Disposable amperometric immunosensing strips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157:H7

https://doi.org/10.1016/j.bios.2008.02.030Get rights and content

Abstract

A disposable amperometric immunosensing strip was fabricated for rapid detection of Escherichia coli O157:H7. The method uses an indirect sandwich enzyme-linked immunoassay with double antibodies. Screen-printed carbon electrodes (SPCEs) were framed by commercial silver and carbon inks. For electrochemical characterization the carbon electrodes were coupled with the first E. coli O157:H7-specific antibody, E. coli O157:H7 intact cells and the second E. coli O157:H7-specific antibody conjugated with horseradish peroxidase (HRP). Hydrogen peroxide and ferrocenedicarboxylic acid (FeDC) were used as the substrate for HRP and mediator, respectively, at a potential +300 mV vs. counter/reference electrode. The response current (RC) of the immunosensing strips could be amplified significantly by 13-nm diameter Au nanoparticles (AuNPs) attached to the working electrode. The results show that the combined effects of AuNPs and FeDC enhanced RC by 13.1-fold. The SPCE immunosensing strips were used to detect E. coli O157:H7 specifically. Concentrations of E. coli O157:H7 from 102 to 107 CFU/ml could be detected. The detection limit was approximately 6 CFU/strip in PBS buffer and 50 CFU/strip in milk. The SPCE modified with AuNPs and FeDC has the potential for further applications and provides the basis for incorporating the method into an integrated system for rapid pathogen detection.

Introduction

In the last 20 years, Escherichia coli O157:H7 has been an important foodborne pathogen in a variety of foods worldwide. This strain of E. coli is classified as an enterohemorrhagic bacterium with the ability to cause hemorrhagic colitis with symptoms such as bloody diarrhoea, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpurea (Ho et al., 2004). The monitoring of E. coli O157:H7 with conventional procedure (Johnson et al., 1995, Meng et al., 2001, Ho et al., 2004) could take 2–3 days. These methods are time-consuming, which delays the introduction of remedial measures. Therefore, a method for rapid detection of this pathogenic micro-organism in food and water would aid the prevention of infection, illness, and economic loss.

The field of electrochemical biosensing has grown rapidly, and biosensors for detecting antigens and nucleic acids have been reported (Bakker, 2004, Mehrvar and Abdi, 2004). Several electrochemical methods for the detection of bacterial contamination are known; for example, the amount of cells electrostatically attaching on carbon electrodes (Morisaki et al., 2000), the detection of glucose catabolism (Palenzuela et al., 2004), or the endogenous enzymatic activity of bacteria (Yemini et al., 2007). Additionally, a broad class of immunosensing techniques with improved specificity also was reported (Patel, 2002, Rasooly and Herold, 2006). Pathogens have been detected using amperometric immunoassay procedures in which the immunoreagents are immobilized on the transducer surface (Zhou et al., 2002, Chemburu et al., 2005, Rao et al., 2006). Reproducible methods of quantifying E. coli O157:H7 were recently studied based on an immunoassay combined with a liposome-based fluorescent-labeling method (Ho et al., 2004), a quartz crystal Au piezoelectric electrode (Su and Li, 2004), a self-assembled monolayer-based surface plasmon resonance sensor(Subramanian et al., 2006), and a fluorescent tracer-digital camera image transport system (Ligler et al., 2007). With these methods, E. coli O157:H7 cells could be detected at concentrations between 102 and 108 colony-forming units (CFU)/ml. However, the expense of the sensor materials or infrequent measuring instruments would limit out-of-laboratory applications for economic and fast screening.

Due to recent developments in biosensor technology, horseradish peroxidase (HRP) are often conjugated with antigen–antibody complex (Zhou et al., 2002, Chemburu et al., 2005, Castaňeda et al., 2007) on the electrodes of immunosensor as reporters which react with substrate to generate electrons to be detected. And the AuNPs-modified electrodes also can be used to amplify the detection signal, improve the electron transducer and reduce the limitation on detection in electrochemical biosensors (Daniel and Astruc, 2004, Willner et al., 2007). Here, we present an approach for the quantification of E. coli O157:H7 that combines amperometric detection with a disposable immunosensing strip. The system was designed for the rapid, sensitive detection of bacterial cells, such as E. coli O157:H7, utilizing AuNPs and ferrocenedicarboxylic acid (FeDC)-modified, disposable screen-printed carbon electrodes (SPCEs).

Section snippets

Apparatus

Cyclic voltammetric and amperometric determinations were performed with a CV50W voltammetric analyzer (Bioanalytical Systems, West Lafayette, IN, USA) connected to a personal computer and a PalmSens/Personal Digital Assistant, hand-held, battery-powered instrument (Palm Instruments, BZ Houten, Netherlands) for data collection and calculation.

Reagents and solutions

G-451 carbon/graphite ink and silver-resin inks were purchased from ERCON (Wareham, MA, USA). Sodium citrate, ethanol, and H2O2 were obtained from Merck

Effect of AuNPs modification of SPCE on electrochemical characters

The results of AuNPs-modified SPCE and an E. coli O157:H7 cell bound to antibodies immobilized on the surface of an AuNPs-modified SPCE were confirmed by scanning electron microscope (SEM) (Supplementary Fig. S1). The size of AuNPs was very uniform and approximately 13 nm in diameter on average. There were 73.6 ± 8.1 AuNPs per 0.04 μm2 of SPCE working surface in five random samplings from the SEM images.

In other studies, FeDC was used as the mediator in the H2O2–peroxidase electrochemical reaction

Discussion

The SPCEs modified with AuNPs and FeDC were investigated in an effort to develop a rapid detection protocol for monitoring E. coli O157:H7 contamination in food. The recognition and sensing procedures for the E. coli O157:H7 immunosensing were designed without pre-incubation or pre-filtration of samples to increase or concentrate the intact cells of the pathogen. After the bacteria were recognized and bound by the first monoclonal anti-E. coli O157:H7 antibodies on the surface of the SPCE

Conclusions

Here, we describe a rapid, specific, and sensitive electrochemical technique for the detection of intact cells of E. coli O157:H7. The technique is based on an indirect sandwich amperometric immunoassay. The disposable AuNPs/FeDC–SPCE immunosensing strips, the key element of this detection system, were fabricated by carbon inks and modified with 13-nm diameter AuNPs and FeDC. Amperometric detection was selected because of its many benefits, including its adaptability for use in miniaturized and

Acknowledgments

The authors thank the Apex Biotechnology Corp. for donating the SPCE. This work was supported by the MoE ATU Programs from the Department of Education, Taiwan.

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