An amperometric enzyme-channeling immunosensor

E L S E V I E R A D V A N C E D TECHNOL O~i'~

Biosensors & Bioelectronics Vol. 12, No. 3, pp. 195-204, 1997 © 1997 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0956-5663/97/$17.00

An amperometric enzyme-channeling immunosensor

J. Rishpon* & D. Ivnitski

Department of Molecular Microbiology & Biotechnology, Tel-Aviv University, Ramat-Aviv 69978, Israel Fax: [972]-3-640-9407 Email: [email protected]

(Received 30 April 1996; accepted 30 September 1996)

Abstract: This paper presents a new disposable amperometric, enzyme-channeling immunosensor for a quantitative, rapid, separation-free enzyme immunoassay (EIA) that can be used in clinical diagnostics, as well as in biomedical, biochemical, and environmental research. The sensor consists of a disposable, polymer-modified, carbon electrode on which enzyme 1 is coimmobilized with a specific antibody that binds the corresponding antigen in a test solution. The solution also contains a conjugate of enzyme 2. An immunological reaction brings the two enzymes into close proximity at the electrode surface, and the signal is amplified through enzyme channeling. The localization of both enzymes on the electrode surface limits the enzymatic reactions to the polymer/membrane/electrode interface. The sensor overcomes the problem of discriminating between the signal that is produced by the immuno-bound enzyme label on the electrode surface and the background level of signal that emerges from the bulk solution. Combining enzyme-channeling reactions, optimizing hydrodynamic conditions, and electrochemically regenerating mediators within the membrane layer of the antibody electrode significantly increased the signal- to-noise ratio of the sensor. The amperometric enzyme-channeling immunosensor enabled the performance of separation-free EIAs without washing steps, resulting in a relatively short assay time of 5-30 rain for the complete immunoassay, compared with at least 1-3 h for ELISA methods. Model systems using peroxidase-antibody, biotin-avidin, viral antigens (CD4-gpl20), and bacteria (Staphylococcus aureus) were investigated. S. aureus cells were detected in pure culture at concentrations as low as 1000 cells/ml. © 1997 Elsevier Science Limited. All rights reserved.

Keywords: separation-free, rapid immunoassay, electrochemical, enzyme immunoassay, antibody electrode, enzyme channeling.

I N T R O D U C T I O N

Modifying quantitative immunoassays for rapid, 'on-the-spot ' performance in the field is important for clinical diagnostics, biochemical and biotechn-

*To whom correspondence should be addressed.

ological research, as well as for environmental monitoring. At present, quantitative immunoassays are usually performed only in major laboratories, which are equipped with complex instru- mentation and highly qualified technical staff. A device operating in such remote sites as a phys- ician's office, an emergency ward, or in field testing of environmental pollutants should not

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J. Rishpon & D. Ivnitski Biosensors & Bioelectronics

only provide fast, sensitive measurements but also be simple to operate and inexpensive. In this respect, an electrochemical device presents an attractive option. Consequently, devices that com- bine the high sensitivity and the relative sim- plicity of electroanalytical techniques with the high specificity of immunoenzymatic assays have shown a powerful analytical capability.

Various types of electrochemical immunosen- sors have been developed (Jenkins et al., 1991; Foulds et al., 1990; Mirhabibollahi et al., 1990; Ngo, 1987; Hadas et al., 1992; Rishpon et al., 1992). The limiting factor in the development of rapid and separation-free electrochemical immu- nosensors, however, is the background noise induced by non-specific reactions. Such noise is most often due to the excess of enzyme-antigen conjugate in solution, which makes it difficult to discriminate between a signal that is obtained from small amounts of immunobound enzyme label and high background levels of signal that emanate from the conjugate in the bulk solution. The most general approaches to overcome the non-specific signal of the conjugate in solution are to link the enzyme label catalytically to an additional system, such as a substrate cycle, or to other enzymes to form 'cascades' (DiGleria et al., 1989; Harris, 1984; Litman et al., 1980).

This viewpoint has led to the development of an enzyme-channeling immunoassay, where an immunological reaction brings two enzymes into immediate proximity at a surface. A novel, separation-free, sandwich-type enzyme immunoassay (EIA) using microporous gold electrodes and self- assembled monolayer/immobilized antibodies was described by Duan & Meyerhoff (1994). An example of a 'pseudo-homogeneous' electrochemical immunoassay without a washing step was described by McNeil et al. (1995). This assay was based on enzyme-channeling with direct electron transfer between an enzyme-conjugated antigen and the electrode.

In previous research, we developed a rapid, one-step, separation-free, amperometric enzyme immunosensor for electrochemically monitoring in situ reactions with rabbit IgG and with human luteinizing hormone in human serum (Ivnitski & Rishpon, 1996). The sensor allowed preferential measurement of surface-bound conjugate relative to the excess enzyme-labeled regent in the bulk sample solution. The amplification of the analytical signal was achieved by employing an anion- exchange PEI-GOD-antibody modified electrode

196

with iodide-ion mediators in solution and a peroxidase labeled conjugate.

When performing mulitple in situ immunoassays, however, one problem that may be encoun- tered is reusing the electrochemical cell after exposure to contaminated samples. A disposable electrochemical cell for use in such measurements would be invaluable. In this report we describe the construction of a disposable, electrochemical cell and polymer-modified, carbon electrode and its application in competition, displacement, and sandwich assays using diverse compounds and microorganisms.

EXPERIMENTAL

Materials

Glucose oxidase (GOD) (EC 1.1.3.4, from Asper- gillus niger, 150 IU/mg), horseradish peroxidase (HRP) (EC 1.11.1.7, RZ 3.1), goat anti-horseradish peroxidase, Staphylococcus aureus (formalin treated), bovine serum albumin (BSA), biotin, glutaraldehyde (50%) and polyethylenimine (50%) (PEI) were obtained from the Sigma Chemical Company (St Louis, MO). Recombi- nant, soluble CD4 and gpl20 proteins were obtained from American Bio-Technologies, Inc. (USA). GOD-conjugated egg-white avidin, Crom- Pure rabbit IgG, whole-molecule (RblgG), Affini- Pure goat anti-rabbit IgG (c~RblgG) and horseradish peroxidase-conjugated Affini-Pure goat anti- rabbit IgG (c~-RblgG-HRP) were obtained from the Jackson Immuno Research Laboratories, Inc. (West Grove, PA). Other chemicals of analytical grade were obtained from standard sources.

Preparation of the PEI membrane antibody electrode

Disposable graphite electrodes in cylindrical form (made from pencil lead, HB 0.9 mm) were used as the working electrodes. The electrodes were cleaned in methanol, rinsed with double-distilled water, dried, and then dipped for 15 min in a 0.2% methanol solution of PEI and air-dried for 8 h. The electrodes were washed with methanol to remove excess unbound polymer and then immersed for 4 h in a 2.5% glutaraldehyde solution (pH 7.0). GOD and antibody were immobilized covalently to PEI film on the graphite electrode surface. The electrodes were then immersed

Biosensors & Bioelectronics Amperometric enzyme-channeling immunosensor

in 0.1 M phosphate buffer (pH 7) containing GOD (0.5 mg/ml) and antibody (0.5 mg/ml) and incubated for 8 h at 4°C. Unreacted aldehyde groups were blocked by soaking the electrodes for 1 h in a solution of 0.1 M glycine in 0.1 M phosphate buffer (pH 7). The electrodes were washed and then stored in 0.1 M phosphate buffer (pH 7) at 4°C. The activity of HRP was monitored spectro- photometrically at 403 nm, using an extinction coefficient of 1 × 105 1 mol -t cm -~ (Chen et al.,

1984). Protein concentration was determined by UV absorbency at 280 nm using an extinction coefficient of 2.16 × 1051 mol -~ c m -1.

Scanning electron microscopy

Electron microscopy of S. aureus cells on the electrode surface was performed with a JEOL- 840a scanning electron microscope with an accel- eration voltage of 25 kV, a magnification of 10,000, and an objective aperture of 1/xm.

Electrochemical measurements

Figure 1 shows the schematic layout of the electrochemical cell. All electrochemical measurements were performed in the three-electrode cell (Vsolution=0.3ml) with a rotating cylindrical graphite antibody working electrode (0 .9mm diameter), a graphite ink counter, and Ag/AgC1 ink reference electrodes, using 0.1 M acetate buffer (pH 5.6) containing 0.1 M NaCl and 3 mM KI. Standard electrochemical equipment for amperometric measurements can be applied in the monitoring of the sensor response. We used an EG & G Par 273 potentiostat interfaced to a PC 486 computer system with PAR M270 software. The rotation of the graphite electrode was performed with a Pine Instruments rotator and with an MSRS speed controller. The kinetic measurements were performed at a fixed electrode poten- tial (0 .0V versus Ag/AgC1) without washing steps. Cyclic voltammetry was conducted at a 20 mV/s scan rate with the RbIgG electrode.

Separation-free, amperometric immunoassay protocol

A disposable antibody graphite electrode, with both immobilized GOD and antibody (or avidin) on the PEI film, was placed in a cell (V = 0.3 ml) containing 0.1 M acetate buffer (pH 5.6) with 0.1 M NaC1, 3 mM KI, and the sample of analyte

and conjugate. The electrode was then rotated at a controlled speed (100-500 r.p.m.) and preincu- bated for 5-10 min. After a fixed period of incubation, the background current was stabilized for 2 min at 0-0 mV versus Ag/AgCI at 20°C. Glu- cose (0.01 ml, 0.01 M) was added to the cell, and changes in the current were monitored continuously and recorded for 1 min. The slope of the binding curve (A//At), which was directly visualized on a computer screen, was proportional to the amount of analyte present in the sample. A blank experiment was performed with the working electrode that was modified only by GOD and BSA. The concentrations of glucose and mediator in the buffer were chosen so that the current change was close to zero in the absence of a bound conjugate on the electrode surface.

GP120 assay procedure

The graphite electrode, with both immobilized CD4 and GOD on the PEI film, was placed in a cell (V=0.3 ml) containing 0.1 M acetate buffer (pH 5-6), 0.1 M NaC1, and 3 mM KI. Aliquots (10/~1) of purified gpl20 at various concentrations in the interval 100-500 ng/ml or buffer alone (control) were injected into the cell and allowed to react with immobilized CD4 on the electrode surface at a controlled speed (500 r.p.m.) for 4 min at 0.0V versus Ag/AgC1. Conjugate (10/~1) (gpl20-HRP 10/xg protein/ml) was then added to the same cell, and after the background current reached stabilization (2 min), 10/xl of 100 mM glucose was added to the solution, and the catalytic current was monitored for 1 min.

Determination of S. aureus

The enzyme-channeling immunoassay of S. aureus is based on the principle of a sandwich assay. The working graphite electrode was coated by a PEI film with immobilized GOD and RblgG via a glutaraldehyde linker, and remaining sites were blocked with 0.1 M glycine as described above. Standard solutions of S. aureus (102-106 cells/ml) were prepared in PBS.

Two procedures were tested in the S. aureus

assay; four replicates of each procedure were performed.

(1) The RblgG electrode was placed in the

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J. Rishpon & D. lvnitski Biosensors & Bioelectronics

rotor

W

/ "

k

r--y ---C

polystyrene tube

test solution

5 m m

J

Fig. 1. Schematic layout of the electrochemical cell. W, working pencil lead electrode; C, counter electrode; R, reference electrode.

(2)

sample solution containing 0.1 M acetate buffer, 0.1 M NaCi, 3 mM KI, and 0.01% BSA. An aliquot (10/xl) of a S. aureus standard solution was added, and the RbIgG-GOD electrode was rotated at 1000r.p.m. for 10 rain at room tempera- ture. Then the rotation speed was changed to 100r.p.m., and an aliquot (10/xl) of RbIgG-HRP (4/xg/ml) was added to the cell. After 10 min of incubation, an aliquot (10/xl) of glucose (0.1 M) was added to the cell, and the response to glucose was recorded. A control experiment was performed using a BSA- and GOD-modified working electrode. The second procedure included separation and washing steps. The RbIgG-GOD electrode was incubated directly in the standard solutions. Immediately after the incubation with the bacteria, the electrode surface was thrice rinsed for 2 min per rinse. The next step was incubating the electrode with the

RblgG-peroxidase conjugate. The electrode was placed in the electrochemical cell containing 0-1 M acetate buffer, 0.1 M NaC1, 3 mM KI, and 130 ng/ml RblgG-HRP conjugate and incubated for another 10 min at 1000r.p.m. Five replicates of this procedure were performed. The background signal was measured and stabilized at 0.0 V versus Ag/AgC1 for 2 min at a controlled speed, typically 100r.p.m. At this point 10/xl of glucose (0.1 M) was added to the cell, and the catalytic current was monitored continuously for 1 min. The bacterial concentration in the stock suspension was counted with a hemocytometer (BS748, UK).

RESULTS AND DISCUSSION

Figure 2 shows the principle of the pseudo-homogeneous enzyme-channeling immunoassay with

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PEI

E L E C T R O D E

(pencil lead)

I

G O D - - - \

\

Glucose +02 ) - ~ H202 + gluconic acid

/ ~ I V ~ e _ AB ~ A g - H R P \

FILM '~ , I2+ 2 H 2 0 " ~ b "-xa--

AB H202 + 2I" +2H +

PEI F I L M

Fig. 2. Schematic illustration of the principle of the separation-free, amperometric enzyme-channeling immunoassay with immobilized GOD and antibody on the PEl-modified electrode surface.

amperometrical detection. The active surface of the immunoelectrode consists of an antibody and glucose oxidase, which are coimmobilized on a PEI membrane. The test solution contains the antigen (the analyte), the HRP conjugate, and iodide. An immunological reaction brings the HRP conjugate into close proximity with the GOD on the electrode. The glucose solution reacts with GOD on the electrode surface and the product of this enzymatic reaction, H202, reacts with iodide ions in the presence of a surface-bound, peroxidase label. The iodine thus formed is monitored at the electrode. The concentration of H202, which is high at the electrode surface, is diluted when leaving the electrode surface to the solution.

The PEI membrane plays a crucial role in

discriminating between the analytical signal (resulting from the immunocomplex at the electrode) and the background signal (resulting from the reaction of the HRP conjugate with H202 that leaches out from the electrode surface to the solution). Figure 3 shows the cyclic voltammograms of a bare electrode and a PEI-modified electrode. Clearly, the iodine/iodide electrode reaction is significantly enhanced in the PEI- modified electrode because the iodide accumulates in the cationic polymer. Consequently, accumu- lation in the PEI membrane further increases the discrimination between the reaction of iodide ions with the HRP in the solution and at the electrode. The electrochemical response also depends on the concentration of the PEI in the organic solution that is used for the membrane preparation, show-

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70 l I I I I I I i

50

30

-10

- 30

3

-50 2 2 5 3 2 5 4 2 5 5 2 5 6 2 5

<

0

-40

-80

- 1 2 0

- 1 6 0

I I I

gl

I I I I J

188 192 196 2 0 0 2 0 4 2 0 6

t (sec)

Fig. 4. The amperometric responses of BSA (curves 1 and 2) and antiHRP (curves 3 and 4) electrodes to the addition of peroxidase: (1, 3) 8.6 ng/ml; (2,4) 17.2 mg/ml. The electrodes are poised at 0.0 V versus SCE at 500 r.p.m. The other conditions are the same

as in Figure 2.

E vs. SCE (mV)

Fig. 3. Cyclic voltammograms obtained for 0.28mM iodide ions at the (1) unmodified and (2-5) PEl-modi- fled graphite electrodes in 0.1 M acetate buffer (pH 5.6) containing O.1M KC1. Electrode rotation speed, 500r.p.m; scan rate, 20mV/s. The electrodes were dipped for 15 min in methanol solution of" (2) 0.1%,

(3) 0.3%, (4) 0.5% PEI and (5) 2% PEI.

ing a maximum at 0.3% in methanol. Higher concentrations lead to diffusion limitations that impede the mass transport in the PEI membrane.

Tatsuma & Watanabe (1993) have shown that in immobilized enzyme systems, the total rate of enzymatic reactions on the solid surface is increased because of internal diffusional limitations. In our case, the reason for the increasing enzymatic rate reaction is the effect of accumu- lation, the cyclic regeneration of redox mediators (I2/I-), and the diffusion limitation within the PEI film on the electrode surface. All three factors greatly increase the specific analytical signal of the sensor.

Detect ion of surface bound peroxidase

The first and simplest model system used was HRP/antiHRP. Figure 4 shows typical dynamic amperometric responses of BSA- and antiHRP- modified electrodes at 0-0 mV versus Ag/AgCI after preincubating with HRP for 5 min and then adding 10/xl of 0.1 M glucose. The BSA-GOD electrode was used as control for non-specific

binding. The time required to obtain the analytical signal after addition of glucose is 10 s. As shown, the current changes were insignificant in control experiments with BSA-modified electrodes (curves 1 and 2). The large catalytic current occurring at the Ab-modified electrode (curves 3 and 4) indicates the preferential measurement of surface-bound peroxidase relative to the excess enzyme in the bulk solution. The optimum concentrations of the conjugate and glucose, at which maximum discrimination between signal and background was obtained, were 40-100 ng/ml for the conjugate and 2-4 mM for glucose.

Detect ion of biotin

Biotin-avidin in a competition assay was the second system to be checked. The calibration curve for biotin, shown in Figure 5, has a sigmoid form. The electrode was rotated at 100 r.p.m, for 10min in the biotin sample, which was long enough for efficient binding of biotin to immobilized avidin and is probably due to the strong binding constant of avidin and biotin (K a : 1015 M-l).

Experiments performed with concentrations of biotin higher than 200ng/ml gave no current changes upon adding glucose, indicating that non- specific binding of the conjugate was not operat- ive. The blank signal in the absence of the biotin- HRP conjugate is equivalent to the signal for the assay containing 225 ng/ml biotin.

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Biosensors & Bioelectronics

5

~ 3

A

I I I I

50 100 150 200

Biotin (ng/ml)

I

250

Fig. 5. Calibration curve for the biotin determination by means of separation-free competitive enzyme immunoassay using avidin-GOD electrodes. The Av-GOD electrodes are poised at -0.1 V versus Ag/AgCl in O. I M acetate buffer (pH 5.6) with O.15MNaCI, 3 mM KI, 2 mM glucose and 212 ng/ml Biotin-HRP,

100 r.p.m.

Detection of HIV antigen

The first step in HIV pathogenesis involves the specific interaction between the HIV glycoprotein gpl20 and the cell surface antigen CD4 (Capon & Ward, 1991). GP120, an HIV envelope glycoprotein, is recognized as an indicator for the detection of HIV infection (NIH AIDS, 1995). We applied this knowledge in our preliminary investigations concerning the possibility of directly monitoring the CD4-gpl20 interaction on the electrode surface. In our experiments, a PEI- coated graphite electrode with coimmobilized CD4 and GOD was first incubated with gpl20, and then with a fixed amount of gpl20-HRP. Figure 6 indicates the amperometric response of the CD4 electrodes in the absence (1) and in the presence (2,3) of two concentrations of gpl20 in the sample. The maximum current signal was observed in the absence of gpl20 in the sample. The current response decreases according to the concentration of gpl20. These preliminary results show that quantitative measurement of gpl20 using CD4-modified electrodes is possible.

Amperometric enzyme-channeling immunosensor

<

-4

-8

-12

-16

I I I I ! I I I

0 5 10 15 20 25 30 35

t (s)

Fig. 6. Typical response curves for the separation-free amperometric competition type assay: (1) O gpl20 in buffer; (2) lOOng/ml gpl20 in buffer," (3) 500ng/ml gpl20 in buffer [gpI20-HRP] = 330 ng/ml. Other con-

ditions are described in the Experimental section.

Detection of S. a u r e u s

An example of an enzyme-channeling, amperometric immunoassay in a sandwich format is shown for S. aureus in pure culture. As protein A is a major cell wall component of S. aureus, it can be detected by binding the cells to an electrode surface that has been modified by IgG (Huet et al., 1990). For the enzyme immunoassay, we used the graphite electrodes that were coated by PEI film with coimmobilized RbIgG and GOD. In the first procedure, the S. aureus cells were detected in 30 min using a separation-free sandwich assay. As the rate-limiting step in EIA is associated with the kinetics of immunobinding of the cells to the antibody, the diffusion-limited, mass transport of the cells to the antibody electrode surface was enhanced by preincubating for 10 min and then rotating the IgG electrodes at 1000 r.p.m. The RbIgG-HRP conjugate was added in a second step. The results in Figure 7 show that the current changes were related to the changes in cell concentration, ranging from 1.0x 103 to 1-0 x 104 cells/ml. (The least detectable dose was the same for the two formats.) The control experiment, without the capture antibody, confirms the absence of non-specific adsorption of S. aureus on the electrode. It is clear that saturation occurs at concentrations higher than 5000 cells/ml. Figure 8 shows the results obtained by using the second procedure with washing and separation, and we can see that under these conditions, the

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i i l control

-10

-20 10 3

-30 I- glucose

-40 -

10 4

-50 5.10 3

-60 I I I I I

20 60 100 140 180

t (s)

Fig. 7. Calibration curve for the S. aureus cells using RblgG electrodes in a separation-free format. Cell concentration [cells/ml] is shown in the figure. The control experiment was performed using a BSA electrode and

104 cells.

035

0.30

0.25

0.20

~', 0.15

0.10

0.05

0.00 i i i I i I I

0 1 2 3 4 5 6

Log IS. aureus] cell/ml

Fig. 8. Calibration curve for S. aureus cells with separation and washing steps

current changes depended on the changes in cell concentration, ranging from l 0 3 to l 0 6 cells/ml. In the low concentration range, the sensitivity (the least detectable dose) was the same for both formats. Non-specific binding of the RblgG-HRP

conjugate to the electrode surface in both formats was consistently low. As shown, the separation- free immunosensor is more effective at low concentrations, as the dynamic range obtained in the second format was much larger. It should be noted, however, that the antibodies used in the S. aureus assay were polyclonal, and thus, the RblgG-HRP in the solution can compete with the RblgG on the electrode. Presumably, the saturation effects that were observed in the separation-free assay are anticipated by the binding of all the RblgG-HRP to the bacteria in the solution. We believe that the use of monoclonal antibodies will increase the dynamic range of the measurement. Experiments with monoclonal antibodies are in progress. These measurements were done separately in series. Only a limited number of samples and electrodes could be tested in one set of experiments. The general effect of the separation-free immunoassay was obviously demon- strated. To determine accurately the precision of the system, however, a large number of measurements should be made simultaneously. The development of a system using a multiplexer, which enables simultaneous measurements of bacterial cells in real samples, is now in progress.

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The results presented here were confirmed by direct examination of the cell adhesion of S. aureus to the IgG-modified electrode surface, with the help of scanning electron microscopy (Figure 9 (A,B)). The bacterial cells can be seen on the electrode surface after incubation in the presence of the bacteria. We found that cell adhesion to the IgG-modified surface was signifi- cant, and it increased with the amount of cells in the incubation sample. At the same time, the degree of adhesion of these cells to unmodified electrode surface was low.

CONCLUSIONS

We have investigated the possibility of using a new disposable amperometric, enzyme-channeling immunosensor for rapid, quantitative EIA of dif- ferent compounds, including microorganisms, without a washing step of the conjugate. The

!

immunoassay is performed with an amperometric immunosensor, using a disposable cell and electrodes. The sensor enables preferential measurement of surface-bound conjugate relative to the excess, enzyme-labeled reagent in the bulk sample solution. The complete immunoassay is carried out in 10-30 min compared with at least 1- 3 h for ELISA methods. The data shown here demonstrate the applicability of the sensor for the quantitative detection of biotin, HIV antigen, and S. aureus. The immunosensor might also be use- ful for characterizing binding reactions via the estimations of the affinity constant and the dis- sociation-rate constant from the slopes of the associated current changes. Work now in progress is aimed at using monoclonal antibodies as well as disposable multi-electrodes to carry out simultaneous immunoassays in parallel and in small volumes. Our investigations will be directed at optimizing such parameters as sensitive and reproducible assays.

ACKNOWLEDGEMENT

The authors wish to thank the European Union- AVICENNE Program for financial support.

Fig. 9. Adsorption of S. aureus cells to a RblgG modified electrode surface observed by electron microscopy. (A) Electrode surface with no bacteria; (B) S. aureus cells adsorbed to the electrode surface. Magnification,

×10,000.

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