A label-free, microfluidics and interdigitated array microelectrode-based impedance biosensor in combination with nanoparticles immunoseparation for detection of Escherichia coli O157:H7 in food samples

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Abstract

A microfluidic flow cell with embedded gold interdigitated array microelectrode (IDAM) was developed and integrated with magnetic nanoparticle-antibody conjugates (MNAC) into an impedance biosensor to rapidly detect pathogenic bacteria in ground beef samples. The flow cell consisting of a detection microchamber and inlet and outlet microchannels was fabricated by bonding an IDAM chip to a poly(dimethylsiloxane) (PDMS) microchannel. The detection microchamber with a dimension of 6 mm × 0.5 mm × 0.02 mm and a volume of 60 nL was used to collect bacterial cells in the active layer above the microelectrode for sensitive impedance change. MNAC were prepared by conjugating streptavidin-coated magnetic nanoparticles with biotin-labeled polyclonal goat anti-E. coli antibodies and were used in the separation and concentration of target bacteria. The cells of E. coli O157:H7 inoculated in a food sample were first captured by the MNAC, separated, and concentrated by applying a magnetic field, washed, and then suspended in mannitol solution and finally injected through the microfluidic flow cell for impedance measurement. This impedance biosensor was able to detect as low as 1.6 × 102 and 1.2 × 103 cells of E. coli O157:H7 cells present in pure culture and ground beef sample, respectively. The total detection time from sampling to measurement was 35 min. Equivalent circuit analysis indicated that the bulk medium resistance, double layer capacitance, and dielectric capacitance were responsible for the impedance change due to the presence of E. coli O157:H7 cells on the surface of IDAM. Sample pre-enrichment, secondary antibodies on the microelectrode surface, and redox probes were not required in this impedance biosensor.

Introduction

The Centers for Disease Control and Prevention [1] estimates that foodborne diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5000 deaths in the United States each year. USDA Economic Research Services [2] reported that medical costs, productivity losses, and costs of premature deaths for diseases caused by major foodborne pathogens total to $6.9 billion per year. Among all pathogens, Escherichia coli O157:H7 is a leading cause of foodborne illness. Based on a 1999 estimate, 73,000 cases of infection and 61 deaths occur in the United States each year due to E. coli O157:H7 only [1]. As the loss caused by E. coli O157:H7 is enormous in terms of medical cost and product recall, it is extremely important to rapidly and specifically detect E. coli O157:H7 in food products.

Until recently, most biosensors studied for pathogenic bacteria detection are label-dependent immunosensors that use labeled secondary antibodies to convert the antibody/antigen interaction into detectable optical or electrochemical signals. Label-free biosensors such as quartz crystal microbalance and surface plasmon resonance have attractive advantages with respect to speed, cost, and simplicity of operation. Impedance technique is yet another rapid and inexpensive alternative for label-free biosensors. Traditionally, macro-sized metal rods or wires were used as electrodes immersed in a medium to measure impedance [3], [4], [5]. Several electrode geometries have been developed during the last decades to add functionalities, improve sensitivities, and lower the detection limits of impedance techniques. Microelectrodes fabricated using lithographic techniques have been of great interest because they typically have higher sensitivities than macroelectrodes, since macroelectrodes have a semi-infinite linear diffusion profile resulting in a greater depletion of reactants in contrast to the microelectrodes which has spherical diffusion profile favoring a greater rate of reactant supply [6]. Among microelectrodes, interdigitated array microelectrodes (IDAM) present promising advantages in terms of low ohmic drop, fast establishment of steady-state, rapid kinetics of reaction, and increased signal-to-noise ratio [7], [8]. IDAM are increasingly used for impedance measurement of bacterial cells during enrichment growth (impedance microbiology) [9], [10], [11], [12], [13] by capturing bacterial cells to the antibodies immobilized on the surface of electrodes (faradic impedance method) [14], [15], [16], [17], [18], by using immunomagnetic particles for the separation of target bacterial cells followed by impedance measurement [19], [20], or by using dielectrophoresis (DEP) for the capture of cells on the surface of electrodes (dielectrophoretic impedance) [21], [22], [23], [24], [25]. In a study, redox probe was used for the detection of bacterial cells captured by antibodies immobilized on the surface of electrodes. Yang et al. [17] used open indium–tin oxide IDAM immobilized with anti-E. coli antibodies for capture of E. coli O157:H7, and impedance measurement was performed in the presence of ferriferro-cyanide. The detection range of the biosensor was 4.36 × 105 to 4.36 × 108 cfu/mL. Characterization of stepwise process of surface modification and bacterial cell capture was studied simultaneously with atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6]−3/−4 as a redox probe [18]. Radke and Alocilja [15], [16] used an open IDAM chip for the direct detection of E. coli O157:H7 cells in food samples by immobilizing anti-E. coli antibodies on the surface of IDAM for the capture of target bacteria. Impedance measurement was performed by immersing electrode array into the samples of E. coli O157:H7 suspended in peptone water. The biosensor was found to be sensitive for the detection of E. coli O157:H7 ranging from 104 to 107 cfu/mL. The sensor surface was selective for the detection of target bacteria, E. coli O157:H7 in presence of non-target bacteria, Salmonella infantis. Although the use of antibodies is very common for capturing bacterial cells on the surface of electrodes, however, dielectrophoresis has also been claimed for the concentration of target bacteria from medium or a mixture of other bacteria [22], [26]. Suehiro et al. [23] described a technique for the quantitative estimation of E. coli suspended in low conductivity mannitol solution by simultaneous concentration and detection of cells by dielectrophoreis and impedance method, respectively. Based on the results, 105 cfu/mL of E. coli O157:H7 were detected in 10 min. In the same system, live and dead bacteria were differentiated by their response to frequency of applied voltage [25]. It was found that the viability of the cell governed the response of the cell to DEP. At 1 MHz, viable E. coli cells showed strong dependency on DEP and thus were concentrated between the electrodes and resulted in an impedance change. At this frequency, dead bacterial cells were not concentrated by DEP and thus were not able to cause change in impedance as compared to live bacterial cells.

To further enhance the capability of IDAM in impedance sensing, microfluidic flow cells can be added to the IDAM to achieve a fully integrated microchip for a broad range of applications including dielectrophoresis and impedance detection [27], [28]. The advantages of combining microfluidic flow cells with embedded IDAM are: high detection sensitivity, small volume handling, low contamination during bacterial growth, achieve concentration of cells, and rapid detection of small number of cells. As the surface to volume ratio increases in the microfluidic flow cells with embedded IDAM, the distance that conductive ions must diffuse to reach the sensor surface decreases, thus resulting in a rapid kinetic reaction [29]. Generally, the impedance measurement can be performed by capturing bacterial cells with antibodies immobilized on the surface of the electrodes. One major problem associated with antibody immobilization is the low capture efficiency (CE) of the immobilized surface. Due to this, the functional surface area (where target bacterial cells are detected) of the detection electrode is not optimally utilized. For example, anti-E. coli antibodies immobilized on the surface of indium tin oxide coated glass electrodes showed only 16% CE for E. coli O157:H7 [30] and the CE of anti-Salmonella antibodies functionalized on roughened glass surface was less than 1% for Salmonella [31]. The problem of low CE can be resolved by developing impedance techniques that do not require immobilization of biosensing material on the surface of electrodes. Instead, magnetic particles coated with specific antibodies can be used to capture target bacteria.

Magnetic particles are useful in separating target cells from a mixture of bacteria and food matrix and also help to concentrate separated cells into a very small volume with the help of a magnetic field. The sensitivity of the impedance detection can be improved by collecting bacterial cells attached with magnetic particles in the active layer of electrodes using a magnetic field [32]. The microelectrodes scan a region called “active layer”, which is few microns above the surface of electrodes with maximum strength of electric field. Microfluidic flow cell with embedded IDAM can be fabricated at very small depth (equal to active layer) to collect bacterial cells in the active layer of microelectrodes.

In this paper we report a label-free impedance biosensor coupled with magnetic nanoparticles immunoseparation for the direct detection of pathogenic bacterial cells in food samples. Magnetic nanoparticle-antibody conjugates (MNAC) were used to separate target cells from a mixture of bacteria and food matrix, and to concentrate separated cells into a desired volume suitable for the impedance measurement that was performed in a specially designed microfluidic flow cell with embedded IDAM. The dimensions of the microchannel were specially designed to collect bacterial cells captured by MNAC in the active layer of the IDAM. The biosensor did not use any surface immobilization of antibodies, sample pre-enrichment, and redox probe. Mannitol solution with low conductivity was used to minimize the effect of medium on direct detection of bacterial cells. An electrical equivalent circuit was proposed to analyze the various parameters involved in the impedance measurement of target bacteria using the microfluidic flow cell with embedded IDAM.

Section snippets

Culture and plating of bacteria

Frozen stock of E. coli O157:H7 (ATCC 43888) was maintained in brain heart infusion broth (BHI, Remel Inc., Lenexa, KS) at −70 °C. The culture was harvested in BHI maintained at 37 °C for 18–22 h. For enumeration, pure cultures were serially diluted in 0.01 M, pH 7.4 phosphate buffered saline (PBS) and surface plated on sorbitol MacConkey (SMAC) agar (Remel Inc., Lenexa, KS), which was incubated at 37 °C for 20–22 h.

Chemicals and reagents

PBS (0.01 M, pH 7.4) was obtained from Sigma–Aldrich (St. Louis, MI). Bovine serum

The equivalent circuit

The experimental data of an impedance biosensor for the detection of E. coli O157:H7 cells attached to MNAC is represented by an equivalent circuit shown in Fig. 2(a) [17], [35]. This circuit consists of two double layer capacitors (Cdl), one for each set of the electrodes, connected in series with a bulk medium resistor (Rs), and a dielectric capacitor (Cdi) connected in parallel with Rs and Cdl. Constant phase element is used to model the double layer capacitance of the electrodes. This is

Conclusions

The present study showed a novel label-free impedance biosensor for the direct impedance measurement of bacterial cells without using radox probe or antibodies on the surface of electrodes. The microfluidic flow cell with embedded IDAM was used to collect bacterial cells in the active layer of electrodes to minimize the effect of particulates in the bulk solution for sensitive impedance measurement. The label-free impedance biosensor was able to detect as low as 1.6 × 102 and 1.2 × 103 cells of E.

Acknowledgment

The project was funded by the Food Safety Consortium sponsored by USDA/CSREES.

Madhukar Varshney has a PhD in biological engineering obtained at the University of Arkansas in 2006. He is currently a postdoctoral associate at Cornell University. His research interest is biosensors for bacteria, prion and other biomolecule detection.

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    Madhukar Varshney has a PhD in biological engineering obtained at the University of Arkansas in 2006. He is currently a postdoctoral associate at Cornell University. His research interest is biosensors for bacteria, prion and other biomolecule detection.

    Yanbin Li obtained his PhD in agricultural engineering from Penn State University in 1989. He is a professor of biological engineering at the University of Arkansas. His research focuses on biosensors for detection of foodborne pathogens and chemical residues and microbial prediction modeling and simulation.

    Balaji Srinivasan is a PhD student in the Department of Mechanical Engineering at the University of Arkansas. His research interests include the design, simulation, fabrication and characterization of MEMS based biosensors.

    Steve Tung obtained his PhD in mechanical engineering from University of Houston in 1992. He is an associate professor of mechanical engineering at the University of Arkansas. His research focuses on bioMEMS and biosensors.

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