A New Approach to Electrochemical Immunoassays Using Conducting

Chapter 4

A New Approach to Electrochemical Immunoassays Using Conducting Electroactive Polymers 1

Downloaded by STANFORD UNIV GREEN LIBR on October 11, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch004

Omowunmi A. Sadik

Intelligent Polymer Research Laboratory, Department of Chemistry, University of Wollongong, New South Wales 2522, Australia The problem of generating a rapid, sensitive and reversible electrochemical s i g n a l w i t h antibody-antigen ( A b - A g ) interactions has previously been addressed. It was shown that the use of antibodies immobilized i n conducting electroactive polypyrrole matrices, with pulsed amperometric detection, and flow injection analysis, provides a unique solution to this problem. A sub-ppm detection limit for the target protein thaumatin was obtained, and a high selectivity towards other non-target proteins was realized. These encouraging results have resulted i n further scrutiny of the principle of the mechanism involved as reported i n this paper. Evidence from electrochemical quartz crystal microbalance studies ( E Q C M ) and cyclic voltammetry confirmed that a reversible mass increase was obtained in the presence of the antigen. The results showed that the application of alternating voltage waveforms induced changes i n the conducting polymers such that a detectable interaction w i t h a target analyte (antigen) was obtained in a reversible manner. Thus the detection method resulted i n a reusable i m m u n o l o g i c a l sensor that responded within a time scale of minutes. The use of antibodies i n electrochemical sensing technologies promises a degree of selectivity previously unattainable (1,2). In practice, however, some difficulties arise which affect the generation of a sensitive analytical signal and the reversibility of the antibodyantigen (Ab-Ag) interaction. Several attempts at overcoming these problems include: the use of potential measurements, indirect amperometric immunoassay, as w e l l as direct measurements of changes in capacitance at the sensor surface. Current address: National Exposure Research Laboratory, U.S. Environmental Protection Agency, P.O. Box 93478, Las Vegas, NV 89193-3478 0097-6156/96/0646-0037$15.00/0 © 1996 American Chemical Society

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


38

ENVIRONMENTAL IMMUNOCHEMICAL METHODS

In most of these cases, the procedures do not address the A b - A g reversibility issue. Conducting electroactive polymers (CEPs) represent a new class of electrode materials which are polymeric and yet conductive. These polymers can be switched from a highly conductive state to a resistive state by controlling the electrode potential. This electrochemical conversion involves mass and charge transport in the polymer film. The i m m o b i l i z a t i o n of specific molecules capable of substrate recognition is carried out d u r i n g polymerization (3). The resulting CEP-based biosensors have been proposed for direct and continuous detection of l o w concentrations of organic species i n process streams, environmental samples, and biological fluids (3,4). The use of C E P modified electrodes in the detection of simple inorganic ions, halogenated acetic acids, and other s m a l l organics has been demonstrated (5-7). Recently, it has been shown that the use of pulsed amperometric detection ( P A D ) provides a sensitive and selective analytical signal for complex biological molecules such as proteins (8 -11). The fundamental idea is that a protein molecule can selectively bind to a specific biological molecule incorporated into a conducting polymer membrane assembly. The b i n d i n g event leads to a change i n the surface nature of the polymer matrix upon the application of pulsed potentials. Thus, the transduction can be quantitatively measured. One unique advantage of this approach is that it is generic enough to be applicable to the interactions of enzymes, antibodies, receptors, or cells. A t the same time, it is sensitive enough to meet the analytical requirements for biosensors. The immunological biosensor research described here utilizes a n o v e l p u l s e d amperometric detection m e t h o d o l o g y for the generation of a useful analytical signal involving A b - A g interactions. The overall objective is the development of a simple antibody-based analytical tool which utilizes the intrinsic signal generation capability of the antibody to reversibly detect antigen (analyte) i n real time without the use of enzyme or optical labels. In the course of this work, the incorporation of antibodies into conducting polymers was probed. The electrochemical control of the A b - A g interaction i n effecting electrical signal generation was investigated. Furthermore, the issue of the effects of the applied pulsed potential producing changes in the structure of the polymer matrix, and the impact of the protein binding steps in giving rise to an analytical signal was also addressed. Experimental P o l y p y r r o l e (PP) electrodes were prepared by galvanostatic electropolymerization of pyrrole monomer from an aqueous solution


4. SADIK

39

New Approach to Electrochemical Immunoassays

containing antibody (Ab) as previously reported (8-10). The procedure can be represented by equation 1:


H

H

(1)

The characterization of the PP/Ab-containing film was carried out by means of electrochemical methods such as cyclic voltammetry, and quartz crystal microbalance ( E Q C M ) . The P P / A b films were used as detectors for the antigen by applying a unique pulsed waveform and measuring the resulting current signals.

Results & Discussion The new sensing system requires that antibodies be incorporated into a conducting polymer matrix, and bioactivity be maintained. The polymer produced as such was conductive and electroactive as shown by the cyclic voltammograms recorded for the p o l y p y r r o l e / a n t i thaumatin ( P P / A T H A U ) electrode (Figure 1). The responses observed are due to oxidation/reduction of the polymer and there was no change i n the voltammogram obtained w h e n the polymer was exposed to the antigen. This sensing element was then used in a flow injection analysis (FIA) system (Figure 2). The analysis involved the movement of analyte in a stream of eluent through the detection cell. The residence time in the detection cell was short (less than 1 min); hence the signal generation was fast. In order to achieve a rapid signal generation, a pulsed potential waveform was employed (Figure 3). E i and E2 were chosen such that A b - A g interactions were encouraged at E i and then discouraged at E2. The frequency (pulse width ti and t2) was such that the A b - A g interaction d i d not reach a stage where it became irreversible. The electrical signal was obtained by repetitive sampling of the current at a specific current sampling time (at tj). Pulsing to a more positive potential produced a small response w h i c h d i d not increase i n magnitude, whereas the signal increased i n magnitude as the potential was pulsed to more negative values. A well defined (non-tailing) and reproducible signal response was obtained during FIA using the pulsed potential routine as shown in Figure 4. The response obtained was very rapid (in minutes), sensitive (about 3-4 orders of magnitude) and reproducible current responses (± 3% over 10 injections) were observed. The selectivity towards other test proteins was investigated by the injection of these proteins into the flow stream. A s shown in Figure 5, responses to these other proteins were obtained at the P P / A T H A U electrode.



40


Ε (V)

Figure 1. Cyclic voltammogram obtained using PP/ATHAU. Electrolyte was 0.05 M Phosphate buffer (pH 7.4). Scan Rate = 100 mV/sec. Polymerization solution contained 0.5 M Pyrrole and 100 mg/1 Anti-thaumatin solutions made up in distilled water, current density = 0.5 mA/cm , growth time = 5 minutes. 2

Recorder

Eluent Pump

Polymer Detector

Figure 2. Flow Injection Analysis System


4. SADIK


rH

Ο Downloaded by STANFORD UNIV GREEN LIBR on October 11, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch004

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m

p


41

42



2 .0

A

(a)

(b)

(c) 10

I

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Figure 4. Typical F I A signals recorded for the injection of 50 m g / L thaumatin solutions using P P / A T H A U electrode at different pulse potentials. Ei=0.00V, E = (a) 0.2 V , (b) 0.4 V , (c) 0.6 V , (d) 0.9 V .Other conditions as i n Figure 1. 2

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Figure 5. Response obtained by injecting various proteins. T H A U = Thaumatin, B S A = Bovine serum a l b u m i n , H S A = H u m a n serum albumin, C H Y M = Chymotrypsin. Other conditions as i n Figure 1.



4. SADIK

43


H o w e v e r , the signal was m u c h lower i n magnitude than that recorded for the same concentration of thaumatin. It may be possible that these responses arose from non-specific interactions on the polymer backbone. The reversibility of the A b - A g signal was also considered. This was confirmed with the use of E Q C M . The P P / A T H A U electrode was coated on quartz crystal by galvanostatic electrodeposition. Changes in the mass of the quartz crystal following the oxidation/reduction of the attached electroactive polymer films can be g i v e n by the Sauerbrey equation (12) which relates changes i n resonant frequency of the quartz crystal to mass changes in the film:

Af = ^ H L . ^ l

(2) 3

where ρ is the density of quartz (2.68 g / c m ) , μ Ϊ 5 the shear modulus of quartz (2.947 χ 1 0 dynes cm" , 1 dyne = 10" N), f is the resonance frequency of the unloaded quartz crystal (6 M H z ) , and A is the piezoelectrically active area of the quartz (cm ). The results indicated a mass increase on reduction, and a decrease on reoxidation (Figure 6). A slight increase in mass was recorded for the P P / A T H A U electrode coated on quartz crystal using only 0.05 M phosphate buffer (Figure 6a), but there was a notable increase i n the mass recorded upon the addition of 100 p p m thaumatin (Figure 6 b). The observed mass increase, which was reversible, may be due to the interaction of thaumatin w i t h the anti-thaumatin antibody as the polymer was reduced. Ω

Ω

11

2

5

Q

2

Conclusions A rapid, sensitive and reproducible detection method for antigens based on the use of polypyrrole-antibody with pulsed amperometric measurements was developed for use with F I A systems. A sensitive analytical signal was obtained by using a unique electrical signal generation process available with conducting polymers and a pulsed potential w a v e f o r m . The selectivity was enhanced by direct incorporation of antibody-based bio-recognition sites into the conducting polymer materials. The results obtained w i t h E Q C M experiments showed that the mass increase was reversible. Current studies are being focused on the determination of the mechanism of signal generation as well as the transfer of this biosensing technology onto microelectrodes. Immunosensor methods are therefore recommended for d i r e c t and c o n t i n u o u s m o n i t o r i n g of environmental samples.



44


0.3H

0.2H

1

0.1·

-0.8

-0.4

0

0.4

Ε (VOLT)

(b) Figure 6. Mass versus potential profile using P P / A T H A U electrode in (a) 0.05 M phosphate buffer only, (b) 100 m g / L thaumatin i n 0.05 M phosphate solution, scan rate = 20 m V / s . Other conditions as i n Figure 1.


4. SADIK


Acknowledgment


The author gratefully acknowledges the support of the Australian Government through the a w a r d E M S S f e l l o w s h i p . Invaluable discussions held with Gordon Wallace, Zhao H u i g i n , L i n Y u p i n g , Chee on Too and N o r m Barisci in the early stages of this work are also acknowledged.

Literature Cited 1. 2. 3. 4. 5.

North, J.R., Trend. Bio. Tech., 1985, 7, 180 Nagy, G . , Pungor, E., Bioelectrochem. Bioenerg., 1988, 20, 1 Bidan G . , Sensors & Actuators, B6 (1992) 45. Sadik Ο.Α.,Analytical Methods & Instrumentation (In Press). O m o w u n m i A . Sadik, Gordon G . Wallace, Electroanalysis, 5 (1993) 555. 6. Sadik O . A . , Talaie Α., Wallace G.G., J.Intel. Mat. Syst. and Struc., 4 (1) (1993) 123. 7. O m o w u n m i A . Sadik, Gordon G . Wallace,Electroanalysis, 6 (1994) 860. 8. Sadik Ο. Α., Wallace G . G . , Anal. Chim. Acta., 297 (1993) 209. 9. Sadik Ο. Α., Wallace G . G . , Proc. 207th ACS National Meeting, March 13 - 18, 1994, San Diego, CA., USA., Vol.70 (1994) 178. 10. Barnett D., Laing D.G., Skopec S., Sadik O.A., Wallace G.G., Anal. Lett., 27 (13), (1994) 2417. 11. Sadik O.A., John M.J., Wallace G.G., Barnett D., Clarke C., Laing D.G., Analyst, 119 (1994) 1997. 12. Sauerbrey G., Z. Phys., 155 (1959) 206.


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A New Approach to Electrochemical Immunoassays Using Conducting

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