Hydroquinone Diphosphate as a Phosphatase Substrate in

Article pubs.acs.org/ac

Hydroquinone Diphosphate as a Phosphatase Substrate in Enzymatic Amplification Combined with Electrochemical−Chemical− Chemical Redox Cycling for the Detection of E. coli O157:H7 Md. Rajibul Akanda, Vellaiappillai Tamilavan, Seonhwa Park, Kyungmin Jo, Myung Ho Hyun, and Haesik Yang* Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea S Supporting Information *

ABSTRACT: Signal amplification by enzyme labels in enzyme-linked immunosorbent assays (ELISAs) is not sufficient for detecting a low number of bacterial pathogens. It is useful to employ approaches that involve multiple signal amplification such as enzymatic amplification plus redox cycling. An advantageous combination of an enzyme product [for fast electrochemical−chemical− chemical (ECC) redox cycling that involves the product] and an enzyme substrate (for slow side reactions and ECC redox cycling that involve the substrate) has been developed to obtain a low detection limit for E. coli O157:H7 in an electrochemical ELISA that employs redox cycling. In our search for an alkaline phosphatase substrate/product couple that is better than the most common couple of 4-aminophenyl phosphate (APP)/4-aminophenol (AP), we compared five couples: APP/ AP, hydroquinone diphosphate (HQDP)/hydroquinone (HQ), L-ascorbic acid 2-phosphate/Lascorbic acid, 4-amino-1-naphthyl phosphate/4-amino-1-naphthol, and 1-naphthyl phosphate/1naphthol. In particular, we examined signal-to-background ratios in ECC redox cycling using Ru(NH3)63+ and tris(2-carboxyethyl)phosphine as an oxidant and a reductant, respectively. The ECC redox cycling that involves HQ is faster than the cycling that involves AP, whereas the side reactions and ECC redox cycling that involve HQDP are negligible compared to the APP case. These results seem to be due to the fact that the formal potential of HQ is lower than that of AP and that the formal potential of HQDP is higher than that of APP. Enzymatic amplification plus ECC redox cycling based on a HQDP/HQ couple allows us to detect E. coli O157:H7 in a wide range of concentrations from 103 to 108 colony-forming units/mL.

B

bacteria, although they are less sensitive and selective than the microbiological methods and the DNA amplification methods.20−22 Nevertheless, ELISAs provide very reproducible assay results via enzymatic signal amplification, and the sandwichtype ELISAs are very insensitive to the matrix effect. Detection limits of several techniques for the detection of E. coli O157:H7 are summarized and compared in Table 1. If the sensitivity of ELISAs could be improved, these assays could be used more commonly for detecting bacteria. To improve their sensitivity, it would be useful to employ an approach that entails multiple signal amplification such as enzymatic amplification plus redox cycling. We have previously shown that electrochemical ELISAs combined with redox cycling allow us to carry out ultrasensitive protein detection.23−26 In electrochemical ELISAs, signals depend on the surface concentration of an enzymatic product at a sensing electrode and not on the average concentration in the detection solution. Therefore, the generation of enzymatic products by even low numbers of enzyme labels that are

acterial pathogens cause food poisoning and water contamination that can lead to severe human diseases.1 Rapid and sensitive monitoring of bacteria is essential for ensuring food and water safety and for carrying out timely and accurate disease diagnosis and treatment.2−8 Among the different types of bacterial pathogens, E. coli O157:H7 frequently causes severe health problems because of the potent toxins that it produces.9,10 A low number of E. coli O157:H7 is sufficient to cause serious illnesses such as hemorrhagic diarrhea and kidney failure.11 Conventional bacterium detection is based on microbiological methods including enrichment steps that are carried out through the cultivation of the bacterium.12,13 Although these methods provide reliable and sensitive results, they are time-consuming and laborious. DNA amplification methods such as the polymerase chain reaction are also commonly used for detecting bacteria.14−16 They are highly sensitive and selective, but they must be carried out by trained personnel to avoid misdiagnosis. In recent years, many simple and sensitive label-free detection methods have been developed for detecting bacteria.17−19 However, the measurement of different signals for different samples that contain the same concentration of analyte (i.e., the matrix effect) limits their practical use. Versatile enzyme-linked immunosorbent assays (ELISAs) are also used for detecting © 2013 American Chemical Society

Received: October 4, 2012 Accepted: January 2, 2013 Published: January 17, 2013 1631

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Table 1. Comparison of Techniques for the Detection of E. coli detection method

matrix

enrichment (1 day to 1 apple cider week) immunomagnetic PBS separation + realtime RT-PCR real-time PCR drinking water enrichment (6 h) + raw milk real-time PCR surface plasmon PBS resonance (SPR) impedometric Pepton detection water ELISA + Trisimpedometric buffered detection saline ELISA + PBS + chemiluminescence Tween 20 this work PBS

detection limit (CFU/mL)

detection range (CFU/mL)

low CFUs

13 10−107

14

102

∼102

15

1

16

107

∼107

17

104

104−107

18

6 × 103

6 × 103 to 6 × 107

20

105

105−106

21

103

103−108

EXPERIMENTAL SECTION

Materials. Avidin, biotin, and bovine-serum albumin (BSA) were purchased from Sigma-Aldrich, Co. Heat-killed E. coli O157:H7 (50−95−90), ALP-conjugated goat anti-E. coli O157:H7 IgG (05−95−90), and goat anti-E. coli O157:H7 IgG (01−95−90) were obtained from Kirkegaard & Perry Laboratories Inc. (Gaithersburg, MD). HQ, AP, AA, AN, NP, NPP, [Ru(NH 3 ) 6 ]Cl 3 , [Ru(NH 3 ) 6 ]Cl 2 , and tris(2carboxyethyl)phosphine (TCEP) hydrochloride were obtained from Sigma-Aldrich, Co. HQDP dipyridinium salt was synthesized as stated in Supporting Information. APP monosodium salt hydrate was obtained from Biosynth (Staad, Switzerland). ANP monosodium salt was synthesized by the method reported previously.28 AAP magnesium salt hydrate was obtained from Wako (Osaka, Japan). All of the reagents for the buffer solutions that we used were supplied by SigmaAldrich, Co. A biotin-labeling kit (LK55-10) was purchased from Dojindo Laboratories (Rockville, MD). Biotinylated antiE. coli O157:H7 IgG (01−95−90) was obtained by using the biotin-labeling kit. All of the chemicals were used as received, and all of the aqueous solutions were prepared in doubly distilled water. Indium−tin oxide (ITO) electrodes were obtained from Samsung Corning (Daegu, Korea). The phosphate-buffered saline (PBS buffer, pH 7.4) contained 10 mM phosphate, 0.138 M NaCl, and 2.7 mM KCl. The rinsing buffer (pH 7.6) contained 50 mM tris(hydroxymethyl)aminomethane, 40 mM HCl, 0.05% (w/ v) BSA, and 0.5 M NaCl. The Tris buffer (pH 8.9) contained 50 mM tris(hydroxymethyl)aminomethane and 10 mM MgCl2. Preparation of Immunosensing Layers and Immunosensing Procedures. To obtain avidin-modified ITO electrodes, 70 μL of a carbonate buffer solution (pH 9.6) that contained 100 μg/mL avidin was dropped and placed onto pretreated ITO electrodes. This state was maintained for 2 h at 20 °C, after which the electrodes were washed twice with rinsing buffer. To immobilize biotinylated IgG on avidin, 70 μL of a PBS buffer solution that contained 10 μg/mL biotinylated anti-E. coli O157:H7 was dropped and placed onto the avidinand BSA-modified ITO electrodes. This state was maintained for 30 min at 4 °C, after which the electrodes were washed twice with rinsing buffer. The resulting electrodes were stored at 4 °C before use. For the binding of target to the immunosensing electrodes, 70 μL of PBS buffer solutions that contained different concentrations of E. coli O157:H7 were dropped and placed onto the immunosensing electrodes. This state was maintained for 30 min at 4 °C, and the electrodes were then washed twice with rinsing buffer. Afterward, 70 μL of a PBS buffer solution that contained 10 μg/mL ALP-conjugated anti-E. coli O157:H7 was dropped and placed on the target-treated electrodes, and this state was maintained for 30 min at 4 °C. Following this, the electrodes were washed twice with rinsing buffer. Teflon electrochemical cells were assembled with an ITO electrode, an Ag/AgCl (3 M NaCl) reference electrode, and a platinum counter electrode. Separately prepared Tris buffer solutions that contained HQ, HQDP, AP, APP, AA, AAP, NP, NPP, AN, ANP, TCEP, Ru(NH3)62+, or Ru(NH3)63+ were injected into the vessels of the cells and were mixed well. The exposed geometric area of each ITO electrode was ca. 0.28 cm2. The electrode area of the glassy carbon disk electrode was 0.071 cm2. For the enzymatic reaction, the cells were stored for 10 min at 30 °C, and electrochemical measurements were then

reference

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attached to a sensing electrode can give high electrochemical signals. Electrochemical−chemical (EC) redox cycling23,24 and electrochemical−chemical−chemical (ECC) redox cycling25 offer an additional signal amplification. EC redox cycling can be performed by adding one reductant to a detection solution, and ECC redox cycling can be achieved by adding one reductant and one oxidant. However, the use of a reductant (and an oxidant) can cause many thermodynamically possible unwanted side reactions that should be kinetically minimized.25 One problem with ELISAs is that the stability of enzyme substrates can significantly affect the assay reproducibility. 4Aminophenyl phosphate (APP), which is the most common substrate of alkaline phosphatase (ALP), is unstable during long-term storage.27 Moreover, APP can take part in unwanted ECC redox cycling, which increases background levels of ELISAs.25 Therefore, it is necessary to find proper enzyme substrates for better sensor reproducibility and lower background levels. The formal potential of AP is higher than that of Ru(NH3)63+, which makes it difficult to obtain fast ECC redox cycling, i.e., high signal levels.25 It is also required to find proper enzyme products for higher signal levels. In this study, we compared five substrate/product couples of ALP in terms of signal-to-background ratios in the ECC redox cycling using Ru(NH3)63+ and tris(2-carboxyethyl)phosphine (TCEP) as an oxidant and a reductant, respectively, to find an ALP substrate/product couple that is better than APP/AP. The couples that we examined were APP/4-aminophenol (AP), hydroquinone diphosphate (HQDP)/hydroquinone (HQ), Lascorbic acid 2-phosphate (AAP)/L-ascorbic acid (AA), 4amino-1-naphthyl phosphate (ANP)/4-amino-1-naphthol (AN), and 1-naphthyl phosphate (NPP)/1-naphthol (NP). The chemical structures for these compounds are detailed in Figure S1 in Supporting Information. An ELISA employing the ECC redox cycling that involves HQ was applied for the sensitive detection of E. coli O157:H7, and the detection limit of the ELISA was determined. 1632

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Figure S2 in Supporting Information shows cyclic voltammograms for five enzyme substrates that were obtained at a glassy carbon electrode in Tris buffer (pH 8.9). The voltammogram for HQDP (curve ii of Figure S2a in Supporting Information) was almost similar to that for no substrate (curve i of Figure S2a in Supporting Information), indicating that HQDP oxidation was negligible up to 0.9 V. This behavior was also observed in Tris buffer that contained NPP (curve iii of Figure S2b in Supporting Information). In other cases, APP, AAP, and ANP oxidation occurred within a potential window and started from ca. 0.3 V, 0.6 V, and 0.1 V, respectively (Figure S2 in Supporting Information). These results revealed that the formal potentials of HQDP and NPP were much higher than those of the other substrates. As a result, the difference in formal potential between HQDP and Ru(NH3)63+ and that between NPP and Ru(NH3)63+ are larger than that between APP and Ru(NH3)63+ (i of Figure 1b). The larger difference makes ECC redox cycling via a coupled reaction mechanism more difficult. The high formal potential of HQDP is due to the diphosphorylation of HQ, and the high formal potential of NPP originates from the high formal potential of NP. It was reported that HQDP shows high stability against ester hydrolysis and air oxidation.29 Therefore, it is highly possible that HQDP and NPP do not take part in the ECC redox cycling that employs Ru(NH3)63+ and TCEP. To check for the presence of the ECC redox cycling that involves P, cyclic voltammograms were obtained at bare ITO electrodes in the following separate solutions: Ru(NH3)62+; P; Ru(NH3)62+ and P; and Ru(NH3)62+, P, and TCEP (Figure 2a

carried out using a CHI 405A or CHI 708C (CH Instruments, Austin, TX).

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RESULTS AND DISCUSSION Enzyme Substrate/Product Couple That Shows a High Signal-to-Background Ratio in ECC Redox Cycling. Figure 1a is a schematic diagram of E. coli detection using

Figure 1. (a) Schematic diagram of E. coli O157:H7 detection based on the ECC redox cycling that involves HQ (P), which is enzymatically generated from HQDP (S). (b) Schematic representation of three-step electron transfer (purple lines) and unwanted electron transfer (green and sky-blue lines) in ideal ECC redox cycling. The blank horizontal lines are positioned according to relative positions of the formal potentials of redox couples and the applied potential of an ITO electrode.

enzymatic amplification combined with ECC redox cycling. Enzymatic amplification converts an enzyme substrate (S, here HQDP) into an enzyme product (P, here HQ) that triggers ECC redox cycling. The side reactions including the reaction between OI and RII should be kinetically minimized.25 We showed that the side reactions can be significantly lowered when Ru(NH3)63+ and TCEP are used as the oxidant (OI) and the reductant (RII), respectively.25 Figure 1b shows the relative positions of the formal potentials of the redox couples that take part in ECC redox cycling. To obtain effective ECC redox cycling by three-step electron transfer, the formal potential of the redox couple of Q/P should be more negative or a little more positive than that of the OI/RI couple. Even though the formal potential of the Q/P couple is more positive than that of the OI/RI couple, ECC redox cycling can occur via a coupledreaction mechanism.25 Ideally, ECC redox cycling should not occur in the presence of S and in the absence of P. The ECC redox cycling that involves S is much slower than that which involves P because the formal potential of S (a phosphorylated form of P) is higher than that of P (Figure 1b). However, such a side reaction (the ECC redox cycling that involves S) can substantially increase background levels of sensors. To obtain high signal-to-background ratios, the ECC redox cycling that involves S should be very slow, but the ECC redox cycling that involves P should be very fast. To find an enzyme substrate/ product couple that meets this requirement, the electrochemical behaviors of five enzyme substrates (HQDP, APP, AAP, ANP, and NPP) and five enzyme products (HQ, AP, AA, AN, and NP) were examined.

Figure 2. (a) Cyclic voltammograms that were obtained at bare ITO electrodes (at a scan rate of 20 mV/s) in Tris-buffer solutions (pH 8.9) containing either (i) 1.0 mM Ru(NH3)62+, (ii) 0.5 mM HQ, (iii) 1.0 mM Ru(NH3)62+ and 0.5 mM HQ, or (iv) 1.0 mM Ru(NH3)62+, 0.5 mM HQ, and 2.0 mM TCEP after purging with argon for 10 min. (b) Schematic representation of Ru(NH3)63+-mediated HQ oxidation.

and Figure S3 in Supporting Information). The electrooxidation of Ru(NH3)62+ was fast at the ITO electrodes (curve i of Figure 2a), but the oxidation of HQ was slow (curve ii of Figure 2a). The anodic currents during the anodic scan in a Ru(NH3)62+ and HQ solution (curve iii of Figure 2a) were higher than the sums of the currents in a Ru(NH3)62+ solution (curve i of Figure 2a) and the currents in a HQ solution (curve 1633

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Figure 3. Chronocoulograms that were obtained at 0.05 V at ITO electrodes in air-saturated Tris-buffer solutions (pH 8.9) that contained (a) 1.0 mM Ru(NH3)63+, 1.0 mM S [(i) HQDP, (ii) APP, (iii) AAP, (iv) ANP, or (v) NPP], and 2.0 mM TCEP or (b) 1.0 mM Ru(NH3)63+, 0.10 mM P [(i) HQ, (ii) AP, (iii) AA, (iv) AN, or (v) NP], and 2.0 mM TCEP just after mixing. (c) Schematic of the EC redox cycling that involves (P). (d) Schematic of chemical−chemical redox cycling that involves P.

ii of Figure 2a). The higher currents indicated that Ru(NH3)63+-mediated HQ oxidation (shown in Figure 2b) was substantial in a Ru(NH3)62+ and HQ solution. The anodic currents in a solution of Ru(NH3)62+, HQ, and TCEP (curve iv of Figure 2a) were much higher than those in a solution of Ru(NH3)62+ and HQ (curve iii of Figure 2a), while the anodic currents of TCEP were negligible in this region of potentials. These results clearly showed that the ECC redox cycling that involves HQ (Figure 1a) occurred and allowed higher anodic currents to occur. In the case of AP and AN, the ECC redox cycling that involves P was also considerable (Figures S3a and S3c in Supporting Information). As for AA and NP, the cycling was not substantial (Figures S3b and S3d in Supporting Information). To compare the signal-to-background ratios in the ECC redox cycling that involves different S and P, chronocoulograms were obtained in the presence of different S (Figure 3a) or P (Figure 3b). The data that were obtained in the presence of S corresponds to the background levels, whereas the data that were obtained in the presence of P corresponds to the signal levels. Recently, we showed that, in ECC redox cycling, chronocoulometry was better than cyclic voltammetry in terms of high signal-to-background ratios.25 For chronocoulometry, an applied potential of 0.05 V was selected to avoid the electroreduction of Ru(NH3)63+ and to minimize the direct electrooxidation of S at the ITO electrodes. At this potential, the Ru(NH3)62+ that was generated by ECC redox cycling was readily electrooxidized (see curve i of Figure 2a). The anodic charges in the presence of HQDP (curve i of Figure 3a) and in the presence of AAP (curve iii of Figure 3a) were almost similar to those in the absence of P (data not shown), which indicated that the side reactions and ECC redox cycling that involve HQDP and those that involve AAP were negligible. The anodic charges for ANP and NPP (curves iv and v of Figure 3a) were very high, and those for APP (curve ii of Figure 3a) were not negligible. In Figure 3b, the anodic charges for HQ and AN were very high, but those for AP and NP were relatively small, and those for AA were very small. Table 2 shows the signal-tobackground ratios that were calculated from the charges obtained at 100 s in Figure 3a and 3b. In the case of the

Table 2. Charges That Were Obtained at 100 s in the Chronocoulograms (shown in Figure 3a and 3b) and the Signal-to-Background Ratios That Were Calculated from the Charges components Ru(NH3)63+ + HQDP + TCEP (curve i of Figure 3a) Ru(NH3)63+ + HQ + TCEP (curve i of Figure 3b) Ru(NH3)63+ + APP + TCEP (curve ii of Figure 3a) Ru(NH3)63+ + AP + TCEP (curve ii of Figure 3b) Ru(NH3)63+ + AAP + TCEP (curve iii of Figure 3a) Ru(NH3)63+ + AA + TCEP (curve iii of Figure 3b) Ru(NH3)63+ + ANP + TCEP (curve iv of Figure 3a) Ru(NH3)63+ + AN + TCEP (curve iv of Figure 3b) Ru(NH3)63+ + NPP + TCEP (curve v of Figure 3a) Ru(NH3)63+ + NP + TCEP (curve v of Figure 3b)

charge (μC)

signal-tobackground ratio

12.3 1006

81.8

24.4a 259.8

10.6

12.7 31.0

2.4

762.0 1299

1.7

84.0 173.6

2.1

a

APP may not be highly pure because it readily decomposes into AP, which may have partly contributed to a high background level.

HQDP/HQ couple, the background charge (12.3 μC) was the lowest and the signal-to-background ratio (81.8) was the highest. Therefore, HQDP was chosen as an ALP substrate for E. coli detection using ECC redox cycling. Interestingly, the signal charge (1006 μC) for HQ was much higher than that for AP (260 μC). This result indicates that the ECC redox cycling that involves HQ is faster than the cycling that involves AP. Judging from the anodic and cathodic peak potentials of the cyclic voltammograms of AP, HQ, and Ru(NH3)62+ that were obtained at the glassy carbon electrode (Figure S4 in Supporting Information), the formal potential of BQ/HQ was more negative than that of 4-quinone imine/AP and it was closer to that of Ru(NH3)63+/Ru(NH3)62+ (ii of Figure 1b). 1634

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This result indicated that the ECC redox cycling that involves HQ was more thermodynamically favorable than the ECC redox cycling that involves AP. This seemed to be one reason for the faster reaction of the ECC redox cycling that involves HQ. Figure S5 in Supporting Information shows chronocoulograms that were obtained in Tris buffer (pH 8.9) that contained Ru(NH3)63+, HQDP, HQ, and/or TCEP. Some of the charge values that were measured at 100 s are shown in Table 3. The Table 3. Charges Obtained at 100 s in Chronocoulograms Shown in Figure S5a−S5c of Supporting Information components None (curve i of Figure S5a) HQ (curve iv of Figure S5a) TCEP (curve iii of Figure S5a) HQ + TCEP (curve i of Figure S5c) Ru(NH3)63+ + TCEP (curve i of Figure S5b) Ru(NH3)63+ + HQ + TCEP (curve iii of Figure S5c)

charge (μC) 0.43 7.56 1.31 106.2 4.05 1006

difference (μC) 7.13

amplification factor 1

104.9

15

1001.9

141

difference between the charge in the solution of HQ and the charge in the Tris buffer solution (7.13 μC) is due to the electrooxidation of HQ. The difference between the charge in the solution of HQ and TCEP and the charge in the solution of TCEP (104.9 μC) is due to the electrooxidation of HQ and the EC redox cycling that involves HQ (Figure 3c). This indicates that the signal in the presence of TCEP was increased by an amplification factor of ca. 15. The difference between the charge in the solution of Ru(NH3)63+, HQ, and TCEP and the charge in the solution of Ru(NH3)63+ and TCEP (1001.9 μC) is due to the electrooxidation of HQ, the EC redox cycling that involves HQ (Figure 3c), and the ECC redox cycling that involves HQ (Figure 1a). This indicated that the signal in the presence of TCEP and Ru(NH3)63+ was increased by an amplification factor of ca. 141. These results clearly show that the ECC redox cycling significantly increased the signal. E. coli Detection Using ECC Redox Cycling. The ECC redox cycling that involves Ru(NH3)63+, HQ, and TCEP was applied to E. coli detection. Figure 4a shows chronocoulograms that were obtained at 0.05 V at 106 CFU (colony-forming units)/mL E. coli O157:H7 by using the ECC redox cycling after the final sensing electrodes were incubated for 10 min in a solution of Ru(NH3)63+, HQDP, and TCEP. We reported that the antigen−antibody binding at 4 °C is much better in terms of reproducibility than at 25 °C (room temperature).24 To check this dependence in the case of E. coli detection, two reactions of E. coli-antibody binding were performed at 4 °C or at 25 °C. In Figure 4a, the data for 4 °C was more reproducible than that for 25 °C. Accordingly, all of the concentrationdependent data were obtained after E. coli−antibody binding was carried out at 4 °C. Figure 4b shows chronocoulograms that were obtained for different concentrations of E. coli O157:H7. The charge that was obtained at a higher concentration of E. coli O157:H7 was higher in this range of concentrations. In all the cases, the charge increased almost linearly with increasing time after an initial nonlinear increase. The reason for this was that ECC redox cycling reached a steady state after some time. During 10-min incubation, the chemical−chemical redox cycling that involves HQ (Figure 3d)

Figure 4. (a) Chronocoulograms that were obtained at 0.05 V (a) at 106 CFU/mL E. coli O157:H7 and (b) in a concentration range from 103 to 108 CFU/mL by using the ECC redox cycling after the final sensing electrodes were incubated for 10 min in a solution of 1.0 mM Ru(NH3)63+, 1.0 mM HQDP, and 2.0 mM TCEP. Two steps of E. coli−antibody binding were carried out (a) at 4 °C or 25 °C and (b) at 4 °C. (c) Calibration plot: concentration dependence of the charge at 100 s in panel b. Each concentration experiment was carried out with three different sensing electrodes for the same assay sample. All of the data were subtracted by the mean value determined at a concentration of zero by seven measurements. The dashed line corresponds to 3 times the standard deviation (SD) of the charge at a concentration of zero. The error bars represent the SD of three measurements.

also occurred,25,26 and it contributed to the initial charge increase that was shown in the chronocoulograms. Figure S6 in Supporting Information shows a scanning electron microscopy (SEM) image that was obtained after the sensing surface was treated with a solution of 108 CFU/mL E. coli O157:H7. This image revealed that E. coli O157:H7 was randomly attached to the sensing surface. Figure 4c represents a calibration plot that was drawn using the charge data recorded at 100 s in the chronocoulogram. The calculated detection limit for E. coli O157:H7 was ca. 103 CFU/mL. Moreover, E. coli O157:H7 were detected in a wide range of concentrations from 103 to 108 CFU/mL. As 70 μL of target solutions was used, the 1635

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detection limit corresponds to ca. 70 CFU, which indicates that the immunosensor was ultrasensitive. When the detection limit and detection range are compared to those in Table 1, this method is more sensitive and covers a wider range of concentrations than ELISA-based techniques. Although very low detection limits can be obtained in PCR-based detection, detection limits of 1 and 10 CFU/mL required immunomagnetic separation or an enrichment step before PCR.14,16 In this study, we could obtain a detection limit of 103 CFU/mL without using immunomagnetic separation or an enrichment step. The sensor reproducibility was checked by measuring three chronocoulograms for 104 CFU/mL E. coli O157:H7 on three different dates (Figure S7 in Supporting Information). The charges at 100 s for three data were 1.14 ± 0.08 × 10−4, 1.15 ± 0.02 × 10−4, and 1.18 ± 0.02 × 10−4 C. The similar mean values and low standard deviations indicate that the sensor was highly reproducible. The sensor performance was also tested with apple juice spiked with E. coli O157:H7. Apple juice was mixed with PBS buffer at a ratio of 1 to 1 before the detection. Figure S8 in Supporting Information shows comparison between the charges for the detection in PBS buffer and the charges for the detection in apple juice. Two mean values at each concentration were similar, indicating that the sensor was reproducible and selective.

CONCLUSIONS An advantageous combination of enzyme product and enzyme substrate has been developed to obtain a low detection limit for E. coli O157:H7 in an ELISA that employs ECC redox cycling. Among five substrate/product couples of ALP, the HQDP/HQ couple showed the highest signal-to-background ratio in ECC redox cycling. The ECC redox cycling that involves HQ was very fast, whereas the side reactions and ECC redox cycling that involve HQDP were negligible. The fast ECC redox cycling seems to be due to the small difference in formal potential between Ru(NH3)63+ and HQ, and the negligible side reactions due to the large difference in formal potentials between Ru(NH3)63+ and HQDP. This result shows that the relative positions of the formal potentials of Ru(NH3)63+, enzyme substrate, and product play a crucial role in obtaining high signal-to-background ratios in ECC redox cycling combined with enzymatic reaction. ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (2012R1A1A2006478, 2012-M3C1A1048860, and 2012R1A2A2A06045327). 1636

dx.doi.org/10.1021/ac3028855 | Anal. Chem. 2013, 85, 1631−1636