摘要:

Analyst, June 1996, VoE. 121 (877-881) 877 Development of Ultraviolet-polymerizable Enzyme Pastes: Bioprocess Applications of Screen-printed L-Lactate Sensors* Ingrid Rohm," Meike Genrich," Wendy Collierb and Ursula Bilitewski~~~ a Department of Enzymology, GBF-Gesellschaft fur Biotechnologische Forschung mbH, Mascheroder Weg I , 381 24 Braunschweig, Germany b Biosensor Group, AgResearch, Grasslands Research Centre, Private Bag I 1 008, Tennent Drive, Palmerston North, New Zealand The development of an amperometric L-lactate-specific enzyme electrode fabricated entirely using screen-printing technology is described. The sensor is based on immobilization of lactate oxidase from Pediococcus sp. by printing and subsequent UV irradiation within a polymerizable paste containing different supplements.The sensor optimization is described with respect to the most important features, such as composition and viscosity of the paste, stability, optimum pH and analytical range. The sensor was applied in a flow-through chamber within a flow injection (FI) system based on dialysis to off-line and on-line bioprocess monitoring of Geotrichum candidum cultivations in complex media. The sensor exhibited good operational stability; 4200 single injections (60 measurements per hour) led to a decrease of only 8.7%, the standard deviation being < 1% (n = 60). Using an FI system with direct sample injection (3 X 10-5 1 sample injection volume), the detection limit was 2 X moll-' and the linear range extended up to 1 X 10-3 moll-'. The linear range of the dialysis-based FI system extended from 5.5 x 10-4 to 5 x 10-2 moll-'.The correlation between results obtained by analysis of diluted off-line bioprocess samples with the amperometric device and with a homogeneous photometric assay using lactate dehydrogenase was r = 0.985. By on-line injection analysis of undiluted complex fermentation broth over a period of 12 h prior to the inoculation, no significant loss of sensor response was observed, which demonstrates the good operational stability of the system even with complex real samples. Additional investigations indicated the necessity for pH conformity of the sample and standard solutions. Keywords: Amperometric biosensors; screen-printed lactate oxidase; ultraviolet polymerization; bioprocess monitoring; Geotrichum candidum Introduction In addition to the importance of L-lactate as a major analyte in clinical diagnostics and food technology, it is one of the main parameters in bioprocess technology.During microbial culti- vations, lactate can function as a carbon source, as a fermentation product, or as an end product of anaerobic carbohydrate metabolism. Owing to cell toxicity,' lactate has to be monitored and controlled to achieve the optimum product yield during mammalian cell cultivations2~3. * Presented at the SAC '95 Meeting, Hull, UK, July 11-15, 1995. + To whom correspondence should be addressed. To date, particularly lactate oxidase (LOD) and lactate dehydrogenase (LDH) have been used in biosensor systems for lactate monitoring, owing to the stability and constant quality of these enzymes: (i) Lactate oxidase (LOD) from Pediococcus sp.: this enzyme catalyses the oxidation of L-lactate to pyruvate4 by 0 2 : L-lactate + 0 2 + pyruvate + H202 (ii) Lactate dehydrogenase (LDH) from muscle tissue (EC 1.1.1.27): L-lactate + NAD+ +- pyruvate + NADH + H+ Descriptions of biosensors containing this enzyme involve electrochemical detection principles, with the cofactor NAD+ being entrapped in front of the enzyme electrode by mem- branes.A large number of publications offer various types of immobilization procedures of these enzymes to generate L- lactate-specific electrodes.2.4 Only a few studies have involved L-lactate monitoring of real samples by amperometric enzyme electrodes. Renneberg et a1.6 described the utilization of conventional platinum electrodes covered with membranes in which the enzymes were immobilized in polyurethane or gelatin.White et al.7 presented studies based on rhodinized carbon electrodes applied to mammalian cell cultivation samples. Schalkhammer et aL8 reported the successful applica- tion of microfabricated thin-film electrodes during the monitor- ing of hybridoma cultivations. However, most of the systems described for on-line monitoring of L-lactate are flow injection (FI) systems consisting of small enzyme columns with relative large amounts of enzymes to enhance the working stability during time-consuming bioprocesses.2 In recent years, the applicability and relevance of screen- printed biosensors have been impressively demonstrated by the economic success of amperometric blood glucose sensors.9 Owing to the widespread establishment of screen-printing processes in the microelectronics and printing industries, devices and various basic materials are now commercially available that allow the automated production of electrochem- ical transducers.lo Unfortunately, most of these sensors, which are normally glucose electrodes, cannot be utilized during continuous measurements in flow-through devices with aque- ous carrier solutions owing to the solubility of their enzyme layers containing graphite or carbon particles and water-soluble p ~ l y m e r s . l ' - ~ ~ To overcome this problem, Nagata and co- w o r k e r ~ ' ~ , ~ ~ developed a screen-printable enzyme layer con- taining ferrocene-modified glucose oxidase (GOD) within a mixture of soluble polyvinylpyrrolidone (PVP) and insoluble polyvinylbutyral (PVB), being dried in air.The production of enzyme electrodes with significant operational stability suitable for integration in analytical flow878 Analyst, June 1996, Vol. 121 systems by UV polymerization of a screen-printed GOD layer was reported recently.'* In this paper, we report details of the development and optimization of L-lactate electrodes with suitable measuring conditions within FIA systems, and the application of the system to on-line monitoring of cultivations of Geotrichurn candidum utilizing L-lactate as the carbon source when producing L-lactate oxidase. 19 Experimental Production of Thick-film Transducers Platinum paste (Condutrox 3804; Ferro, Sindelfingen, Germany) was printed using a conventional screen-printing machine (M2; Ekra, Kirchheim, Germany) on fired aluminium oxide ceramics (Laser Tech, Nurnberg, Germany), producing the working electrodes.A silver-palladium paste (No. 7474; Du Pont, Bad Homburg, Germany) was used to print the reference electrode, and the conducting paths and the isolating layer were applied by screen printing of a dielectric paste (No. 5032; Du Pont) (Fig. 1). Each printing step was followed by a firing process at 850°C (Bruce 7354 Control Unit; BTU, Fam- borough, UK) to generate stable and well defined layers with a thickness of 2 X 10-5 m. Eight electrochemical transducers were printed simultaneously. Immobilization A 50 mg amount of UV-polymerizable and water-soluble paste (UVP) was mixed with 2 X 10-5 1 (or 1 X 10-5 1) of LOD A I g - substrate I Pt - working electrodes Ag/Pd - reference electrode Ag/Pd - conducting Path Isolating dielectric Ag/Pd - connectors Fig.1 Screen-printed thick-film transducer (1.27 X X 2.5 X m) suitable for flow-through devices. Each platinum working electrode is 5.3 x 10-3 m2 and the silver-palladium reference electrode is 2.9 X m2. solution [30 (60) mg ml-l in 0.1 moll-' potassium phosphate buffer (pH7.5)]. Different amounts of a porous SiO2 (Syloid) material with a particle diameter of 4 X 10-6 m, graphite, and/ or solutions of DEAE-dextran (20%) and lactitol (50%) in potassium phosphate buffer (pH 7.5) were added and the resulting enzyme paste was screen printed to a thickness of 5 X lO-(j-lO X m on the surface of one platinum working electrode of each electrode system.The second electrode remained untreated to monitor the influence of interferences of components of the medium and to detect possible 'cross-talk' between the electrodes during standard measurements. Polym- erization was performed by UV irradiation with a mercury- vapour lamp (100 W; Osram, Hannover, Germany) for 3-5 s at a distance of 0.1-0.15 m. The UV-enzyme electrodes were stored under dry conditions at 4 "C. Characterization of Viscosity By using a rotating disc viscosimeter (Rotovisco RV 20; Haake, Karlsruhe, Germany), the viscous behaviour of the non- polymerized pastes during increasing and following decreasing rotation velocity was measured, A commercially available UV- polymerizable screen-printable ink was used as a reference (UVLM-Nl; Marabu, Thamm, Germany).FI Manifold Two types of FI systems were used: (i) The device connected via an automated sampling device (ESIP 5441; Eppendorf, Hamburg, Germany) to a bioreactor is shown schematically in Fig. 2. The system contained a dialysis module for removal of components of the medium. It consisted of peristaltic pumps (Meredos, Norten Hardenberg, Germany), an injection valve (V 100; Perstorp Analytik, Rodgau, Ger- many), a dialysis module (GBF, Braunschweig, Germany) and an electrochemical wall jet detector cell with a volume of approximately 3 X 10-5 1 (GBF) containing the screen-printed sensor. This flow through chamber was similar to that described by Gunther and Bilitewski.20 The amperometric measurements were made by using a two-channel potentiostat (Meredos) and applying a constant potential of 6 X 10- V in a three-electrode configuration (two working electrodes, one reference electrode, one auxiliary electrode). A hypodermic needle was inserted as an inlet to the flow-through device operating as an auxiliary electrode.The tubing was made of PTFE (Omnifit, Cambridge, UK) with an inner diameter of 5 X m and the carrier buffer (see below) was pumped at 5 X 10-4 1 min-1. A sample volume of 1 X 10-4 1 was injected. Fig. 2 Layout of FI device used for off-line (without in situ filtration module) and on-line bioprocess L-lactate measurements.Analyst, June 1996, Vol. 121 879 (ii) Additionally, a conventional FI system without a dialysis module and based on direct injection of sample volumes of 3 X 10-5 1 was used for basic experiments during sensor develop- ment.The FI-system was controlled via a PC by the program FIA-FOX 2.0 (GBF),21 which permitted the recording and processing of data by calculating calibration curves and resulting sample concentrations. Fermentations A strain of Geotrichum candidum producing lactate oxidase and screened at the GBF was cultivated in its filamentous habit in a 10 1 strongly stirred bioreactor. The fermentations were carried out in complex medium starting with an L-lactic acid concentra- tion of approximately 10-15 g l-l.I9 Buffers Potassium phosphate buffers (PPB) (0.1 moll-1) from pH 6 to 8, 0.05 mol 1-1 HEPES { 2-[4-(hydroxyethy1)- 1 -piperazino]- ethanesulfonic acid}-NaOH buffers of pH 7 and 8 and Clark and Lubs (C&L) solutions (0.05 mol 1-1 with respect to both KCl and H3B03 and an appropriate amount of NaOH) from pH 7.5 to 10 were utilized as carrier buffers to determine the optimum pH of the sensors.Reagents The following reagents were used: L-lactate oxidase from Pediococcus sp. (LOD) (Boehringer, Mannheim, Germany), L- (+)-lactic acid, lithium salt (Sigma, Deisenhofen, Germany), Si02 particles (Syloid ED 20, ED 40 and ED 378; Grace, Worms, Germany), graphite (fine powder, pure quality; Merck, Darmstadt, Germany), lactitol (4-O-P-~-galactopyranosyl-~- glucitol) (Sigma, Deisenhofen, Germany) and diethanolaminoe- thyl(DEAE)-dextran (Sigma); assay for L-lactic acid, food analysis, UV method (Boehringer). All other chemicals, of analytical-reagent grade, were obtained from Merck.Results and Discussion Modification of Viscosity During the manufacturing process of thick-film electrodes, pastes are pressed through a patterned screen by a squeegee on to a solid surface,22 hence reproducible results can only be obtained if the pastes possess appropriate flowing characteris- tics. These are given by a homogeneous mixture of all constituents of the paste, even if pressure is applied to the paste during the printing process, and by a suitable viscosity of the mixture. However, the latter parameter can vary widely and exact data are dependent on the method chosen for determina- tion. Both parameters were varied, when the basic paste was modified to an enzyme paste and were determined using a rotating disc viscosimeter.Most of the enzyme pastes described in literature contain graphite and therefore its influence on the flow characteristics was evaluated. Though the viscosity increased, it remained within acceptable limits, however, a discontinuous tension was observed when shear forces were increased. This was not found, when graphite was replaced by Si02 particles (Syloid). On the other hand, the addition of enzyme, lactitol, or DEAE-dextran solutions decreased the viscosity. Amperometric Detection of L-Lactic Acid by Screen-printed UV-LOD Electrodes Addition of stabilizers Gibson et al.23 found polymerized alcohols and carbohydrates in certain combinations to be suitable for the long-term stabilization of immobilized enzymes. Thus, the addition of lactitol and DEAE-dextran as supplements to the UV-LOD paste was investigated.Different amounts of each compound were added. The best results were obtained using a final concentration of 5% lactitol and 1% DEAE-dextran in the enzyme paste (Fig. 3). Compared with data obtained using an electrode prepared without stabilizing agents, the response of stabilized sensors exhibited at least a tenfold improved response. An L-lactate concentration of 2 X 10-8 mol 1-1 was obtained as the detection limit (signal-to-noise ratio 3 : l), a concentration of 2 X 10-7 moll-' could be quantified and the linear detection range extended up to 1 X 10-3 mol 1-1. The improved sensitivity could be caused by the formation of a charged and hydrophilic environment within the enzyme layer which improves the diffusion and increases the local concen- tration of the charged L-lactate molecules. Furthermore, carbohydrates are known as protein stabilizers, hence the LOD could be additionally protected.23 The apparent K, of the sensor containing the stabilizers was 2.5 X 10-3 moll-1, which is in good agreement with the value for soluble LOD of 0.7 X mol 1-1.24 This indicates a low diffusion resistance of the immobilized enzyme layer to the analyte molecules and/or an improved enzyme conformation due to the stabilizers.A combination of 5% lactitol and 1% DEAE-dextran within the enzyme paste was used for all subsequent experiments. Optimum pH of screen-printed L-lactate sensors Different types of buffer systems were used as carrier buffers in an FI system with direct sample injection to determine the best carrier solution.Standards were dissolved in each buffer. Similarly to screen-printed UV-GOD sensors, 18 the optimum pH of UV-LOD sensors was found to be 8.5 (Fig. 4). This is 10000 J 4- UVP-LOD with stabilizers -A- UVP-LOD without stabilizer: .---- 104 I . I . I . 1 ' I ' I 0 2 4 6 8 10 L-Lactate/l O - ~ ~ O I I-' Fig. 3 tion with DEAE-dextran (1 %) and lactitol (5%). Sensitivity increase of UV-LOD electrodes by paste supplementa- 140 1 60- -m-0.1 rnol I-'Potassium phosphate -0-0.05 rnol I-'Clark & Lubs -A-0.05 rnol I-'HEPES 40 1 10 pH of carrier Fig. 4 Optimum pH of UV-LOD sensors containing stabilizers (DEAE- dextran, lactitol) within the enzyme layer. Procedure for one buffer to be tested: injection of 1 X rnol 1-I L-lacate; 10 measurements using 0.1 moll-' PPB pH 7.5 as reference carrier, 50 measurements in carrier buffer; detection signal in PPB was set as 100%.8 80 Analyst, June 1996, Vol.121 caused by the combination of two reactions: ( i ) the reaction of LOD with an 0ptimurn2~ at pH 6.5 and (ii) hydrogen peroxide oxidation at thick-film platinum electrodes with its optimum25 at pH 10. Utilization of C&L buffer as the carrier solution generated higher sensor signals than with PPB and HEPES buffers at the same pH values. As mentioned earlier,15 the complexing agent borate in C&L solution could prevent the deposition of metal ions solubilized from the glassy thick-film pastes of conducting and isolating layers. This causes decreased sensor signals due to reduced H202 oxidation.Long-term operational stability in C&L and PPB The long-term stability tests shown in Fig. 5 were carried out in an FI device using direct sample injection. The frequency of measurements was 60 h-1 and temperature control was not applied. Using PPB (pH 7.5) as carrier solution, the signals decreased within 8 h (480 measurements) to 18% of their original height. During the following 120 measurements the signals remained stable, so the experiment was stopped. In contrast, the use of C&L (pH 8) as the carrier buffer improved the stability of the sensor response impressively. During a period of 70 h (4200 detections), the signals decreased by only 8.7% of their original height. The standard deviation was 0.96% for 60 measurements.Before these results were obtained, it was assumed that LOD is more stable in the neutral pH range of PPB owing to its pH optimum of 6.5. However, it could be shown that the decrease in sensor response observed was mainly an effect of PPB on LOD. Earlier detection of H202 in PPB at a blank electrode exhibited a smaller sensor decrease. These data indicate that C&L is suitable as buffer solution for L-lactate determinations with UV-polymerized enzyme layers containing LOD. The basic pH did not affect the enzyme activity, so this buffer was preferred for use during most of the following investigations. Bioprocess Monitoring of Geotrichum canadidum Cultivations Detection range and shape of signals The experiments described below were carried out with an FI system containing a dialysis module to prevent clogging of the enzyme layer by high molecular mass cellular compounds (see Fig.2). Donor and acceptor streams were transported con- tinuously through the dialysis module, where diffusion of analyte took place. The peaks shown in Fig. 6 represent original signals obtained during the calibration of such a dialysis-FI with L-lactic acid standards. The linear range extended from 5 X 10-2 to 4.5 g 1-l. Owing to the sample dilution by dialysis, A 120 r 0 500 1000 1500 2000 2500 3000 3500 4000 2 o L n . I . ' . " " ' " * .I Number of measurements Fig. 5 Long-term operational stability test of UV-LOD sensors contain- ing stabilizers (DEAE-dextran, lactitol) using C&L and PPB buffer systems. Injection of 30 p1 of 10-4 moll-' L-lactate.Decrease in signal height: 8.7% within 4200 detections (C&L), 82% within 480 detections (PPB). saturation could be observed at L-lactic acid concentrations higher than 50 g 1-1. Hence lactate concentrations beyond the linear range from 5 to 50 g 1-1 could be calculated by polynomial regressions. Off-line Samples Filtered samples from a Geotrichum candidum fermentation were diluted 10-fold in C&L (pH 8). This buffer was used also as a donor and acceptor solution in the dialysis-FI. The samples were analysed simultaneously with the bioprocess. After 21.5 h, lactate was fed to the cultivation medium as a feeding experiment. Apart from the influence of temperature, no significant decrease in sensor signals caused by the complex medium could be observed during 28 h of fermentation, as indicated in Fig.7. The results obtained with the Fl[ system showed a good correlation with data given by a photometric assay using lactate dehydrogenase, with a correlation coefficient of 0.985, a standard deviation of 0.804 (n = 21) and a slope of the linear fit of 0.998. On-line measurements For on-line monitoring of a similar Geotrichum candidum cultivation, the FI system was directly connected to an in situ lo" 5 x 10" lo'* 5 x loe2 10'' , I lo-' A Fig. 6 Original signals obtained by L-lactic acid calibration using LOD- UV sensors integrated in a dialysis-based F1 system without a filtration module. Linear range between 5 X 10-2 and 4.5 g 1-1; standard deviation 12% ( n = 4). 20 I */.----4 \ a A A A ,,.aaP \,-A" - 2 3 Addition of L-lactate 7.-a- Sensor stability -0- Photometric assay Off-line FI data - 1100 -80 v, r! - 60 2 ' c r - 4 0 5 0 5 10 15 20 25 Fermentation/h Fig. 7 Bioprocess monitoring with amperometric UV-LOD sensors integrated in a dialysis-based FI system. Sample dilution, 1 + 9 in C&L (pH 8); multi-concentration calibration before inoculation (see Fig. 6); one-point calibration (9 X 10-1 g 1-1) during entire cultivation; n = 16.88 1 Analyst, June 1996, Vol. 121 r ?- 12 - .- 'D 10 - a 8 - o 6 - 4 4 - u) \ .- - filtration module from which sample was transported to the injection valve by a peristaltic pump (Fig. 2), i.e., the samples were injected undiluted without any further treatment. Twelve hours ( = - 12 h) prior to the inoculation ( = 0 h) of the fermenter, the determination of L-lactate started with 15 measurements per hour. Apart from the decrease in signals caused by the fall in room temperature overnight, no decrease in signals was observed even when a complex medium was used as sample.The second (blank) working electrode of the sensor (Fig. 1) showed a response of less than 1% of the enzyme sensor. Therefore, no correction to L-lactate concentrations by differential calculations was applied. Comparison of the on-line data with results obtained by a homogeneous photometric assay showed good agreement (Fig. 8). -I 0 - Conclusion This paper has described the development of amperometric L- lactate-specific biosensors exhibiting satisfactory operational stability in an FI system. To allow the production of enzyme sensors on a larger scale, transducers and enzyme layers were produced by screen-printing technology with hardening of the LOD layer by UV irradiation.The optimization of the enzyme sensor by viscosimetric investigations, addition of various supplements such as stabilizing reagents to the enzyme paste and the search for the best working buffer were described in detail. To demonstrate the applicability of the L-lactate sensors, they were inserted in a dialysis-based FI system for analysis of real samples obtained by off-line and on-line sampling during Geotrichum candidurn fermentations in complex media. The sensors showed good operational stability also in complex solutions and the analytical detection range corresponded well with the analyte concentrations in bioprocess samples.Further it should be possible to adapt the analytical range of the sensor to specific analytical problems by variations of the enzyme paste compositions. This immobilization procedure has also been used successfully to entrap other enzymes, such as glucose Photometric lactic acid assay *A A On-line lactic acid determination + A A M %?A A '&'A, m Addition of L-lactate AA %.j ,"A A A oxidase,'* alcohol oxidase, fructose dehydrogenase, pyranose oxidase, acetylcholinesterase and tyrosinase. Further investiga- tions will be performed to extend the number of practical applications of these screen-printed, UV-polymerizable, am- perometric biosensors. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Ozturk, S.S., Riley, M. R., Palsson, O., Biotechnol. Bioeng., 1992, 39, 418. Bilitewski, U., and Rohm, I., in CRC Handbook of Biosensors: Medicine, Food and Environment, ed. Kress-Rogers, AT1 Sensor Applications, Ripley, Surrey, in the press. Brooks, S. L., Higgins, I. J., Newman, J. D., and Turner, A. P. F., Enzyme Microb. Technol., 1991, 13,946. Hendry, S. P., Higgins, I. J., and Bannister, J. V., J . Biotechnol., 1990, 15, 229. Weaver, M. R., and Vadgama, P. M., Clin. Chim. Acta, 1986, 155, 295. Renneberg, R., Trott-Kriegeskorte, G., Lietz, M., Jager, V., Pawlowa, M., Kaiser, G., Wollenberger, U., Schubert, F., Wagner, R., Schmid, R. D., and Scheller, F., J . Biotechnol., 1991, 21, 173. White, S. F., Turner, A. P. F., Bilitewski, U., Schmid, R. D., and Bradley, J., Anal.Chim. Acta, 1994, 295, 243. Schalkhammer, T., Lobmaier, C., Ecker, B., Wakolbinger, W., Kynclova, E., Hawa, G., and Pittner, F., Sens. Actuators B, 1994, 18-19,587. Cardosi, M. F., and Turner, A. P. F., in The Diabetes Annual, ed. Alberti, K. G . M. M., and Krall, L. P., Elsevier, Amsterdam, 1990, vol. 5 , p. 254. Alvarez-Icaza, M., and Bilitewski, U., Anal. Chem., 1993, 65, 525A. Bilitewski, U., Chemnitius, G. C., Riiger, P., and Schmid, R. D., Sens. Actuators B , 1992, 7, 351. Cardosi, M. F., and Birch, S. W., Anal. Chim. Acta, 1993, 276, 69. Marcinkeviciene, J., and Kulys, J., Biosens. Bioelectron., 1993, 8, 209. Koopal, C . G. J., Bos, A. A. C . M., and Nolte, R. J. M., Sens. Actuators B , 1994, 18-19, 166. Schmidt, A., Rohm, I., Riiger, P., Weise, W., and Bilitewski, U., Fresenius' J . Anal. Chem., 1994, 349, 607. Nagata, R., Clark, S.A., Yokoyama, K., Tamiya, E., and Karube, I., Anal. Chim. Acta, 1995, 304, 157. Nagata, R., Yokoyama, K., Clark, S. A., and Karube, I., Biosens. Bioelectron., 1995, 10, 261. Rohm, I., Kunnecke, W., and Bilitewski, U., Anal. Chem., 1995, 67, 2304. Genrich, M., Thesis, Technical University of Braunschweig, 1996. Giinther, A., and Bilitewski, U., Anal. Chim. Acta, 1995, 300, 117. Stiene, M., and Bilitewski, U., in Proceedings ofthe 7th European Congress on Biotechnology, Nice, France, February 19-23, 1995, Abstracts Book, SocietC Fransaise de Microbiologie, Paris, 1995, vol. 3, p. MEP 174. Wring, S. A., and Hart, J. P., Analyst, 1992, 117, 1281. Gibson, T. D., Hulbert, J. N., Parker, S. M., Woodward, J. R., and Higgins, I. J., Biosens. Bioelectron., 1992, 7, 701. Catalogue, Sigma Chemical, Sigma Chemie, Biochemikalien Orga- nische Verbindungen Diagnostika, Deisenhofen, 1995. Rohm, I., Thesis, Technical University of Braunschweig, 1996. Paper 5108008H Received December 8,1995 Accepted February 21,1996

ISSN:0003-2654    

DOI:10.1039/AN9962100877

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, June 1996, Vol. 121 (883-886) 883 Impedance Sensor for Dissolved Nitrogen Oxide Using a Series Piezoelectric Crystal Device Yuanjin Xu, Changyin Lu,* Yan Hu, Lihua Nie and Shouzhuo Yaot Department of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China An impedance sensor for the determination of nitrogen oxide in aqueous solution is presented. The sensor is realized by using a series piezoelectric crystal (SPC) device, which is constructed by connecting an AT-cut piezoelectric crystal with a probe in series. The probe is filled with an internal electrolyte solution that is separated from sample solutions by a gas-permeable membrane. Such a gas sensor based on the SPC device can overcome the influence of water vapour efficiently and can be used in the determination of nitrite in aqueous solutions.The proposed sensor exhibits a favourable frequency response to 5 x 10-7-1 x 10-3 moll-' nitrite. The limit of detection is 1 X 10-7 mol I-'. Aspects of the sensor preparation are considered. The dynamic range, reproducibility, response time and selectivity of the sensor are also discussed. The proposed sensor was used successfully for the determination of nitrite in smoked pork samples; the results obtained were comparable to those obtained by the official first action AOAC method. The technique described here should find wider application in the development of other gas and enzyme sensors. Keywords: Piezoelectric crystal; impedance sensor; nitrogen oxide sensor; nitrite determination Introduction By using selective absorbent coatings, piezoelectric bulk acoustic wave (BAW) sensors have been developed into a highly sensitive technique for the detection of trace amounts of atmospheric pollutants.1 For the detection of nitrogen oxide in air, Edmonds et a1.2 described the use of a manganese dioxide coated BAW sensor. However, the coated BAW sensor cannot be used directly in the determination of nitrogen oxide in aqueous solution because it is very sensitive to the interference of water vapour. In order to overcome the water vapour response of the coated BAW gas sensor, we report a novel impedance sensor for dissolved nitrogen oxide which is based on a series piezoelectric crystal (SPC) device. In contrast to the coated BAW sensor based on the mass effect of the piezoelectric crystal, the proposed sensor is based on the high sensitivity to solution conductivity of the piezoelectric crystal d e ~ i c e .3 ~ Therefore, the interference of water vapour can be avoided. In this work, an impedance sensor for the determination of nitrogen oxide in aqueous solution was developed. The proposed sensor is not influenced by water vapour; in addition, the sensor has a long lifetime, a simple design and is amenable to miniaturization. * On leave from Hengyang Medical College, Hunan, 421001, China. + To whom correspondence should be addressed. Nitrite, which has long been used in the curing of meat, poultry and fish,7 is able to react with secondary amines to form nitrosamines, a family of compounds in which most of the members are carcinogenic.8 Hence, there has been a growing concern about the possible danger and specific side reactions of sodium nitrite.The use of the proposed sensor for the determination of nitrite in smoked pork samples was investi- gated. The accuracy of the sensor was also evaluated by comparing the results obtained with those obtained by the official first action AOAC m e t h ~ d . ~ Experimental Apparatus and Reagents The experimental apparatus was assembled as shown in Fig. 1. The IC-TTL oscillator has been described previ~usly.~ The frequency output from the oscillator was monitored by an Iwatsu (Shijia Zhiang, China) model SC7201 Universal fre- quency counter and was recorded by a computer [Hewlett- Packard (Avondale, PA, USA), 3001 which was interfaced to the frequency counter.The piezoelectric crystal was a 9 MHz AT-cut 12.5 mm disc with gold electrodes (6 mm in diameter) on both sides. The temperature of the test cell was thermostated at 25 L- 0.1 "C with a water jacket and a temperature controller. Teflon membranes with an average pore size of 0.02 pm were purchased from Jiangsu Electroanalytical Instrument Co. (Jiangsu, China). Absorbance measurements were made with a 75 1G spectrophotometer (Shanghai Analytical Instruments Co., Shanghai, China). An impedance analyser (Hewlett-Packard, 4192A) was used to measure the parameters of the sensor. The probe is shown schematically in Fig. 2(a). The over-all dimensions of the probe were about 15 X 6 X 0.3 mm. Two finely ground platinum foils were used as electrodes. The thin layer between the electrodes was filled with an internal electrolyte solution (about 5 pl) which was separated from the sample solutions by two pieces of gas-permeable membrane Fig.1 Schematic diagram of the experimental system: A, power supply; B, oscillator; C , frequency counter; D, computer; E, piezoelectric crystal; F, test cell; and G, temperature controller.884 Analyst, June 1996, VoE. 121 mounted firmly on both sides of the probe. The cell constant of the probe was determined by measuring the conductance when the probe was filled with a standard KCl solution the conductivity of which was accurately known. All other reagents used were of analytical-reagent grade. Doubly distilled, de-ionized water was used for all solutions. Smoked pork samples were obtained from the local Meat Products Co.Procedures Sensor calibration was performed by immersing the probe into the test cell which contained 5 ml of 0.1 mol 1-1 sulfuric acid solution. The experiment was started by making an addition of sodium nitrite standard solution and the frequency was then recorded with time until a steady frequency was obtained. The smoked pork samples were prepared according to the AOAC m e t h ~ d . ~ Before the measurements, the prepared samples were heated on a steam-bath to eliminate the reductants found in the The nitrite concentrations in the smoked pork samples were measured with the sensor and with the AOAC spectrophotometric method.9 lL L q Cs R Q G Fig. 2 (a) Schematic diagram of the probe: A, B, leading wires of the probe; C, gas-permeable membrane: D, E, platinum electrodes; F, thin layer of internal electrolyte solution; and G, insulation substrate. (b), Equivalent circuit of the SPC: C,, static capacitance; L,, motional inductance; C,, motional capacitance; R,, motional resistance of the piezoelectric crystal; C,, solution capacitance; and G, solution conductance.Fig. 3 various cell constants. Theoretical response sensitivity of the sensor to conductivity with Results and Discussion Theoretical Background The SPC device, which is constructed by connecting an AT-cut piezoelectric crystal and a conductive electrode in series, possesses a high sensitivity to the conductivity and relative permittivity of the solution and a good frequency stability.5.6 The equivalent circuit of the SPC is illustrated in Fig.2(b). The oscillation frequency, F , is expressed as F = F , 1 + ~FoCq(2~FoC, - YG) 4n2F,C,(C, + C,) - ~TCF,C,YG + G2 -XFoCP,Y] (1) I where F , = 1 / 2 n m is the resonant frequency of the crystal, L,, C,, R, and C, are the motional inductance, motional capacitance, motional resistance and static capacitance of the piezoelectric crystal, respectively; G = kX, C is the conductance of the solution, k is the cell constant of the conductive electrode and X is the conductivity; C, = kE + C,, C, is the solution capacitance E is the relative permittivity of the solution and C, is the parasitic capacitance between the leading wires of the conductivity electrode; Y = tan(@, 0 is the oscillator phase. When the SPC device is used in aqueous solutions, the frequency response depends mainly on the solution conductiv- ity since the relative permittivity varies little in dilute electrolyte solutions.The sensitivity to conductivity, i.e., the slope of the plot of frequency F versus conductivity X , can be calculated by differentiating eqn. (1) with respect to X aF nkF;C, (4n2F:C:Y + 4nFoC,G - YG2) (2) The theoretical response sensitivity of the sensor to con- ductivity with various cell constants given by eqn. (2) is illustrated in Fig. 3 for typical parameters. It can be seen that the SPC device still has a highly sensitive response to conductivity in high conductivity solutions. Compared with the direct conductivity measurement method, in which sensitivity is limited and accuracy is poor when foreign electrolytes are present,'* the SPC device can be used in samples that contain large amounts of unreacted foreign electrolytes.Hence, the SPC device can be used as the basis of a practical sensor, in the same -- - - ax [4n2F:C, (C, + C,) - ~TcF,C,YG + G2]2 2.8 1.5 1.0 0 . 5 0.0 0.0 0 - 2 0 . 4 0 . 6 0 . 8 1.0 Sample concentration/I0-4moI. 1-1 Fig. 4 Effect of NaN02 concentration in internal electrolyte solution on sensitivity. Cell constant, 0.0126 m; NaN02 concentrations (moll-'): A, 0; B, C, 5 X and D,885 Analyst, June 1996, Vol. 121 way as the potentiometric and fibre optic pH sensors, to develop gas and enzyme sensors by using additional membranes. When the probe is in contact with a sample, gaseous nitrogen oxide diffuses across the gas-permeable membrane until the partial pressures are equal in the sample and in the thin film of internal electrolyte solution.The nitrogen dioxide that diffuses into the internal electrolyte solution will dissolve in water and reach equilibrium with nitrite' 2N02 + H20 N03- + NO2- + 2H+ (3) Variation of the nitrogen dioxide concentration in the internal electrolyte solution causes a change in the conductance of this solution which alters the oscillation frequency of the SPC device. As the sample nitrite concentration increases, the amount of nitrogen dioxide in the internal electrolyte solution also increases, which results in an increase in the conductance of the internal electrolyte solution and a decrease in the oscillation frequency of the SPC device. Hence, an increase in sample nitrite concentration is measured as a decrease in the oscillation frequency of the sensor.Choice of Buffer Composition The influence of the NaN02 concentration in the internal electrolyte solution on the sensor sensitivity was investigated. The frequency response of the sensor when filled with NaN02 solutions of various concentrations is shown in Fig. 4. It is evident that the sensitivity of the sensor filled with solutions of low NaN02 concentration is higher for samples with a low NaN02 concentration. On the other hand, the sensor filled with solutions of high NaN02 concentration produces curves with a steady, linear response for samples with a high NaN02 concentration. In the following experiments, the probe was filled with 5 X The sensitivity of the sensor is also influenced by the concentrations of unreacted foreign electrolytes which exist in the internal electrolyte solution.The sensitivity of the sensor with various concentrations of foreign electrolyte, KCl, added is shown in Fig. 5. When the KCI concentration reaches 5 X 10-3 mol 1-1, the sensitivity decreases significantly. However, at a KCl concentration of less than 1 X 10-3 moll-1, the influence of the unreacted foreign electrolytes on the sensitivity is slight. mol 1-1 NaN02 solution. 2 . 0 I 1 B 1 . 5 1.0 0 . 5 0.0 8 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1.0 N i t r i t e concentratfon/lfiV m o l . 1-1 Fig. 5 Effect of foreign electrolyte concentration in internal electrolyte solution on sensitivity. Cell constant, 0.0126 m; KC1 concentrations (moll-1): A, 0; B, C, 5 x D, 10-3; and E, 5 x 10-3.The osmotic effect is inherent to the design of the probe in that a semi-permeable membrane separates two solutions, the sample and the internal electrolyte film.*' If the total concentra- tions of dissolved species on the two sides of the membrane are different, an osmotic pressure difference results and water vapour diffuses across the membrane until the water activity is the same on each side. Transfer of water across the membrane results in dilution or concentration of the internal electrolyte solution, which in turn causes the sensor response to drift. Therefore, glycerin was used in the internal electrolyte solution to reduce the osmotic effect. Effect of Cell Constant The cell constant of the probe, k, is an important parameter for the design of an SPC impedance sensor.Various values of k result in a marked difference in the sensitivity to the conductivity (X), and alter the solution capacitance (C,) of the 2.0 1 . 5 1.0 0 . 5 0 . 0 C 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1.8 Nitrite concentration/lB" mol. 1-l Fig. 6 Effect of the cell constant on sensitivity. Internal electrolyte solution: 5 X mol 1-1 NaN02 + 1% glycerin; cell constants (m): A, 0.0038; B, 0.0075; C, 0.0126; D, 0.0189; and E, 0.0273. ~~ ~ ~~~ ~ Table 1 Interferences in the determination of nitrite with the proposed sensor* Interferent Nitrite concentratiodmol 1-1 concentration/ Interferent moll-' Added Found COZ 1 x 10-3 1 x 10-5 1.12 x 10-5 CH~COOH 1 x 10-5 1 x 10-5 1.01 x 10-5 HCOOH 1 x 10-5 1 x 10-5 1.05 x 10-5 HF 1 x 10-5 I x 10-5 1.08 x 10-5 * Mean of three determinations.Table 2 Determination of nitrite in smoked pork samples* Proposed AOAC sensor/ method?/ Sample mg kg-1 mg kg-1 1 72 k 0.26 74 f 0.20 2 59 f 0.21 63 k 0.15 67 & 0.13 3 66 f 0.30 * Mean k standard deviation (n = 3). + Ref. 9.886 Analyst, June 1996, Vol. 121 Table 3 Comparison with a potentiometric nitrogen oxide sensor Response range/ Detection limit/ Response Reference moll-' moll-' time/s* electrode Ref. 3 10 None This work Proposed sensor 5 x 10-7-1 x 10-3 1 x 10-7 Potentiometric sensor 2 x 10-6-1 x 10-2 10-7 370-480 Ag/AgCl 11 * Nitrite concentration changes from 1 X 10-6 to 1 X 10-5 moll-1. probe. When k increases, the sensitivity to the conductivity increases (G = kX); meanwhile, the solution capacitance also increases (C, = k~ + C,) which reduces the sensitivity of the frequency reponse of the sensor to solution conductance.The sensitivity of the frequency response of the sensor is determined by these two opposing factors. As shown in Fig. 6, the sensitivity of the sensor reaches a maximum around k = 0.01 m and decreases significantly when k is larger than 0.02 m. In this work, the cell constant of the probe was 0.0126 m. Response Properties As shown in Fig. 6, the sensor filled with 5 X 10-4 mol 1-1 NaN02 + 1 % glycerin solution exhibits a favourable response to 5 X 10-7-1 X 10-3 moll-' nitrite, with a detection limit of 1 X mol I-'. For six calibration samples in the range 1 X 10-5-1 X 10-4 moll-' nitrite, the regression equation is given AF = 190 + 1.358 X lO7c (4) with a correlation coefficient of 0.961; c is the nitrite concentration of the sample.The curve tends to decline downwards at higher concentrations. The recovery of nitrite added to samples containing 2 X 10-5 mol 1-1 nitrite was 97.2 k 2.5% (n = 5). The response time (to 95% of the steady state) of the sensor is about 5 min for a nitrite concentration change from 1 x 10-6 to 1 x 10-5mol1-l. In order to test the short-term reproducibility of the sensor, the frequency response was measured when the sensor was exposed to repeated concentration step changes between 1 X 10-5 and 5 X 10-5 moll-' nitrite. For five concentration step changes, the standard deviations of the frequency were 3.5 and 7.6 Hz for 1 X 10-5 and 5 X 10-5 mol 1-1 nitrite, which correspond to a standard deviation of nitrite concentration of 2.6 and 1.1 %, respectively.The sensor was used for 4 weeks without replacing the internal electrolyte solution. There is no significant loss of sensitivity if the probe is kept in a dilute nitrite solution when not in use. by Interferences As the samples were prepared in a strongly acidic solution, the interference from ammonium and volatile amines can be ignored. Several potential interferents were tested and the results obtained are presented in Table 1. When the determined nitrite concentration was above 10-5 mol 1-1, no significant interference was caused by dissolved carbon dioxide. Sample Analysis The proposed sensor was used to determine nitrite in smoked pork samples. For comparison, the AOAC method9 was also used to analyse the same samples. The nitrite concentrations were directly evaluated with a calibration graph and compared with those determined by the AOAC spectrophotometric method.The results obtained are given in Table 2. It can be seen that the results obtained with the proposed sensor agree well with those obtained by the AOAC spectrophotometric method. Comparison With a Potentiometric Nitrogen Oxide Sensor As summarized in Table 3, the proposed sensor has several advantages over the conventional nitrogen oxide sensor in that it gives a rapid and sensitive response. Compared with the potentiometric sensor, the proposed sensor does not require either a reference electrode or a fragile glass pH electrode; hence, it is simple in design and amenable to miniaturization.In addition, the proposed sensor offers cost advantages over the potentiometric sensor. Compared with the coated BAW gas sensor, the proposed sensor can overcome the influence of water vapour and can be applied to aqueous samples. The advantages of the proposed sensor should make it an attractive alternative to the sensors currently in use. Financial support from the National Science Foundation and Education Commission Foundation of China is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 Guilbault, G. G., Ion-Sel. Electrode Rev., 1980, 2, 3. Edmonds, T. E., Hepher, M. J., and West, T. S., Anal. Chim. Acta, 1988, 207, 67. Thompson, M., Kipling, A. L., Duncan-Hewitt, W. C., Rajakovic, L. V., and Cavic-Vlasak, B. A., Analyst, 1991, 116, 881, and references cited therein. Yao, S. Z., and Mo, Z. H., Anal. Chim. Acta, 1987, 193, 97. Shen, D. Z., Zu, W. H., Nie, L. H., and Yao, S. Z., Anal. Chim. Acta, 1993, 276, 87. Nomura, T., Takada, K., and Mitsui, T., Bunseki Kagaku, 1992, 41, 309. Fox, J. B., Jr., and Nicholas, R. A., J . Agric. Food Chem., 1974, 22, 302. Sherken, S., J. Assoc. Off. Anal. Chem., 1976, 59, 971. Official Methods of Analysis of the Association of Official Analytical Chemists, ed. Williams, S., Association of Official Analytical Chemists, Arlington, VA, 14th edn., 1984, sects. 24.044-24.045. Reilley, C. N., in New Instrumental Methods in Electrochemistry, ed. Delahay, P., Interscience, New York, 1954, p. 319. Riley, M., in Ion-Selective Electrode Methodology, ed. Covington, A. K., CRC Press, Boca Raton, FL, 1979, vol. 11, ch. 1. Paper 5106408B Received September 28, 1995 Accepted February 8, I996

ISSN:0003-2654    

DOI:10.1039/AN9962100883

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analyst, June 1996, Vol. 121 887 Abraham, Michael H., 5 11 AbramoviC, Biljana F., 401, 425 AbramoviC, Borislav K., 401,425 Abroskin, Andrei G., 419 Acedo Valenzuela, M. I., 547 Adam, S., 527 Adeloju, S. B., 699 Aheme, G. Wynne, 329 Akhtar, M. Humayoun, 803 Al-Othman, Rashed, 601 Alazard, S., 527 Aleixo, Luiz M., 559 Analytical Methods Committee, Andrade, Francisco J., 613 Angeletti, R., 229 AntonijeviC, M. M., 255 Appleton, Mark, 743 Aratake, Sachiko, 325 Araujo, Pedro W., 581 Arias, Juan JosC, 169 Artjushenko, Slava, 789 Bacci, Mauro, 553 Balasubramanian, N., 647 Bannon, Thomas, 715 Bartlett, Philip N., 715 Barnabas, Ian J., 465 Barroso, C. G., 297 Barwick, Vicki J., 691 Baxter, Douglas C., 19 Baya, Maria P., 303 Benedetti, A. V., 541 Benmakroha, Yazid, 521 Biancotto, G., 229 Bilitewski, Ursula, 119, 863, 877 Bjorklund, Erland, 19 Blais, Jean-Simon, 483 Blanco, Marcelo, 395 Bogan, Declan R., 243 Bond, Alan M., 357 Boswell, Stephen M., 505 Bouhsain, Zouhair, 635 Boukortt, Sheriffa, 663 Boutelle, Martyn G., 761 Boyd, Damien, 1R Brereton, Richard G., 441, 575, 58 1, 585, 65 1 Brinkman, Udo A.Th, 61 Bni, E.R., 297 Buchet, Jean-Pierre, 663 Burgot, Jean-Louis, 43 Bye, Ragnar, 201 Callej6n Mochbn, M., 681 Cammann, K., 527 Cao, Zhong, 259 Carbonnelle, Philippe, 663 Cardoso, A. A., 541 Cardwell, Terence J., 357 Carmona, Pedro, 105 Casajus, Rocio, 8 13 Casella, Innocenzo G., 249 Cassidy, Richard M., 839 CaviC-Vlasak, Biljana A., 53R Cela, R., 297 Cepas, Juana, 49 Ceramelli, Giuseppe, 2 19 Cerdi, A., 13 Cerdi, V., 13 Chan, Wing Hong, 531 Chatergoon, Lutchminarine, 373 Chen, Guo Nan, 37 Chou, Shu-Fen, 7 1 Christian, Gary D., 601 Christie, Ian, 521 Cirovic, Dragan A., 575, 581 Coello, Jordi, 395 Cole, S.Keith, 495 Collier, Wendy, 877 Copeland, D. D., 173 573 CUMULATIVE AUTHOR INDEX JANUARY-JUNE 1996 Corbella Tena, R., 459 Corti, Piero, 219 Cosano, J., 83 Craston, Derek H., 177 Crosby, Neil T., 691 Croteau, Louise G., 803 Crump, Paul W., 871 Crumrine, David S., 567 Cullen, Michael, 75 Cullen, William R., 223 Daenens, Paul, 857 de Jong, Dirk, 61 de Jong, Gerhardus J., 61 de la Guardia, Miguel, 635 de Lacy Costello, Benjamin P. J., de Oliveira Neto, Graciliano, 559 Dean, John R., 465 Demidova, M. G., 489 Demir, Cevdet, 651 Deng, Zhiping, 671 Desai, Mohamed, 521 Desimoni, Elio, 249 Destradis, Angelo, 249 Devi, Surekha, 807 Dilleen, John W., 755 Dodd, Matthew, 223 Dolmanova, Inga F., 431 Dreassi, Elena, 219 Dumasia, Minoo C., 65 1 Dumschat, C., 527 Dunemann, Lothar, 845 Economou, Anastasios, 97 Eigendorf, Guenter K., 223 Eikenberg, Oliver, 119 El-shahat, Mohamed F., 89 El-Shorbagi, Abdel-Nasser, 183 Elbergali, Abdallah K., 585 Ellwood, Jo A., 575 Emara, Samy, 183 Emteborg, Hkan, 19 Endo, Masatoshi, 391 Escobar, Rosario, 105 Evans, Phillip, 793 Facer, M., 173 Fallon, Michael G., 127 Fawaz Katmeh, M., 329 Fearn, Tom, 275 Fell, Gordon S., 189 Femandes, Julio Cesar B., 559 Ferreira, Valdir S., 263 Fielden, Peter R., 97 Fillenz, Marianne, 76 1 Fitzgerald, Catherine A., 7 15 Fleet, Ian A., 55 Forster, Robert J., 733 Forteza, R., 13 Francis, John M., 177 Frech, Wolfgang, 19 Fugivara, C.S., 541 Fukasawa, Tsutomu, 89 Fung, Yingsing, 369 Gail, Ferenc F., 401, 425 Galeano Diaz, T., 547 Gamble, Donald S., 289 Gao, Xiao Xia, 687 Garrigues, Salvador, 635 Genrich, Meike, 877 Giannousios, A., 413 Giersch, Thomas, 863 Glennon, Jeremy D., 127 Godinho, Oswaldo E. S . , 559 Goosens, Elise C., 61 Gordon, Derek B., 55 Greer, James C., 715 Grol, Michael, I19 Groves, John A., 441 Guiberteau Cabanillas, A., 547 Guiraum PCrez, A., 681 793 Gurden, Stephen. P., 441 Hadjiivanov, K., 607 Hammerich, Ole, 345 Hansen, Elo H., 31 Harrison, Iain, 189 Hartnett, M., 749 Hauser, Peter C., 339 Hayashi, Yuzuru, 591 Hayashibe, Yutaka, 7 Hendrix, James L., 799 Hemhdez, Oscar, 169 Hu, Yan, 883 Hulanicki, Adam, 133 Hyland, Mark, 705 Ibrahim, Naaim M. A., 239 Inagawa, Jun, 623 Irwin, G. W., 749 Ishida, Yasuyuki, 853 Ishihara, Masahito, 39 1 Isomura, Shinichi, 853 Itumaga, Hortensia, 395 Ivanova, Elena K., 419 Iwatsuki, Masaaki, 89 Jackson, Laurence S., 67 Jaselskis, Bruno, 567 Jiang, Chongqiu, 317 JimCnez, Ana Isabel, 169 JimCnez, Francisco, 169 JimCnez-Prieto, Rafael, 563 Jimknez Sgnchez, J. C., 681 Kalish, N.K., 489 Karayannis, Miltiades I., 435 Karlsson, Lars, 19 Kettling, Ulrich, 863 Kimbrough, David Eugene, 309 Kimoto, Takashi, 853 Kindness, Andrew, 205 Knoll, M., 527 Korda, T. M., 489 Kozik, Andrzej, 333 Kratochvil, Byron, 163 Kuznetsova, Vera V., 419 Kwong, Daniel W. J., 531 Lan, Zhang-Hua, 21 1 Lancashire, Susan, 75 Lancia, Antonio, 789 Lawrence, Chris M., 755 Lee, Albert W. M., 531 Legouin, Beatrice, 43 Lewenstam, Andrzej, 133 Li, Hao, 223 Lightbody, G., 749 Lin, Hui-Gai, 259 Lipkovska, N.A., 501 Lison, Dominique, 663 Littlejohn, David, 189 Lonardi, S., 219 Lopez Carreto, Maria, 33R Lopez-Cueto, Guillermo, 407 Lord, Gwyn A., 55 Loukas, Yannis L., 279 Lowry, John P., 761 Lowthian, Philip, 743 Lowy, Daniel A., 363 Lu, Bin, 29R Lu, Changyin, 883 Lu, Zheng, 163 Lund, Walter, 201 Luo, Yongyi, 601 Luque de Castro, M. D., 83 Lyons, Michael E. G., 715 McAdams, Eric T., 705 McAlemon, Patricia, 743 McAteer, Karl, 773 MacCraith, Brian D., 785, 789 McDonagh, Colette M., 785 McEvoy, Aisling K., 785 MacLachlan, John, 11R McLaughlin, James A., 705 McNaughtan, Arthur, 1 IR Madsen, Gary L., 567 Maines, Andrew, 435 Maj-Zurawska, Magdalena, 133 Mannaert, Erik, 857 Marr, Iain L., 205 Marshall, William D., 289, 483, Martin, Patricia, 495 Martinez-Lozano, Carmen, 477, Maspoch, Santiago, 395, 407 Masujima, Tsutomu, 183 Mathiasson, Lennart, 19 Matsuda, Rieko, 591 Meaney, Mary, 789 Melios, Cristo B., 263 Mieczkowski, Jbzef, 133 Mihajlovic, R., 255 MilaEiE, Radmila, 627 Mills, Andrew, 535 MilosavljeviC, Emil B., 799 MitroviC, Bojan, 627 Mizgunova, Ulyana M., 431 Mo, Songying, 369 Moane, Siobhan, 779 Mocak, Jan, 357 Mohamed, Ashraf A., 89 Molina, Marina, 105 Monaf, Lela, 535 Monaghan, John J., 55 Montelongo, F.Garcia, 459 Moollan, Roland W., 233 Moore, Andrew, 67 Mosello, R., 83 Mottola, Horacio A., 211, 381 Mulcahy, David, 127 Muller, Beat, 339 Munro, C. H., 835 Murphy, William S., 127 Nakamura, Masatoshi, 469 Nakamura, Motoshi, 469 Nakanishi, Masami, 853 Newton, R., 173 Nie, Lihua, 883 Nielsen, Steffen, 3 1 Nolte, Joachim, 845 Obendorf, Dagmar, 351 ObradoviC, Danilo M., 401 Odman, Fredrik, 19 Ohtani, Hajime, 853 O’Keeffe, Michael, lR, 779 O’Kennedy, Richard, 243,767, Olmi, Filippo, 553 Oms, M.T., 13 O’Neill, Robert D., 761, 773 Oniciu, Liviu, 363 Orlando, Andrea, 553 Osipova, Nataliya V., 419 Ostaszewska, Joanna, 133 Owen, Susan P., 465 Packham, Andrew J., 97 Papadopoulos, C., 413 Paradowski, Dariusz, 133 Pardue, Harry L., 385 Parsons, Patrick J., 195 Paulls, David A., 831 Pedrero, Maria, 345 Perez-Bendito, Dolores, 49, 563, Perez-Bustamante, J. A., 297 PCrez-Ruiz, Tomis, 477, 813 Pergantis, Spiros A., 223 Perruccio, Piero Luigi, 219 Pihlar, Boris, 627 Pingarron, Jose, 345 Piperaki, Efrosini A., 11 1 Piro, R.D. M., 229 817 813 29R 33R888 Analyst, June 1996, Vol. 121 Pitre, K. S., 79 Poe, Russell B., 591 Poole, Colin F., 51 1 Power, J. F., 451 Prodromidis, Mamas I., 435 Proinova, I., 607 Proskumin, Mikhail A., 419 Qu, Yi Bin, 139 Quevauviller, Ph., 83 Quinn, John G., 767 Rae, Bruce, 233 Rader, W. Scott, 799 Raghunath, A. V., 825 Rahmani, Ali, 585 Ramanaiah, G. V., 825 Ratcliffe, Norman M., 793 Razee, Saeid, 183 Redbn, Miguel, 395 Regan, Fiona, 789 Reimer, Kenneth J., 223 Reinartz, Heiko W., 767 Rigby, Geraldine P., 871 Rios, Angel, 1 Rodriguez Delgado, M. A., 459 Rodriguez-Medina, JosC F., 407 Rohm, Ingrid, 877 Rubio, Soledad, 33R Ruzicka, Jaromir, 601 Sakslund, Henning, 345 Saleh, Gamal A., 641 Salinas, F., 547 San Martin FemAndez-Marcote, SAnchez, Ma.J., 459 Santos, Jose H., 357 Sanz, Antonio, 477 M., 681 Sato, Hidetoshi, 325 Satyanarayana, K., 825 Sayama, Yasumasa, 7 Schafer, E. A., 243 Schmid, Rolf D., 863 Schoppenthau, Jorg, 845 Scobbie, Emma, 575 Seibert, Donna S., 5 11 Sekino, Tatsuki, 853 Shah, Rupal, 807 Shanthi, K., 647 Shijo, Yoshio, 325 Shukla, Jyotsna, 79 Shulman, R. S., 489 Silva, Manuel, 49, 563 Siskos, Panayotis A., 303 Slater, Jonathan M., 743, 755 Slavin, Walter, 195 Sloth, Jens J., 31 Smith, Clayton, 373 Smith, Dennis C., 53R Smith, Robert F., 67 Smith, Roy, 321 Smith, W. E., 835 Smyth, Malcolm R., 779, lR, 29R Smythe-Wright, Denise, 505 Sokalski, Tomasz, 133 SolujiC, Ljiljana, 799 Stathakis, Costas, 839 Stegman, Karel H., 61 Stevenson, Derek, 329 Stone, David C . , 671 Stradiotto, Nelson R., 263 Stuart, Iain A., 11R Stubauer, Gottfried, 35 1 Subramaniam, K., 825 Suffet, I.H. ‘Mel’, 309 Sukhan, V. V., 501 Suliman, Fakhr Eldin O., 617 Sultan, Salah M., 617 Sumodjo, P. T. A., 541 Sweedler, Jonathan V., 45R Szklar, Roman S., 321 Tam, Wing Leong, 53 1 Tan, Yanxi, 483 Tang, Bo, 317 Tang, Shida, 195 Tegtmeier, M., 243 TepavEeviC, Sanja D., 425 Thomaidis, Nikolaos S., 11 1 Thompson, Michael, 275, 285, Thomes, R. D. , 243 Timperman, Aaron T., 45R Tomas, Virginia Tomas, Virginia, 477, 813 Torgov, V. G., 489 Townshend, Alan, 831 Troccoli, Osvaldo E., 613 Tsuge, Shin, 853 Tudino, Mabel B., 613 Tzouwara-Karayanni, Stella M., Ubide, Carlos, 407 Uehara, Nobuo, 325 Vadgama, Pankaj, 435,521, 871 Vaggelli, Gloria, 553 Valcarcel, Miguel, 1, 83 Vassileva, E., 607 Verbeek, Alistair, 233 Villegas, Nuria, 395 Vos, Johannes G., 789 671, 53R 435 VukanoviC, B., 255 Walker, P.J., 173 Wallace, G. G., 699 Walsh, James E., 789 Walsh, Peter T., 575 Wang, Bin-Feng, 259 Wang, Chen, 317 Wang, Jin, 289, 817 Wang, Joseph, 345 Wang, Ke-Min, 259, 531 Wang, Shi-Hua, 259 Watanabe, Kazuo, 623 Wheals, Brian B., 239 White, P. C., 835 Whiting, Robin, 373 Wickstram, Torild, 201 Wilmot, John C., 799 Wittmann, Christine, 863 Woolfson, A. David, 71 1 Wu, Weh S., 321 Xin, Wen Kuan, 687 Xu, Xue Qin, 37 Xu, Yuanjin, 883 Yamada, Shinkichi, 469 Yao, Shouzhuo, 883 Yu, Ru-Qin, 259 Ziinker, Kurt, 767 Zanoni, Maria Valnice B., 263 Zaporozhets, 0. A., 501 Zhang, Fan, 37 Zhang, Xiaogang, 3 17 Zhi, Zheng-liang, 1 Zhou, Dao-Min, 705 Ziegler, Torsten, 119 Zolotova, Galina A., 431 NOMINATIONS FOR THE 1997 BENEDETTI-PICHLER AWARD The American Microchemical Society is soliciting nominations for the prestigious 1 997 Benedetti-Pichler Award. The award, established in 1966, is given annually to recognise outstanding achievements in microanalytical chemistry. The award consists of a plaque and expenses to attend the Eastern Analytical Symposium in Somerset, New Jersey, in November 1997 to receive the award at a session to honour the awardee. Nominations or further information, including at least two supporting letters should be sent no later than October 30, 1996 to: Dr Robert G. Michel Department of Chemistry University of Connecticut Storrs, CT 06269 USA Tel : +1 203 486 3 143; Fax : +1 203 486 2981; E-mail : MICHEL@UCONNVM.UCONN.EDU

ISSN:0003-2654    

DOI:10.1039/AN9962100887

出版商:RSC    

年代:1996

数据来源: RSC