AbstractâBacillus cereus (B. cereus) is a bacterial pathogen, which is more commonly found in milk and milk products and it is of particular concern to the infant ...
Label-free detection of Bacillus cereus DNA hybridization using electrochemical impedance spectroscopy for food quality monitoring application Vijayalakshmi Velusamya, Khalil Arshaka, Cathy Yangc, Lei Yuc, Olga Korostynskaa, Kamila Oliwa- Stasiakb, Catherine Adleyb a
b
Electronic and Computer Engineering Department, University of Limerick, Ireland Department of Chemical and Environmental Sciences, University of Limerick, Ireland c Department of Chemistry and Biochemistry, Rowan University, USA
Abstract—Bacillus cereus (B. cereus) is a bacterial pathogen, which is more commonly found in milk and milk products and it is of particular concern to the infant formula industry. However, it can also be found in turkey, beef, rice, mashed potatoes and vegetable sprouts and causes diarrhoeal and emetic type of food poisoning. Therefore, real-time detection of B. cereus is vital for food quality monitoring. The prime intention of this paper is to pioneer the design and fabrication of a single-strand (ss) DNA biosensor without modifying the ss-probe DNA. The conducting polypyrrole modified surface is used as an immobilization matrix. The polypyrrole (PPy) film is formed on the gold electrode surface by electrochemical polymerization of pyrrole with MgCl2 as a doping electrolyte. 1µg of 20-mer ss-probe DNA specific for the B. cereus have been immobilized on the PPy film by physioadsorption. Increase in electron transfer resistance was observed after the immobilization of the probe compared to the impedance spectra obtained for the polypyrrole modified surface. After the hybridization of the 20-mer target DNA, a further increase in impedance was noted and it is due to the addition of negative charges to the PPy/ss-DNA probe modified surface in the form of complementary DNA. Control experiments were performed to prove the specificity of the biosensor in the presence of 21-mer non complementary oligonucleotide and no unspecific bonding with the immobilized probe was observed. The performance of the DNA sensor proved to be effective in terms of selectivity, sensitivity and reproducibility of hybridization events. Keywords: Bacillus cereus; biosensor; DNA; polypyrrole; impedance spectroscopy; food quality monitoring.
I.
INTRODUCTION
The current area of interest in the development of biological sensors are greatly focused on the design and fabrication of handheld real-time DNA sensors due to their numerous applications such as the clinical analysis, food quality monitoring for the detection pathogenic micro-
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organisms, and in biosecurity. The detection technique employed in the DNA biosensors can be optical [1-4], electrochemical [5-7] or mass [8, 9] based. Among the other detection techniques, the electrochemical based detection method is more advantageous not only because they are simple and cost effective but also they can be easily miniaturized for real-time handheld use without sacrificing their sensitivity and selectivity. In electrochemical DNA biosensors, detection is based on the variation in the electrical properties of the DNA modified electrode before and after hybridization, which may be due to the change of double–layer capacitance, electronic transfer resistance, impedance or current. In most of the DNA biosensors which produce either electrical or photonic signals when immobilized and/or to detect the hybridization with the target, the probes are either modified by electrochemically active substances or labelled; which includes, organic dyes, metal nanoparticles, thiol-linked, enzyme labelled, biotin labelled, fluorescent and radioactive labelled probes [2, 4, 10, 11]. Although, these indirect (labelled) methods proved to be effective they are complex, expensive and time consuming and also may cause DNA damage which in turn affect the DNA recognition. Thus, the direct or the label-less DNA has become extremely beneficial and can be readily employed for the real-time detection. Despite the advantages of the label-less DNA probes, a modified electrode surface is required to immobilize them which, can attract and hold the DNA molecules effectively onto it. The materials being used to modify the electrode (Au, Ag, Pl, C, ITO and so on.) surface includes the carbon nanotubes, conducting polymers, metal nanoparticles and composites of various electroactive materials etc. Also modification of the electrode surface has to be simple, rapid and one step procedure. Owing to the above requirements, the conducting polymers are suitable not only because they can be easily synthesized but also due to their good stability and excellent electrical properties. In particular, polypyrrole (PPy) is one of the most attractive conducting polymers with some special electrical properties. These properties originate from the fact that PPy is an intrinsically conducting polymer
and can be synthesized to have conductivities up to 1000 S/cm which approaches the conductivity of metals. Electrical conduction in PPy is the result of electron movement within delocalized orbitals and positive charge defects known as polarons [12]. PPy is extensively used for the immobilization of the biomolecules especially, DNA. It can be simply synthesized from its monomer by one-step electropolymerization and the other important factor is that it is electroactive in neutral environments and can be more easily deposited from neutral pH aqueous solutions of pyrrolemonomers [13]. Moreover, DNA can form a strong bond with PPy based on the interchanging of dopant DNA molecules [14]. The two factors which significantly play a key role in the development of a DNA biosensor are the immobilization matrix and the method of immobilization. The biosensor features like sensitivity, specificity and reproducibility are significantly important and it should not be compromised by improper immobilization. In DNA sensors, detection of a hybridization event is essential and it also depends on the state of the immobilized single-stranded (ss) DNA probes onto the transducer surface. Therefore, precise care should be taken to immobilize the ss-DNA probes onto the suitable substrates. In this paper, we reported the impedance labelless detection of DNA hybridization with the physio-adsorbed oligonucleotide probe immobilized on the PPy modified gold electrode. Here, attention is given to food quality monitoring emphasizing on the bacterial pathogen, Bacillus cereus. II. MATERIALS AND METHODS A. Materials Pyrrole and MgCl2 solution was purchased from SigmaAldrich, USA. All the reagents were analytical grade and used without further purification. Specific sequences for the B.cereus were designed in our laboratory [15] and its synthetic form was purchased from Integrated DNA technologies, USA. All stock solutions were prepared from distilled water and a concentration of 1µg/ µl from the stock solution was made using 10mM Tris-HCl buffer, pH 7.2. The capture probe and the target probe are 20-mer oligonucleotides and the non complementary probe is 21mer in length. Table.1 shows the sequence of the oligonucleotides probes used in this work. Table: 1 Function
Sequence
Probe (5’-3’)
ATC GCC TCG TTG GAT GAC GA
Complementary (5’-3’)
NonComplementary (5’-3’)
TCG TCA TCC AAC GAG GCG
AT AAA ATC GAT GGT AAA GGT TGG
B. Instrumentation A three-electrode cell comprising a gold working electrode (2-mm diameter), a platinum wire counter electrode and a Ag/AgCl reference electrode were used. All electrochemical measurements were carried out using Autolab PGSTAT302N (Eco Chemie, The Netherlands) with the FRA module interfaced for impedance measurements. C. Gold electode surface modification and electrochemical sysnthesis of Polypyrrole The Au electrode surface was polished to mirror finish prior to use sequentially with 1, 0.3, and 0.05 µm α-Al2O3 paste, and rigorously rinsed with distilled water following each polish. Before surface modification, the bare electrode was scanned in 0.1M MgCl2 in 10 mM Tris-HCl buffer ( pH 7.2) between -0.3 and 0.8 V until a reproducible cyclic voltammogram (CV) was obtained. Electropolymerization of PPy was performed by CV, involved the immersion of an Au disk electrode into a solution of 0.1M pyrrole containing the doping electrolyte 0.1M MgCl2. A cyclic potential from -0.3 to 0.80V (versus Ag/AgCl) for 20 cycles at a scan rate of 50mVs−1 was applied. The Au electrode coated with PPy film was then washed with distilled water and dried under nitrogen gas. Prepared PPy-coated electrode was analysed by impedance spectroscopy. All impedance measurements were performed in an analysis buffer consisting of 0.1M MgCl2 and 10mM Tris-HCl buffer, with a pH of 7.2. An ac amplitude of 5mV was used and the data were collected in the frequency range l0 KHz -100 mHz taking six points per decade. The Nyquist Plot for the measured complex impedance (Z) versus frequency was recorded. D. Immobilization of capture DNA Prior to immobilization, the PPy coated electrode was transferred to a 20mM MgCl2 and 10mM Tris–HCl buffer (pH 7.2) for electrochemical oxidation of the PPy layer at 0.5V (versus Ag/AgCl) for 600s. After that, 1µg/ µl of the ss-caputure probe were physisadsorbed onto the PPy film on the Au electrode. The ss-DNA immobilized surface was washed with distilled water to remove unadsorbed DNA and dried under nitrogen gas. The ss-DNA/PPy film was later analysed for impedance measurements. E. Hybridization of the target DNA 1µg/ µl of the ss- target probe were physisadsorbed onto the capure probe immobilized PPy film. The hybridized Au/PPy/DNA film was washed with distilled water to remove unadsorbed DNA and dried under nitrogen gas and analysed for impedance measurements. III. RESULTS AND DISCUSSION A. Polymerization of Pyrrole During an electropolymerization of pyrrole, the forming polymer backbone was charged positively. This positive charge is compensated by anions from the electrolyte solution via an incorporation of these anions into the
polymer backbone. The electrochemical polymerization can be presented by the following general scheme: n(PY) +nyA-
→ [(PY)y+A-y]n + n(2+y)e-
components. The complex impedance is the sum of Z’ and Z”.
(1)
where n is the degree of polymerization, y is the charge of a polymer unit (or the doping level) [16], and A- is the dopant ion (MgCl2). Therefore, from equation (1) it is clear that for one monomer unit formation (2+y) electrons are required. The impedance spectra of the conducting polymer were used to evaluate film conductivity and charge the transport at the PPy film/electrolyte interface. Fig. 1 shows the Nyquist diagram for the PPy films prepared by CV. As seen in Fig. 1, the impedance spectrum of the modified electrode has two charge-transfer steps. The first semicircle represents the ohmic resistance of the electrolyte solution at the electrolyte–PPy interface (Rs). The second semicircle represents the charge transfer resistance at the PPy– electrode interface (Rct). The frequency-dependent semicircle impedance curves were observed at the high frequency range followed by a straight line.
Figure 2. Nyquist plot of (a) the PPy film, (b) PPy/ss-DNA capture immobilized surface, (c) After the hybridization with complementary target DNA.
Fig.3 shows the Randle equivalent circuit which is generally used to model the complex impedance of the electrochemical reactions. It consists of the ohmic resistance of the electrolyte solution, Rs in connection in series with parallel elements of double layer capacitance, Cdl, of the polymer/electrolyte interphase, and Faraday impedance, Zf.
Figure 1. Nyquist plot of the PPy film synthesized by CV
B. Immobilization and hybridization of the DNA Fig.2 shows the ac impedances were measured for the Au/PPy/DNA film before and after the target DNA hybridization and the corresponding Nyquist Plot. As seen from fig.2, the diameter of the semicircle increased after the capture DNA probe was immobilized on the PPy film and a further increase in diameter of the semicircle was observed after hybridization. The increase in impedance is due to presence of the DNA probe at the PPY surface, which blocks the passage of chloride ions at the PPY/solution interface. The results indicate that the addition of negative charge to the surface of the electrode, in the form of complementary oligonucleotide, further blocks the chloride ion exchange which, yield to the increase in impedance after hybridization. In the impedance spectra, Z’ is the real component mainly from resistance and Z” is the imaginary components from capacitance, inductance and other distribution
Figure 3. The Randle equivalent circuit model
The semicircle is formed by the parallel elements Cdl and Zf versus frequencies. Zf often comprises serially connected charge-transfer resistance, Rct, and Warburg impedance, Zw, was originally developed by Warburg in 1899 to describe a finite diffusion process resulting from the diffusion of ions from the electrolyte to the surface of the electrode. The results prove that the electronic properties of the PPy film was changed after the immobilization and the hybridization event, Since Cdl and Rct depend on the dielectric and electrocatalytic properties at the electrode/electrolyte interface, and electrochemical reactions occurred at the Au/PPy/DNA surface. Therefore, the change in impedance can be used to detect the DNA hybridization for the DNA biosensor application. C. Specificity and reproducibility of the system Fig.4 shows the hybridization event with non complementary. The specificity of this DNA protocol was investigated by varying the target DNA sequence. A non
complementary sequence was used to hybridize with the immobilized capture DNA probe and no unspecific bonding with the immobilized probe was observed. The non complementary target DNA had only a negligible effect on the charge transfer resistance.
ENEF500. The presented research work is funded by SFISTTF award. REFERENCES [1]
The performance of the DNA sensor proved to be effective in terms of selectivity, and reproducibility of hybridization events.
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Figure 4. Nyquist plot of (a) the PPy film, (b) PPy/ss-DNA capture immobilized surface, (c) After the hybridization with non complementary DNA.
IV. CONCLUSION The novel use of conducting polymer for sensor fabrication has allowed the development of a DNA biosensor. The application of this paper is focused on the food quality monitoring for the rapid detection of foodborne pathogens. The hybridization event was clearly distinguishable from complementary and non complementary sequences using impedance spectroscopy. Electrochemical DNA detection is more beneficial due to its rapid performance and cost effective and simple procedure. We have shown that, the impedimetric detection of labelless DNA is possible and it proved to be effective. The sensor surface showed high specificity since no unspecific bonding was observed. The non complementary target DNA had only a negligible effect on the charge transfer resistance. Moreover, the total duration for impedimetric DNA detection was significantly reduced which was less than 5 minutes and the overall duration to perform this assay took lees than 30 minutes.
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ACKNOWLEDGEMENT This project is funded by Science Foundation Ireland (SFI) Research Frontiers Programme, ID no: 07RPF-
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