Biosensor Design and Application - ACS Publications - American

Chapter 6

Immunoelectrodes for the Detection of Bacteria

Downloaded by UCSF LIB CKM RSCS MGMT on November 29, 2014 | http://pubs.acs.org Publication Date: October 23, 1992 | doi: 10.1021/bk-1992-0511.ch006

Judith Rishpon, Yigal Gezundhajt, Lior Soussan, Ilana Rosen-Margalit, and Eran Hadas Department of Biotechnology and Molecular Microbiology, Faculty of Life Science, Tel-Aviv University, Ramat-Aviv 69978, Israel

This work describes a rapid and sensitive method for the determination of bacteria. The method is based on an enzyme-tagged immuno-electrochemical assay. Antibodies are immobilized on disposable carbon felt disc electrodes and are used to capture antigens in test solutions. After a short incubation with a second antibody, which is labeled with the enzyme alkaline phosphatase, the activity of the enzyme electrode thus formed is measured. This enzyme reacts with the substrate p-aminophenyl phosphate and the product of this enzymatic reaction, p-aminophenol, is detected amperometrically. The use of rotating electrodes significantly shortens the incubation times and, together with the computerized electrochemical system, results in extremely high sensitivity. The results obtained with Staphylococcus aereus and Escherichia coli cells show that the system can detect as low as 10 cell/ml in less than 10 minutes. There is a great need for fast and reliable devices capable of detection and identification of various types of bacteria. Traditional microbiological methods are based on cultural techniques which are tedious and slow. A considerable effort has been invested in development of detection methods based on immunological interactions. These methods, which generally involve labeling of antibodies with radioactive, fluorescent or enzymatic label, have been shown to be quite effective, giving specific and quantitative detection of target antigens. During the last decade several attempts were made to combine the specificity of antibody- antigen interaction with the high sensitivity, wide dynamic range, and simplicity of electroanalytical methods. These attempts led to the development of electrochemical immunosensors, several of which detect the corresponding antigen at extremely high sensitivity (1-5). 0097-6156/92/0511-0059$06.00/0 © 1992 American Chemical Society

In Biosensor Design and Application; Mathewson, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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In electrochemical enzyme immunoassays, an antigen or an antibody is ordinarily tagged with an enzyme and the enzymatic reaction is monitored by a potentiometric or amperometric electrode (6-13). The amplification obtained by enzyme catalysis is particularly advantageous for the detection of very low concentrations (14-15). A semihomogenous amperometric immunsensor for protein A - bearing Staphylocococcus aureus in foods has been recently reported by Mirhabibollahi et al. (16). These authors report a quantitative assay in the range 10 to 10 cfu/ml. Recently, some reports have described the use of an electrode surface as both the immunological solid phase and as the electrochemical detector (17-19). In a previous publication we have described preliminary results obtained with an immunsensor based upon the enzymatic reaction of alkaline phosphatase with p-aminophenyl phosphate (20). In that system, glassy carbon served both as the solid phase in the immunorecognition reaction and as an amperometric electrode for oxidation of p-aminophenol formed by the enzymatic reaction. Glassy carbon electrodes were also used in a heterogeneous immunoassay using glucose oxidase as an enzyme label (1718). By using a rotating disc electrode, Huet et al. (21) have recently shown that mass transfer to the solid phase, consisiting of antibodies immobilized on the glassy carbon, is a key step in heterogeneous immunassay.


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In a preliminary report (Hadas, E., Sousan, L . , Rosen-Margalit, I., Farkash, A . and Rishpon J., J. Immun. Assay, paper submitted) we have shown that by varying the nature of the carbon surface and increasing the number of the antibody binding sites we could considerably extend the detection limit of the electrodes. This was made possible by the use of electrodes made of carbon felt and by the covalent binding of the antibodies to the carbon surface via a spacer. The importance of a spacer arm in ligand coupling has been documented by several reports (22). A n adequate spacer promotes the effective utilization of active sites with a minimum degree of blockage and allows for flexibility and mobility of the antibody molecule as it protrudes into the solvent In this work the specific sequence employed was to bind biotin via an aliphatic spacer (hexamethylene-diamine) to the electrode surface, and use the biotin end to form a biotin/avidin/biotin bridge to the antibody. The use of a biotin /avidin/biotin bridge is a well known strategy in immunochemistry (23-24), and lately the same bridge has been applied to bind an enzyme to an electrode surface (25). The present paper describes the application of that system to the detection of extrememly low concentrations of bacteria. Materials and Methods Materials. l-Cyclohexyl-3-(morpholino-ethyl)carbodiimide metho-ptoluenesulphonate (CCD), avidin, N-hydroxy-succinimide biotin (NHS-biotin) and Protein A from S. aureus (Cowan strain) were obtained from Sigma (USA). 1, 6 Diaminohexane, ( H M D ) was obtained from Fluka A G (Germany). P-aminophenyl phosphate (APP) was synthesized as described previously (26). Biotin rabbit anti-mouse IgG, alkaline phosphatase-conjugated rabbit antimouse IgG, and alkaline phosphatase - conjugated goat anti-rabbit IgG were obtained from Jackson Immunoresearch Laboratories (USA). Formalin-fixed Staphylococcus aureus (Staph. A) was obtained from Sigma. In Biosensor Design and Application; Mathewson, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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Escherichia coli HB101 (a non pathogenic strain of E. coli) was obtained from M . Greenberg of the Department of Biotechnology and Microbiology, Tel Aviv University. Carbon felt sheets ( R V G 1000) were obtained from Le Carbon Lorraine (France). Carbon felt discs (5 mm diameter) were cut from the carbon felt sheets.


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Preparation of rabbit a-E. coli antibodies. 1 ml of 1.5 xlO HB101 cells together with 1 ml complete Freund's adjuvant were injected into rabbits. Injections were repeated four times every 3 weeks. However, after the first injection, 1 ml of incomplete Freund's adjuvant was substituted for the complete adjuvant. Blood from the rabbit ear was then collected in a test tube, kept 1 hour at room temperature and stored at 4°C overnight. It was then centrifuged and the antibody level of the supernatant was determined using standard E L I S A techniques. The antibodies were precipitated by addition of ammonium sulphate at 4°C, followed by centrifugation at 10000 rpm. The precipitate was then dissolved in PBS and dialyzed in PBS for 48 hours at 4°C.

Preparation of mouse a E. coli antibodies. The method used was similar to that used with the rabbit a E . coli, but the volume injected was 0.5 ml and the concentration of the bacteria cell was 1.5xl0 per injection. The injections were repeated once a week and the blood was drawn from the mouse eye. 6

Biotinilization of the Antibodies. 0.1ml of NHS-biotin in D M S O was added to a solution of lmg/ml antibody (affinity purified rabbit antimouse IgG or anti HB101 prepared in rabbits) in 0.1M, p H 8, carbonate buffer. The mixture was stirred at room temperature for 4 hours, dialyzed against PBS at 4°C, centrifuged, and the supernatant collected. Immobilization of Antibodies. The carbon felt discs were immersed in a solution of 1M H M D and 500 mg/ml C C D in water, the p H was adjusted to 5 (with HC1), and the discs were incubated for 16 hours at ambient temperature with shaking followed by a thorough washing with PBS. The discs were then immersed in a solution containing 0.1M, p H 8, phosphate buffer 1 mg/ml N-hydroxy-succinimide-biotin (previously dissolved in dimethylformamide) and incubated for 16 hours at ambient temperature with shaking. The discs were then washed well with PBS, immersed in a solution containing avidin 50 μg/ml avidin in PBS and incubated for 10 minutes with shaking. Finally the discs were immersed in a solution contained PBS and the biotilinated antibody. In the case of Staph. Α., biotinilated rabbit antimouse Ig antibodies were used, while in the case of E. Coli, the biotinilated antibodies prepared were used. The immersed discs were incubated for 10 minutes with shaking and washed extensively with PBS. Immunoelectrochemical Assay. Prior to use the carbon felt discs were mounted on a housing made of a teflon cylinder containing a concentric platinum wire and a teflon cap with a stainless steel mesh (Figure 1). The carbon felt disc mounted in this assembly served as both heterogeneous phase



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for antigen (analyte) capture and as the working electrode in the amperometric measurement. The assay was performed with 5-10 electrodes simultaneously. The immunoelectrochemical assay consists of three steps: 1. Bacteria Capture: Each of the antibody coated electrodes was immersed in 1 ml of the antigen (Staph. A, protein A or E. coli at different concentrations) and rotated at about 1300 rpm for 5 minutes (unless otherwise specified), followed by gentle washing in PBS. 2. Conjugate binding: For the analysis of Staph. A and protein A , the electrodes were then introduced into a solution containing A P conjugated rabbit antibody (Sigma A P rabbit antimouse IgG, diluted 1:250 2%), Tween 20 and 1% B S A in PBS, and rotated for 5 minutes at 1000 rpm. For the E. coli analysis, the electrodes were introduced into a solution containing mouse or rabbit IgG anti HB101, together with 2% Tween 20 and 1% B S A in PBS. The electrodes were then rotated at 1300 rpm (unless otherwise specified) for 5 minutes, washed gently in water and introduced into a solution containing A P rabbit anti-mouse IgG or A P conjugated goat anti-rabbit and rotated for 5 minutes at 1300 rpm. 3. Electrochemical determination of enzyme activity: After being gently washed in water the electrodes were transferred into the electrochemical cell containing 5 ml of substrate solution (3.7 mg/ml) of P A P P in 50 m M carbonate buffer (pH 9.6) and rotated at about 500 rpm. The P A R 273 potentiostat was used for the electrochemical measurements. The antibody electrode was employed as the working electrode and a standard calomel electrode and a platinum mesh were used as the reference and counter electrodes, respectively, in an amperometric measurement. The working electrode potential was held at 0.22V vs. S C E . A computerized electrochemical system described earlier (27) was employed for current reading and signal averaging. The system was capable of simultaneous readings of several working electrodes in the same substrate solution, using a single reference electrode, a single counter electrode and a multiplexer (28). The electrochemical cell was thermostated at 37°C unless otherwise specified. Measurements were automatically repeated until readings were stabilized, which usually occurred within 1 minute. A complete scheme of the immunelectrodes is presented in Figure 2. Results Detection of protein A bearing bacteria: Figure 3 presents results obtained with the immunoelectrodes for the determination of the bacteria Staph. A. A s a comparison, the same immunoenzymatic system was employed in a regular E L I S A test. The results of the latter assay are shown in Figure 4. It is evident


RISHPON ET AL.



Pt Wire

Stainless Steel Net

Felt Disc

Figure 1. Schematic layout of the carbon felt electrode configuration.

Figure 2. Scheme showing the immunoelectrode structure.


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