Royal Society of Chemistry for financial support (Journals Grants for International Authors) for the visit of Dr P. N. Nesterenko to the University of Plymouth where part of this investigation was performed. References 1 M. P. Tsyurupa, L .A. Maslova, A. I. Andreeva, T. A. Mrachkovskaya and V. A. Davankov, React.Polym., 1995, 25, 69; and references cited therein. 2 N. A. Penner, P. N. Nesterenko, M. M. Ilyin, M. P. Tsyurupa and V. A. Davankov, Chromatographia, 1999, in the press. 3 N. A. Penner, P. N. Nesterenko, A. V. Khryashchevskii, T. N. Stranadko and O. A. Shpigun, Mendeleev Commun., 1998, 23. 4 A. V. Khryashchevskii, T. I. Tikhomirova, P. N. Nesterenko and V. I Fadeeva, J. Anal. Chem., 1997, 52, 429. 5 M. Streat and L. A. Sweetland, Trans. Inst. Chem. Eng., 1998, 76, 115. 6 M. Streat and L. A. Sweetland, Trans. Inst. Chem. Eng., 1998, 76, 127. 7 R. M. S. Sutton, S. J. Hill and P. Jones, J. Chromatogr. A, 1997, 789, 389. 8 N. Masque, R. M. Marce and F. Borrull, J. Chromatogr. A, 1998, 793, 257. 9 P. R. Haddad and A. L. Heckenberg, J. Chromatogr., 1984, 300, 357. Paper 9/02449B Anal. Commun., 1999, 36, 199–201 201

ISSN:1359-7337    

DOI:10.1039/a902449b

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Electrochemical control of solid phase micro-extraction using unique conducting polymer coated fibers Thompson P. Gbatu,a Ozcan Ceylan,a Karen L. Sutton,a Judith F. Rubinson,a Ahmed Galal,b Joseph A. Carusoa and Harry B. Mark, Jr*a aDepartment of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, OH, 45221-0172, USA b Department of Chemistry, Faculty of Science, United Arab Emirates University, P.O. Box 17551, Al-Ain, United Arab Emirates Received 15th March 1999, Accepted 15th April 1999 The use of a solid phase micro-extraction (SPME) method with poly(3-methylthiophene) coated platinum micro-fiber electrodes to extract arsenate ions from aqueous solutions without derivatization is described.The fibers were fabricated by cycling the working electrode between 20.20 and +1.7 V (vs. Ag/AgCl) in an acetonitrile solution containing 50 mM 3-methylthiophene monomer and 75 mM tetrabutylammonium tetrafluoroborate (TBATFB) electrolyte.All electrochemical procedures (extraction and expulsion) were conducted in a three-electrode system. After fabrication, the conducting polymer film was immersed in the sample solution and converted to its oxidized, positively charged form by applying a constant potential of +1.2 V with respect to Ag/AgCl reference electrode. Arsenate ions migrated into the film to maintain electroneutrality. Upon subsequent reversal of the potential to 20.60 V vs. Ag/AgCl, the polymer film was converted to its reduced, neutral form and the arsenate ions were expelled into a smaller volume (200 mL) of de-ionized water for analysis using flow injection with inductively coupled plasma mass spectrometric (ICPMS) detection.Introduction Chemical speciation becomes important for trace metal analysis since, from a risk assessment perspective, it is not sufficient to quantitate the total elemental content of samples to define toxicities. For example, the determination of different forms of arsenic in the environment is critical because of the different levels of toxicity.Arsenate (AsO4 32) and arsenite (AsO22) are present in surface waters, in ground waters, in soils, in plant tissues, and in animal tissues1 and are highly toxic. In general, the principal analyzed compounds for the speciation of arsenic are arsenite, arsenate, monomethylarsonic acid (MMA) (CH3AsO3H2), dimethylarsenic acid (DMA) [(CH3)2AsO2H], arsenobetaine [(CH3)3As+CH2COO2] and arsenocholine [(CH3)3As+CH2CH2OH,Br2] and these have varying toxicities.2,3 Therefore, for concise evaluation of the risks associated with the exposure of biological systems to arsenic, precise and accurate methods for the identification and quantitation of arsenic compounds must be evaluated.Among the many methods available for determining inorganic and organic arsenic compounds are liquid chromatography,2–4 gas chromatography5–11 and capillary zone electrophoresis.12–14 Even though these methods give good sensitivities when coupled to element-specific spectrometric detectors such as ICP-AES and ICP-MS, they usually include difficult and time consuming sample preparation steps involving extraction and pre-concentration.A more efficient method is necessary for the extraction/preconcentration step of the analysis. Recently, solid phase micro-extraction (SPME)15–18 using silica and bonded phase fiber membranes19–21 as well as crown ethers22 has been the subject of investigations by a number of groups for extraction and pre-concentration of ionic and nonionic organometals and metal ions from aqueous solutions.Although these techniques have proven useful for the extraction of non-ionic organometals, there are several disadvantages when performing extractions of ionic organometals and metal ions. These include the need for derivatization of the species or the fiber surface to enhance the coating/water or coating/air partition coefficient of the ionic organometals,21, 23 the need for using highly selective and expensive crown ethers for metal ions,22 the length of time needed for both extraction and desorption (release) of the species from the coating when nonthermal desorption techniques such as HPLC solvents are used, and the possibilities of fouling the coating surface by matrix components.Even though derivatization methods in conjunction with SPME have considerably reduced the sample preparation time for these compounds compared to previously used classical liquid/liquid and other forms of extractions,21 they still have major drawbacks.Foremost is the need for expensive and sometimes toxic reagents and solvents. Also, side reactions involving the derivatizing reagents and matrix components such as metals, ions, ligands, and other hydrophobic compounds found in environmental samples tend to complicate derivatization. Another problem associated with derivatization is its effect on sensitivity.For example, NaBH4 volatilizes inorganic As(iii) and As(v) by formation of arsine (AsH3), and methylarsenic acids by the formation of methylarsenic(iii) hydrides.24 However, different arsenic compounds have different optimum pH values for derivatization. The result is a difference in sensitivity when derivatization reactions are carried out at constant pH. In fact, this is a common problem in the simultaneous derivatization of several compounds. Conducting polymers are a class of polymers that have electronic conductivity. The types, methods of preparation and applications are discussed elsewhere.25–31 Conducting polymer films such as poly(3-methylthiophene) (P3MT), may be doped when exposed to an aqueous solution containing an analyte anion at a particular oxidizing potential.The dopant may be expelled when the polymer film is reduced to its neutral form by applying a constant reduction potential for a set period of time.This paper presents preliminary results for the improvement of extraction and pre-concentration, without derivatization, of arsenate ions from aqueous solutions using such conducting polymers. Experimental Preparation of conducting polymer micro-fiber electrode Prior to the polymerization of 3-methylthiophene (3-MT), the micro-fiber electrode surface was conditioned (micro-fiber Anal. Commun., 1999, 36, 203–205 203electrode: 200 mm 3 1.5 cm Pt wire; reference electrode: Ag/ AgCl; and auxiliary electrode: 2 32 cm2 platinum sheet) in a10 mM aqueous H2SO4 solution.The conditioning process was performed with a Model 173 potentiostat/galvanostat (EG & G, Princeton Applied Research, Princeton, NJ) with an applied potential of 21.6 V vs. Ag/AgCl for 2 min. After conditioning of the platinum micro-fiber electrode, 3-methylthiophene was polymerized on the surface to P3MT by repeatedly cycling the electrode (100 mV scan21) between 20.20 and +1.75 V32 in an acetonitrile solution containing 50 mM 3-MT and 75 mM tetrabutylammonium tetrafluoroborate (TBATFB) electrolyte using a BAS-100 electrochemical analyzer (BAS, West Lafayette, IN).Typical cyclic voltammetric conditions for the electropolymerization of 3-MT included the following: working electrode, 200 mm 3 1.5 cm Pt wire; reference electrode, Ag/ AgCl; auxiliary electrode, 2 32 cm2 Pt sheet; scan rate, 100 mV scan21; CV cycle, 20.2 V to +1.7 V.The following conclusions, which are in agreement with the work of Galal32 may be drawn: (i) 3-MT is oxidized at +1.65 V to form the corresponding polymer. (ii) Upon reversing the direction of the sweep from the first oxidation wave, a corresponding cathodic wave for the reduction of the polymer film is observed in the region from +0.20 to +0.60 V. (iii) The subsequent anodic sweeps revealed the growth of the polymer film at a lower potential ( ~ Epa +1.6 V). The anodic peak potential occurring at ~ +1.20 V is attributed to the oxidation of the polymer film deposited during subsequent cycles.The formation of a polymer layer over the substrate was indicated by an increase of the current at a potential of ~ +1.65 V, the oxidation potential of the monomer.33 Flow injection ICP-MS A reciprocating pump (Dionex Corporation, Sunnyvale, CA) and a 6-port Rheodyne injector (Rheodyne, Cotati, CA) with a 10 mL loop were connected to an ELAN 6000 ICP-MS (Perkin- Elmer Sciex, Toronto, Canada) by means of a 46 cm long PEEK tube (0.01 in id).A cross flow nebulizer (Perkin-Elmer Sciex) with a Scott type spray chamber (Perkin-Elmer Sciex) was used. Reagents 3-MT (99+% purity, Acros Organics, Bridgewater, NJ), TBATFB (99% purity, Acros Organics), acetonitrile (HPLC grade, Fisher Scientific, Fairlawn, NJ), sulfuric acid (96% pure, Fisher Scientific) and dibasic sodium arsenate (Na2AsO4·- 7H2O) (Matheson, Coleman and Bell, Cincinnati, OH) were used as purchased.Results and discussion Preparation of conducting polymer micro-electrode fiber Fig. 1 is a scanning electron micrograph (SEM) of the polymer film on the platinum after polymerization. The surface of the ~ 5 mm thick polymer film has a ‘bumpy’ appearance. This is in agreement with other investigators32 who found that when P3MT films were grafted as thin films (102–103 Å) on the electrode, the surface was very homogenous, but when the polymer thickness was increased to a few microns, a ‘bumpy’ deposit rather than a smooth film was obtained.These morphological changes may be explained in terms of structural defects, such as cross-linking, b- versus a-coupling of the thiophene units, and the reticulation associated with it.32,34 Flow injection ICP-MS After the fabrication of the conducting polymer film on the platinum micro-fiber electrode, it was rinsed with de-ionized water and dried under a stream of N2 gas for a few seconds.Afterwards, it was immersed in 20 mL of a 100 mg L21 aqueous solution of AsO4 32 and converted to its positively charged form by application of a potential of +1.2 V vs. Ag/AgCl. Arsenate ions (AsO4 32) migrated into the polymer film to maintain electro-neutrality. Upon reversal of the potential to 20.6 V vs. Ag/AgCl, the polymer was converted back to its neutral hydrophobic form and the arsenate ions were expelled into a smaller volume (200 mL) of de-ionized water. 10 mL aliquots were injected via flow injection into the ICP-MS, using deionized water as the carrier solvent.It was determined that for a 10 min extraction period, approximately 210.4 ± 2.3 pg As was extracted from the 100 mg L21 aqueous solution into the film. This figure was obtained from consideration of the first extraction of three films fabricated in very similar fashion. Attempts to determine the linear dynamic range of the film proved difficult because of a decrease in the film uptake ability during successive extractions.This phenomenon is explained below. Fig. 2 is a chronoamperometric plot of the expulsion of the arsenate ions from the polymer film when a potential of 20.60 V vs. Ag/AgCl was applied. It may be seen that the expulsion is completed in a very short period of time; approximately 10 s in an aqueous matrix. Previous electrochemical and X-ray photoelectron spectroscopy (XPS) studies have shown that less than 5% of doping anions remain in the polymer matrix on reduction.32 Shortening of this expulsion time further can Fig. 1 Scanning electron micrograph (SEM) of poly(3-methylthiophene) film fabricated by cyclic voltammetry (working electrode: 200 mm 31.5 cm Pt wire; reference electrode: Ag/AgCl; auxiliary electrode: 2 3 2 cm2 Pt sheet); scan rate: 100 mV scan21; CV cycle: 20.2 to +1.7 V. Fig. 2 Chronoamperometry of the doping and undoping of poly(3- methylthiophene) film with AsO4 32. Doping potential: +1.2 V; undoping potential: 20.6 V. 204 Anal. Commun., 1999, 36, 203–205probably be obtained by more extensive optimization of the synthesis conditions and is the subject of current studies. Typical flow injection peaks obtained from ICP-MS analysis of aliquots of the expelled arsenate are shown in Fig. 3. At least three injections of aliquots from each extract were carried out as shown. A closer look at the flow injection peaks (A–K) of Fig. 3 shows a decrease in the amount of arsenate (AsO4 32) taken up during successive extractions. Considering the uptake and expulsion cycle of each film, it seems that the morphology of the film changes during each cycle.This trend was observed in three polymer films fabricated under similar conditions. Even though it has been shown that electrochemically synthesized polythiophenes showed high stability and doping/undoping reversibility in aqueous medium,32,35 it is known that, in the case of poly(methylthiophenes) (PMT), the film expands and contracts when doped and undoped, resulting in conformational changes during each cycle. This change is attributed to the loss of the fibrillar nature of the outer layer of the film36 where the bulk of the doping occurs.Therefore, it is postulated that the decreasing doping ability of the film may be attributed to these factors. Efforts to increase the reproducibility on multiple extractions by optimization of the synthesis conditions37 are presently underway.These include variation in film thickness and conditioning protocols before each extraction, among others. As the uptake and expulsion process involve a redox process in the polymer, there is a definite possibility of valence change of redox analytes, such as the various arsenic species, during these processes. This would eliminate the speciation capability in the analyses. This effect is being investigated. However, because of the availability of polymers with a wide range of redox potentials, it is very likely that such problems could be circumvented. Conclusions The potential use of poly(3-methylthiophene) film to extract and pre-concentrate anionic species such as arsenate from aqueous solutions without derivatization, and desorb or expel directly into an HPLC or flow injection system for analysis, has been demonstrated.There are several advantages of such an extraction and pre-concentration technique over previously established methods.First of all, it eliminates the need for expensive and sometimes toxic reagents and solvents. Second, by eliminating the derivatization step, the problem of sensitivity encountered when the species that are being simultaneously derivatized have different reaction rates at a particular pH or temperature is removed. Finally, extraction and pre-concentration of these species from aqueous solutions, without derivatization ultimately greatly reduces analysis time. References 1 W.R. Cullen and K. J. Reimer, Chem. Rev., 1989, 89, 713. 2 P. Morin, M. B. Amran, M. D. Lakkis and M. J. F. 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ISSN:1359-7337    

DOI:10.1039/a901991j

出版商:RSC    

年代:1999

数据来源: RSC