1998
Anal. Chem. 1985, 57, 1998-2002
+
a weak acid such as carbonic acid (COz HzO)would not yield dramatic pH changes. This was observed in the case of the ISFETs prepared with valinomycin. Unfortunately, a t this point it is not clear why devices prepared with PVC membranes without valinomycin and KTPB or silicone rubber membranes without valinomycin typically displayed larger responses to COz,although one can speculate that the presence of valinomycin influences, in some way, the partitioning of membrane species into the gate region, and, therefore, the initial pH of this region. The above model was substantiated by additional experiments using ISFETs which were first treated by dip coating the gate region in an aqueous sodium bicarbonate solution. Once dried, these devices possessed a thin f i i of solid sodium bicarbonate on the gate and were then recoated with the PVC or silicone rubber membranes. Following initial conditioning in aqueous solutions, all of these ISFETs displayed large and rapid responses to COP We conclude that the bicarbonate layer rehydrates during the conditioning step and forms a more well-defined layer (with higher pH) between the gate and polymeric membranes. Recent literature and conference reports (4, 12, 13) regarding the performance of potassium ISFETs for continuous in vivo measurements have been contradictory. Some reports suggest that such devices are stable (12) while others clearly suggested significant drift problems (13). Obviously interference from organic acids can be ruled out at physiological pHs (Le., the acids are present as anions at pH 7.4 and do not partition into the PVC or silicon rubber membranes) (14). However, based on the observations reported here, variations in normal blood pCOz levels could pose a major stability problem resulting in erroneous potassium values. While numerous questions remain regarding the exact composition and structure of the membrane-gate interface and the extraneous potentials generated in this region as a result of COP and organic acid diffusion, there is little doubt that such diffusion can interfere with the performance of all ISFETs coated with polymeric membranes. Therefore, we urge researchers to more carefully examine this selectivity issue before suggesting that such ISFETs behave exactly as conventional ISE arrangements in terms of selectivity and other response properties.
Registry No. PVC, 9002-86-2; K, 7440-09-7; acetic acid, 6419-7; benzoic acid, 65-85-0; carbon dioxide, 124-38-9. LITERATURE CITED Arnold, M. A.; Meyerhoff, M. E. Anal. Chem. 1984, 55, 20R-48R. Treasure, T.; Band, D. M. J. Med. Eng. Technol. 1977, 1 , 271-273. Linton, R. A. F.; Lim, M.; Band, D. M. Crlt. Care Med. 1982, IO, 337-340. McKinley, B. A.; Houtchens, B. A.; Janata, J. Ion-Sel. Electrode Rev. 1984, 6 , 173-208. Janata, J.; Huber, R. J. I n "Ion-Selective Electrodes In Analytical Chemistry"; Frelser, H., Ed.; Plenum Press: New York, 1980; Chapter 3. Cheung, P. W., Neuman, M. R., Fleming, D. G., KO, W. H., Eds. "Theory, Deslgn, and Biomedical Applications of Solid State Chemical Sensors"; CRC Press: West Palm Beach, FL. 1978. Slbbald. A.; Covlngton, A. E.; Cooper, E. A. Clin. Chem. (Winston-Sa/em, N.C.) 1983, 2 9 , 405-406. Schepei, S. J.; de Rooij, N. F.; Koning, G.; Oeseburg, B.; Zijlstra, W. G. Med. 8/01. Eng. Comput. 1984, 2 2 , 6-11. Czaban, J. D.; Cormier, A. D.; Legg, K. D. Clin. Chem. (Wlnston-Sa/em, N.C.)1982, 28, 1936-1945. Severinghaus, J. W.; Bradley, A. F. J . Appl. Physlol. 1958, 73, 515-520. Yasuda, H. I n "Polymer Handbook"; Brandrup, J., Immergut, E. H., Eds.; Interscience: New York. 1966; pp V13-V24. McKinley, B. A.; Staffle, J.; Jordon, W. S.; Janata, J.; Moss, S. D.; Westenskow, D. R. Med. Instrum. 1980, 14, 93-97. Jordon, W. S.; Busey, B. R.; Janata, J. I n "Proceedings of the Symposium on Biosensors"; Potvin, A. R., Neuman, M. R., Eds.; IEEE: New York, 1984 pp 59-62. Kobos, R. K.; Parks, S. J.; Meyerhoff, M. E. Anal. Chem. 1882, 5 4 , 1976-1 980.
Eric J. Fogt Darrel F. Untereker Marye S.Norenberg Energy Technology Division Medtronic, Inc. Brooklyn Center, Minnesota 55430 Mark E. Meyerhoff* Department of Chemistry University of Michigan Ann Arbor, Michigan 48109
RECEIVED for review March 8,1985. Accepted April 15,1985. M.E.M. gratefully acknowledges the National Institutes of Health for supporting part of this work (Grant No. GM
28882-04).
Pulse Immunoassay for Candida albicans Sir: Immunoassay is based on the specific binding reaction of antigen and antibody. Various antigens including proteins, peptides, drugs, and microorganisms are determined by immunoassay. Immunoreaction results in the formation of antigen-antibody adducts. The amount of agglutinated adducts is measured by visual inspection, turbidimetry, or weighing after centrifugation. When the amount is too small to be detected by these methods, radioisotopes, enzymes, and fluorescence dyes are used for amplification of these reactions (1,2). Usually, however, the agglutinating reaction proceeds very slowly. Sometimes it requires 1-2 days to complete the reaction. Therefore, the acceleration of the agglutination is required for rapid immunoassay. The promotion of the agglutinating reaction is expected by increase of contact frequency between antigen and antibody. Mechanical stirring may not be effective for a minute amount of reaction solution. Increase of temperature is also not effective for biologically active substances including antibodies. 0003-2700/85/0357-1998$01.50/0
On the other hand, it was formerly observed that conducting or nonconducting particles suspended in various fluids formed linear linkages under electric field (3-6). These particles are aluminum powder, carbon powder, potato starch, polystyrene particles, red blood cells, and yeast cells. These electric field effects were considered promising for the increase of contact frequency between antigen and antibody. In the case of antigen, with a size of micrometers, the agglutination is composed of two steps: (1)binding of antibody on a particle (antigen) and (2) binding of another particle with the antibody-bound particle. The electric pulse probably accelerates the second step and increases the total reaction rate. Similar effects were expected in the case of small antigens (e.g., protein, peptide) and antibodies immobilized on a large particle (e.g., latex bead). In the present study, a novel immunoassay using an electric pulse is proposed and applied to the immunoassay of Candida albicans as a typical case in which the antigen has a size of 0 1985 American Chemlcal Society
ANALYTICAL CHEMISTRY. VOL. 57, NO. 9, AUGUST 1985
1999
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reactor. several micrometers. C. albicam is a pathogenic yeast and the determination of ita eoncentration has clinical imprtxmce (7-9). Since yeast cells were known to form linear linkages under electric field, the immunoreaction of C. albicans and antibody was expected to be accelerated. EXPERIMENTAL SECTION Materials. Candida albicans was cultured in a medium ewtainingglueose(l%),peptone(l%),andyeaatertraet(O.S%) for 17 h a t 37 "C, pH 6.5. The cella were coUected and suspended in distilled water after washing twice with distilled water. Antiserum to C. albicans was obtained from Sank0 Pure Chemical Co. and dialyzed against distilled water for 10 h before use. Reactor of Immunoreaction. Figure 1shows the readorn used for the immunoreaction. The slit reactor was composed of a couple of lead foil electrodes (electrode distance, 1mm;electrode thickness, 25 pm) and a slide glass. The cuvette reactor was constructed with glass plates. A couple of lead foil electrodes (3 x 1em2) were attached on the inside surface of the cuvette. The distance of the electrodes was 1 mm. Observation of Cell Agglutination under Electric Field. A drop of cell suspension was put on the slit reactor (Figure 1, A), covered with a cover glass, and observed under a microscope (x200). Then electric pulses were applied with a pulse generator. Reversible agglutination and dispersion of microbial cells were observed under electric field and ita removal. Immunoreaetion under Electric Field. One hundred microliters of C. nlbicnns suspension and 40 pL of antibody were mixed in the cuvette reactor with a syringe. Then electric pulses were applied for a certain period. Immediately after the electric pulse was stopped, an aliquot of reaction solution was t r a n s f e d on a slide glass and photographed through a microscope. Five photographs were taken for each sample. In a similar manner, the immunoreaction was performed without electric pulse as a control. A reaction solution containing C. albicans and its antibody was placed in the cuvette reador and mixed with a syringe and allowed to stand for the 881118 period as described above. Then an aliquot of the reaction solution was observed through the microscope and photographed. Nonspecific Agglutination under Electric Field. In order to check the irreversible agglutination due to electric pulses, a cell suspension was exposed to electric pulses in the ahsence of antibody. After the same reaction period as described above, an aliquot was placed under the microscope and photographed in the same manner. Survey of Parameters for Electric Pulse Conditions. A drop of cell suspension was put on the slit reactor and photopaphed through a micromope. Then electric pulaes were applied to the reador. After a certain period, the agglutinations formed were photographed under the electric field. Effectsof pulse height and frequency on the agglutination rate were investigated as defined as below. Investigation 01 Nonspecific Agglutination Caused by
Flpxa 8. Unear aggkfthatbn d C . ablcans under elscflc Mid. p u b freqwncy 111 = 8 kHr. Pulse wldm 7 = 20 ps. pulse helgM H = 100 V.
Protein. lnstead of antibody, human serum albumin (HSA) was added to a cell suspension in order to check the effect of nonspecific protein agglutination. A reaction solution (140 pL) containing cells (6.0 X 10' ceUamL-') and HSA (2.9 rng of proteinml-') was injected in the cuvette reactur and exposed to electric pulses. After the electric pulse was stopped. an aliquot was taken out and photographed through a microscope. Definition of Agglutination Rate. The extent of agglutination observed under the electric pulse application or after the electric pulse was stopped was represented by the agglutination rate (AR)as defined by the following equation:
where N. is total number of n-cell agglutinations. In the prewnt study, the lower limit of n in the denominator was fixed at 5 for convenience. RESULTS AND DISCUSSION Reversible Agglutination Generated by Electric Pulse. C.albicans cells are homogeneously distributed as shown in Figure 2. When electric pulses were applied to the suspension, linear linkages were formed within several seconds as shown in F w 3. The extent of linear linkage formation depended on electric pulse conditions as described below. Linear agglutinations. however, dispersed immediately after the removal of the electric pulse. Alternation of patterns as shown in Figures 2 and 3 w a s repeatedly observed. Effects of Electric Pulse Conditions on the Formation of Linear Agglutination. Figure 4 shows the effects of electric pulse height on the formation of linear agglutination
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 9. AUGUST 1985
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100
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Flgure 6. Irreversible agglutination caused by immunoreaction under electric field: (A) C. albicans 6 X lo’ cells.mL-’. antibody 2.3 mg of protein.mL-‘; (B) C. albicans 6 X lo’ cells.mL-’, antibcdy 2.9 mg of protein.mL-’.
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a t 8 kHz. No agglutination was observed below 20 V. Above 20 V, the extent of agglutination increased with increasing . . pulse height. Furthermore effect of the freauencv is shown in Fieure 5. TIEformation of linear agglutination iderea~edwith in& frequency. From thee results, the pulse height and frequency were adjusted a t 100 V (1kV cm-’) and 8 kHz, hereafter. I m e r s i h l e Agglutination C a d by Immunoreaction under Electric Field. When the cell suspension was exposed to electric pulses in the presence of antibody, some agglutinations remained even after the removal of electric pulse as shown in Figure 6. In Figure 6A, concentration of antibody was 2.3 mg of proteinml-’, while it was 2.9 mg of proteinml-’ in Figure 6B. Figure 7 was obtained by counting the number of agglutinations and the number of cells in each agglutination in Figure 6B. Adapting this result to eq 1,AR was estimated as 60%. Similarly, AR was 15% for Figure 6A. Time Course of Immunoreaction. Immunoreaction was performed with or without electric pulse. Without electric pulse, AR increased gradually and reached about 10% in 20 min as shown in Figure 8. In contrast, AFt increased sharply and reached 50% in 5 min with electric pulse. On the other band, without antibody, AR also increased under electric field,
0 r
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Agglutination histogram of Figure 66. The height of each bar represents the total number of cells belonging to the respective size of agglutionatbns; e.g., the bar at size 5 means there are three agglutinatlons each of whlch is composed of five cells. the bar at size 10 means there is only one agglutination composed of 10 cells. Definition of agglutination rate is described in the text. Figure 7.
though it was about 10% in 20 min. These results clearly demonstrate that electric pulse accelerated immunoreaction. From the time courses obtained above, the reaction time was adjusted to 5 min hereafter. Effect of Human Serum Albumin on Agglutination under Electric Field. Under electric field, a certain amount of protein is probably denatured due to heat generated. Since denatured protein often causes irreversible agglutination, the effect of HSA on the cell agglutination under electric field was investigated.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
2001
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