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Efficient separation and quantitative detection of Listeria monocytogenes based on screen-printed interdigitated electrode, urease and magnetic nanoparticles
Food Control xxx (2016) 1e7
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Efficient separation and quantitative detection of Listeria monocytogenes based on screen-printed interdigitated electrode, urease and magnetic nanoparticles Dan Wang a, Qi Chen a, Huiling Huo b, Shanshan Bai a, Gaozhe Cai a, Weihua Lai c, Jianhan Lin d, * a MOA Key Laboratory of Agricultural Information Acquisition Technology (Beijing), China Agricultural University, 17 East Qinghua Road, Beijing, 100083 China b Hebei Province Institute of Veterinary Drug Control, 19 Yangtze River Avenue, Shijiazhuang, 050035 China c State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047 China d Modern Precision Agriculture System Integration Research Key Laboratory of Ministry of Education, China Agricultural University, 17 East Qinghua Road, Beijing, 100083 China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2016 Received in revised form 1 September 2016 Accepted 2 September 2016 Available online xxx
Rapid screening of pathogenic bacteria contaminated foods is the key to prevent and control the outbreaks of foodborne illness. In this study, an impedance biosensor was developed using immunomagnetic nanoparticles for efficient separation and concentration of the Listeria monocytogenes cells, urease for amplifying the weak signal, and screen-printed interdigitated electrode for quantitative measurement of the impedance change of the catalysate. The magnetic nanoparticles (MNPs) coated by the monoclonal antibodies (MAbs) were used to separate the Listeria cells from the background and concentrate them in small volume of PBS. Then, the gold nanoparticles (GNPs) modified with the urease and the polyclonal antibodies (PAbs) were used to react with Listeria to form the MNP-Listeria-GNP sandwich complexes. The complexes were re-suspended with the urea to catalyze the hydrolysis of the urea into ammonium ions and carbonate ions, which were measured by the electrode. A new equivalent circuit was designed for simulation of the biosensor with a good fitting result. Under the optimized conditions, a linear relationship between the impedance changes and the concentrations of Listeria from 1.9 103 to 1.9 106 CFU/mL was obtained. The limit of detection of this biosensor was 1.6 103 CFU/mL and the recovery of the spiked lettuce sample ranges from 94.7% to 103.8%. This proposed biosensor was developed at much lower cost than our previous studies and could be more applicable for in-field detection of foodborne pathogens. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Impedance biosensor Screen-printed interdigitated electrode Immuno-magnetic nanoparticles Urease Listeria monocytogenes
1. Introduction In the recent two decades, foodborne diseases caused by pathogens have become a global public health issue due to their significantly increasing occurrences (Law, Ab Mutalib, Chan, & Lee, 2014). It was reported by WHO that an estimated 600 million, i.e., almost 1 in 10 people in the world, got ill after eating contaminated food and 420,000 people died every year, resulting in a loss of 33 million healthy life years. Bacteria are the most common foodborne pathogens, accounting for 91% of the total outbreaks of foodborne
* Corresponding author. E-mail address: [email protected] (J. Lin).
illness in the US (Yang & Bashir, 2008). According to the statistics of National Health and Family Planning Commission of China, bacteria were responsible for over 50% of the total number of food poisoning in the past decade. Listeria monocytogenes is one of the most common and dangerous foodborne pathogenic bacteria, which exists in many kinds of food, such as meat, dairy products, aquatic products, vegetables, and so on. Infection of Listeria can lead to gastroenteritis, septicemia, meningitis, abortion or other symptoms, and the mortality rate of people at high risk is 30% (Rocourt, Jacquet, & Reilly, 2000). Many countries conduct “zero-tolerance” policy for the presence of Listeria monocytogenes in ready-to-eat foods (Kanayeva et al., 2012). The current methods for detecting foodborne pathogenic
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Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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bacteria mainly include the culture method based on the physical and chemical differences of bacteria (biochemical reaction, selective medium, etc.) (Zhao, Lin, Wang, & Oh, 2014), the PCR methods based on nucleic acid amplification (conventional PCR, quantitative PCR, LAMP, etc.) (Mori, Kanda, & Notomi, 2013; Xu et al., 2016), and the immunological methods based on the reaction of antigen and antibody (ELISA, immune-chromatographic test strip, etc.) (Tu et al., 2016). Culture is the gold standard method with the highest sensitivity and accuracy, but it is time-consuming (2e4 d) and laborious due to multiple steps of separation, cultivation and identification of the bacteria (Xu, Wang, & Li, 2016). The PCR methods are fast (3e6 h) and sensitive, but they require complicated nucleic acid extraction procedures and expensive equipment. The immunological methods are fast (2e4 h) and relative easier, but their detection sensitivity is often not high. For instance, the detection limit of ELISA ranges from 103 to 105 CFU/mL (Kanayeva et al., 2012), and to obtain a detectable signal, it requires 16e24 h to enrich the bacteria (Xu et al., 2016). Therefore, it is very important to develop sensitive and low-cost methods for rapid screening of the bacteria in contaminated foods. As an alternative, various biosensors have drawn increasing attention due to their simplicity, short time and high sensitivity in the detection of foodborne pathogenic bacteria. Impedance biosensor is a type of electrochemical biosensor, relying on the measurement of the electrochemical impedance change on the interface of an electrode under an alternating perturbation with a constant bias, and has shown the advantages over the conventional biodetection methods, such as compact design, rapid detection, relatively low cost and ease to integration (Daniels & Pourmand, 2007; Pejcic, De Marco, & Parkinson, 2006). Interdigitated array microelectrodes are often used in the development of impedance biosensors and have demonstrated for sensitive detection of bacteria (Etayash, Jiang, Thundat, & Kaur, 2014; Liu, Settu, Tsai, & Chen, 2015; Pal, Sharma, & Gupta, 2016). The commonly used strategy of an impedance biosensor is to immobilize the specific antibodies onto the surface of the electrode for capturing the target bacteria, thus resulting in an increase in the impedance of the electrode measured by an impedance meter or electrochemical workstation (Lum et al., 2012; Wang et al., 2009). However, this strategy has three main drawbacks: (1) The immunoreaction between the target and the antibodies is a solid-liquid reaction, so the capture efficiency is often as low as 35% (Wang et al., 2015); (2) The procedure
of immobilizing the antibodies onto the electrode is complex, so it requires well-trained technicians; (3) The electrode is often difficult to clean thoroughly, so it can hardly be reused. These greatly limit the practical applications of the impedance biosensors. Therefore, simple and low-cost impedance biosensors should be promising for bacterial detection in the food industry. In this study, a low-cost and simple impedance biosensor combining immune magnetic separation, urease catalysis with electrochemical impedance analysis was developed based on our previous research (Chen et al., 2015). As shown in Fig. 1, the streptavidin modified magnetic nanoparticles (MNPs) were conjugated with the biotinylated monoclonal antibodies (MAbs) against Listeria, and then used for the specific separation of the Listeria cells in the sample, resulting in the forming of the MNPListeria complexes. The complexes were then incubated with the gold nanoparticles (GNPs), which were modified with the urease and the polyclonal antibodies (PAbs) against Listeria, to form the MNP-ListeriaeGNP-Urease sandwich complexes. The urease in the complexes were used to catalyze the hydrolysis of the urea into carbonate ions and ammonium ions, thus resulting in an increase in the ion strength of the solution and a decrease in its impedance. The impedance was measured using a low-cost screen-printed interdigitated electrode (SPIE) instead of the expensive interdigitated array microelectrodes in our previous studies for quantitative determination of the amount of the Listeria cells in the sample. 2. Materials and methods 2.1. Materials and reagents Phosphate buffer saline (PBS, 10 mmol L1, pH 7.4), gold chloride acid (HAuCl4$3H2O), sodium citrate, streptavidin, urease (E.C.3.5.1.5, Type III, 15,000e50,000 units/g solid), urea (NH2CONH2), ammonium carbonate ((NH4)2CO3), and 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC$HCl) were purchased from Sigma-Aldrich (St. Louis, MO). The biotinylated monoclonal antibodies and the polyclonal antibodies against Listeria monocytogenes were developed by Nanchang University and obtained from Zodolabs Biotech (Nanchang, China) for specific binding with the Listeria cells. Bovine serum albumin (BSA) from EM Science (Gibbstown, NJ) was prepared in PBS for blocking. Tween-20 from Amresco (Solon, OH) was prepared in PBS for lubricating. The magnetic nanoparticles
Fig. 1. The principle of the impedance biosensor for rapid detection of foodborne pathogenic bacteria.
Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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were purchased from Allrun Nano (PM3-020, Shanghai, China) and modified with the monoclonal antibodies for immunomagnetic separation of Listeria. The deionized water was produced by Millipore Advantage 10 (18.2 MU cm, Billerica, MA) for preparation of all the solutions. 2.2. Fabrication of the screen-printed interdigitated electrode The screen-printed interdigitated electrode was designed as shown in Fig. 2, and fabricated by DropSens (Asturias, Spain). The electrode is consisted of three pairs of concentric circle-shaped interdigitated gold fingers on a ceramic substrate with the length of 34 mm, the width of 10 mm and the height of 1 mm. The width of each finger and the gap between two adjacent fingers are 200 mm. The outer diameter of the electrode is 5.4 mm and the effective working area is about 12 mm2. The holder for housing the electrode was fabricated using a 3D printer (Objet 24, Stratasys, Eden Prairie, MN) with an inverted truncated cone-shaped well for holding the aqueous solution and a USB port for connecting with the impedance analyzer to measure its impedance. The depth, the base circle's diameter, the top circle's diameter of the well are 6 mm, 7 mm and 18 mm, respectively, and the volume of the well is 783 mL. 2.3. Bacteria culturing and counting Prior to culture, Liseria monocytogenes (ATCC13932) was frozen stored at 20 C with 20% glycerol, and revived by streaking on Luria-Bertani (LB) agar plates. After a colony was cultured in LB medium (Aoboxing Biotech, Beijing, China) at 37 C for 12e16 h with shaking at 180 rpm, the bacteria culture generally had the concentration of ~109 CFU/mL and were serially diluted with PBST (PBS with 0.05% Tween20) to ~103 CFU/mL. For bacteria counting, the bacteria samples were serially diluted with sterile PBS to ~103 CFU/mL and triplicate diluents (100 mL) were surface plated on the LB agar plates. After incubating at 37 C for 22e24 h, the bacterial colonies were counted to calculate the concentration of bacteria culture. 2.4. Preparation of immuno-MNPs and immuno-GNPs The anti-Listeria monoclonal antibodies were conjugated onto the surface of the MNPs based on streptavidin-biotin binding. 1.5 mg of the carboxyl monodisperse MNPs with the diameter of 180 nm and the Fe content of 10 mg/mL were covalently bound with 100 mg of streptavidin by the activation reaction with EDC, which is the same as previously reported protocol (Mao et al.,
3
2016). Then, the streptavidin-coated MNPs were mixed with 100 mg of biotinylated MAbs and incubated for 45 min at 15 rpm. After washing with PBS twice to remove the surplus MAbs, the MAbs modified MNPs were finally suspended in PBS containing 1% (w/v) BSA and 0.02% (w/v) NaN3 at a final concentration of 1.5 mg/ mL (Fe content) or 100 mg/mL (monoclonal antibodies). The immuno-MNPs were stored at 4 C and magnetically separated to remove the storing solution prior to use. The synthesis of GNPs and the modification of the polyclonal antibodies and the urease onto the GNPs had been reported in our previous paper (Chen et al., 2015). Briefly, the GNPs were obtained by adding 0.1% sodium citrate into boiling 0.01% HAuCl4$3H2O and heating for 10 min. Then, the PAbs (3.6 mg/mL, prepared by DI water) and the urease (10 mg/mL, in 5 mM MES, prepared by DI water) were successively added into the GNPs with the incubation time of 5 min and 1 h, respectively. Finally, the GNPs were blocked by 1% (w/v) PEG 20,000 for 30 min and 10% (w/v) BSA for 30 min, respectively, and centrifuged at 10,000 rpm/min for 15 min to remove the surplus PAbs and urease and dissolved in the DI water. The immune-GNPs were stored at 4 C and ready for use.
2.5. Bacteria separation and detection The Listeria cells were first conjugated with the immune-MNPs for separation from the sample (pure culture or spiked lettuce sample), then conjugated with the immune-GNPs for the form of MNP-Listeria-GNP complexes, and finally detected using the SPIE to measure the impedance of the catalysate of urea catalyzed by urease. First, 50 mL of the immuno-MNPs was incubated with 450 mL of the sample containing Listeria at different concentrations (PBS as negative control sample) at 15 rpm for 45 min to form the MNP-Listeria complexes (magnetic Listeria) and magnetically separated for 2 min using a super strong magnetic separator with a maximum strength of 1.65 T and a mean gradient of 90 T/m, which has been reported in our previous study (Lin, Li, Li, & Chen, 2015). After the magnetic Listeria cells were re-suspended with 190 mL of the PBST, they were incubated with 10 mL of the immuno-GNPs at 15 rpm for 30 min to form the MNP-Listeria-GNP complexes (urease-Listeria). The urease-Listeria cells were magnetically separated and washed with 200 mL of the deionized water containing 0.05% Tween-20 one time and the deionized water three times, respectively, to remove the unbound GNPs and conductive ions. Finally, the urease-Listeria cells were re-suspended and incubated with 300 mL of the urea at the concentration of 100 mM at 15 rpm, and magnetically captured against the wall of the tube to pipette 100 mL of the catalysate for impedance measurement.
Fig. 2. (a) The screen-printed interdigitated electrode and its 3D printed holder with USB port; (b) the structure diagram of the electrode (in mm).
Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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2.6. Impedance measurement and analysis The electrochemical impedance spectra (EIS) of the catalysate were measured using the impedance analyzer (E4980A, Agilent, Santa Clara, CA) and the screen-printed interdigitated electrode. For all the impedance measurements, a sinusoidal alternating potential with an amplitude of 5 mV, a direct-current bias of 0 V and a frequency range of 100 Hze1 MHz was applied on the SPIE. The impedance changes were calculated at the characteristic frequency of 7 kHz. Besides, the bode plots and an equivalent circuit were used to interpret the EIS data for better understanding. 3. Results and discussions 3.1. Optimization of the enzymatic catalysis time The enzymatic catalysis time for the urease to catalyze the hydrolysis of the urea into the catalysate (carbonate ion and ammonium ion) (Vial, Prevot, & Forano, 2006) is a key factor to the sensitivity of this proposed biosensor since it has a great impact on the amount of the catalysate. Different enzymatic catalysis times ranging from 5 min to 60 min were applied for the incubation of the urease-Listeria cells at the concentration of 106 CFU/mL with the urea at the fixed concentration of 100 mM and the impedance of the catalysate was measured using the SPIE. As shown in Fig. 3, the impedance decreases since more non-conductive urea is catalyzed into the conductive ammonium ions and carbonate ions, while the enzymatic catalysis time increases. The increasing rate of the catalysate, i.e., the decreasing rate of the impedance, in the period of 5e30 min is obviously faster than that in the period of 30e60 min. Thus, the enzymatic catalysis time was optimized to be 30 min after trading off the time and the sensitivity. 3.2. Comparison of the sensitivity of the SPIE and the microelectrode The sensitivity of the impedance measurement of the catalysate is another key factor to the sensitivity of the proposed biosensor. In our previous study, an interdigitated array microelectrode (IDAM) with the finger width and space of 15 mm was used for the impedance measurement, but it was fabricated using photolithography and wet etching at a much higher cost than the proposed SPIE. The sensitivity of these two electrode for the impedance measurement were compared using different concentrations (1e100 mM) of ammonium carbonate (the simulation of ammonium ions and carbonate ions). As shown in Fig. 4, both electrodes
Fig. 3. The impedance measured at the characteristic frequency of 7 kHz for different enzymatic catalysis time ranging from 5 min to 60 min in the detection of 106 CFU/mL of Listeria monocytogenes.
Fig. 4. Comparison of the sensitivity of the proposed screen-printed interdigitated electrode and the previously-used interdigitated array microelectrode for the impedance measurement of ammonium carbonate.
have shown a good linear relationship between the impedance of the electrodes and the concentration of ammonium carbonate, and the slope of the linear fitted curve for the IDAM is a smaller than that of the SPIE, indicating that the IDAM is less sensitive than SPIE. Besides, the cost of the IDAM is around 10 times more than that of the SPIE. However, the relative errors of the IDAM are obviously less than those of the SPIE, indicating the stability of the IDAM is better than that of the SPIE. Considering the sensitivity, the cost and the stability, the SPIE is probably more suitable for practical applications of bacteria contaminated food screening. 3.3. Characterization of electrochemical impedance spectra The electrochemical impedance spectra of the proposed impedance biosensor were collected for determining the characteristic frequency, building the calibration model between the impedance measured at the characteristic frequency and the concentration of the catalysate, and analyzing the equivalent circuit for better understanding the electrochemical responses. Fig. 5 (a) shows the impedance of the proposed biosensor for the detection of Listeria at the concentrations ranging from 103 CFU/mL to 106 CFU/mL and negative control. Comparing to the impedance spectrum of the negative control, the impedance spectra of the Listeria sample at a higher concentration have a larger change. The maximum impedance changes of each sample occurs almost at the same frequency range of 200 Hz to 20 kHz, and the impedances measured at the low frequency range from 200 Hz to 2 kHz has a relative deviation of 5.5% and is larger than those at the middle frequency range from 2 kHz to 20 kHz with a deviation of 3.2%. Thus, the characteristic frequency of the biosensor was selected as 7 kHz. Fig. 5 (b) shows the phase angle of the proposed biosensor for the detection of Listeria at the concentrations ranging from 103 CFU/ mL to 106 CFU/mL and negative control. At the low frequencies (100 Hze5 kHz), the phase angles range from 0 to 10 , indicating that the biosensor is like a resistor; at the middle frequencies (5 kHze500 kHz), the phase angles decreases from 10 to 90 ; and at the high frequencies (500 kHze1 MHz), the phase angles are close to 90 , indicating that the biosensor is like a capacitor. Besides, it can also been seen that the phase angle shifts towards the high frequency direction, while the concentration of Listeria increases. To interpret the electrochemical impedance spectra, a new equivalent circuit was used to simulate the biosensor. As shown in Fig. 5c, the equivalent circuit is consisted of two double layer capacitors (Cdl), two electron transfer resistors (Ret), and a solution
Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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(Cs) was used to represent the impedance of the solution since the resistance of the solution is much larger due to the long distance of electron transfer between the fingers. Cdl stands for the effect of the ions on the capacitance at the electrode/solution interface; Ret stands for the resistance of the electron transfer from the interface to the solution; and Cs stands for the dielectric characteristics of the solution. To verify the fitting of the equivalent circuit to the biosensor, the measured impedance data (magnitude and phase angle) obtained from the impedance spectrum for the detection of Listeria at the concentration of 104 CFU/mL and the fitted impedance data calculated by equation (1) are compared in Fig. 5d.
Z ¼ Cdl ==Ret þ Cdl þ Cdl ==Ret ¼ 2Ret =ð1 þ juRet Cdl Þ þ 1=ðjuCs Þ (1) where, u is the angular frequency. The mean relative errors between the measured data and the calculated data are 2.12% for impedance and 2.62% for phase angle, respectively, indicating that the equivalent circuit has a good fitting to the proposed biosensor. To further understand the change of the equivalent circuit at the detection of Listeria at different concentrations, the impedance simulation software, ZMAN, was used to calculate the values of the elements in the equivalent circuit and shown in Table 1. When the concentration of Listeria increases, Cdl remains almost unchanged or less changes; Ret has a significant decrease from 96.1 kU to 52.8 kU; and Cs also has an obvious increase from 35 nF to 87 nF. The increase of Cs from 35 nF to 87 nF can only cause an impedance decrease of 389 U at the characteristic frequency of 7 kHz based on the calculation of 1/(juCs), however, the decrease of Ret from 96.1 kU to 52.8 kU can cause a much larger impedance decrease of 28.2 kU based on the calculation of 2Ret/(1þjuRetCdl). Thus, the impedance changes of the biosensor for different concentrations of Listeria were mainly attributed to Ret, resulting from the ionic strength change of the catalysate. 3.4. Detection of Listeria monocytogenes in pure culture and spiked lettuce sample The separation efficiency of the Listeria cells have been demonstrated in our previously report to be over 95% for the pure cultures and over 85% for the spiked lettuce samples, respectively (Chen et al., 2015). In this study, different concentrations of Listeria monocytogenes were serially diluted with PBS and detected using this proposed biosensor. TEM imaging was used to check the forming of the MNP-Listeria-GNP complexes (see Fig. 6a). As shown in Fig. 6b, a good linear relationship between the impedance change (DZ) measured at the characteristic frequency of 7 kHz and the concentration (C) of Listeria from 1.9 103 to 1.9 106 CFU/mL are obtained and can be expressed by DZ ¼ 11631*log (C) e 82,274 (R2 ¼ 0.97). DZ is calculated by subtracting the impedance of the bacterial sample from that of the negative control. To evaluate the applicability of this proposed biosensor for the detection of Listeria in real samples, Listeria at different concentrations of 2.0 103 to 2.0 106 CFU/mL were prepared and added into the Listeria-free Fig. 5. (a) The electrochemical impedance spectra for the detection of Listeria at the concentrations from 103 to 106 CFU/mL and negative sample; (b) The phase angle spectra for the detection of Listeria at the concentrations from 103 to 106 CFU/mL and negative sample; (c) The equivalent circuit for simulation of the proposed biosensor; (d) Comparison of the measured impedance and phase angle data with the calculated ones.
capacitor (Cs). In the interface of the finger and the solution, the double layer capacitor (Cdl) in parallel with the electron transfer resistor (Ret) was used to represent the impedance of the interface, and in between the two adjacent fingers, only a solution capacitor
Table 1 Simulated value of the elements in equivalent circuit for impedance measurement of the control and the samples at different concentrations. Listeria Conc.(CFU/ml)
Cdl(pF)
Control 1.9 103 1.9 104 1.9 105 1.9 106
50.0 52.4 55.2 52.2 52.4
± ± ± ± ±
Ret(kU) 3.3 4.6 2.3 3.5 4.4
95.4 96.1 83.7 64.9 52.8
± ± ± ± ±
Cs(nF) 2.5 3.4 1.4 1.7 1.7
35.1 35.4 52.2 64.8 87.7
± ± ± ± ±
4.0 5.5 4.9 6.9 1.8
Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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tests on the mixture of Listeria and E. coli O157:H7 at the concentration of 104 CFU/mL, only E. coli O157:H7 at the concentration of 104 CFU/mL, and only Listeria at the concentration of 104 CFU/mL were conducted, and the results showed that the impedance change of the mixture of Listeria and E. coli O157:H7 (29.1 kU) was close to that of only Listeria (28.9 kU) and the impedance change of only E. coli O157:H7 (2.5 kU) was close to that of the negative control (1.3 kU), indicated that this proposed biosensor had a good specificity. This might be attributed to the following reasons: (1) most of the E. coli O157:H7 cells in the mixture were not captured and separated by the MNPs and only few of them (<3%) (Chen et al., 2015) were non-specifically bound onto the MNPs; (2) The few E. coli cells did not react with the GNPs modified by the PAbs against Listeria, and no or trace urease were captured for catalyzing the hydrolysis of urea, resulting in very small change in the impedance of the SPIE. 4. Conclusion In this study, we have developed and demonstrated an efficient separation and quantitative detection method for rapid detection of Listeria using the screen-printed interdigitated electrode, the urease and the polyclonal antibody modified gold nanoparticles, and the monoclonal antibody coated magnetic nanoparticles. This proposed impedance biosensor has a good linear relationship between the impedance and the concentration from 103 to 106 CFU/ mL and can detect Listeria as low as 1.6 103 CFU/mL within 3 h. The recovery of the spiked lettuce samples ranges from 94.7% to 103.8%. The use of the low-cost screen-printed electrode makes it more promising in in-field screening. Acknowledgments Fig. 6. (a) The TEM image of a Listeria monocytogenes cell conjugated with the monoclonal antibodies coated magnetic nanoparticles and the polyclonal antibodies and the urease modified gold nanoparticles; (2) The linear relationship between the impedance change measured at the characteristic frequency of 7 kHz and the concentration of Listeria (N ¼ 3).
lettuce samples, followed by three parallel tests of the spiked lettuce samples using the proposed biosensor. The mean relative error for the parallel detection is 10.16%, indicating that the biosensor has a good repeatability. The recoveries for the Listeria with the concentrations of 2.0 103 CFU/mL, 2.0 104 CFU/mL, 2.0 105 CFU/ mL, and 2.0 106 CFU/mL were 94.7%, 103.8%, 100.6%, 93.7%, respectively, and the mean recovery was 98.2%, indicating the feasibility of the proposed biosensor for practical applications of foodborne bacteria detection. To obtain the detection limit of this proposed biosensor, ten negative control samples were tested using this biosensor with an average impedance value of 186.4 ± 1.3 kU. The detection limit was determined by three times of signal to noise ratio and was calculated as 1.6 103 CFU/mL. Also, this proposed biosensor was compared with some recently-reported Listeria detection methods and shown in Table S1 in the supplemental material. It can be seen that the LOD for this work is close to or lower than the previously reported methods and the detection time for this work is less than these reported methods. The specificity of this proposed biosensor depends on the monoclonal antibodies and the polyclonal antibodies against Listeria. Since these anti-Listeria antibodies have been tested with good specificity to more than 15 common foodborne pathogenic bacteria by Mao and Huang (Huang et al., 2015; Mao et al., 2016), only E. coli O157:H7 was used in this study as the non-target bacteria to test the specificity of this proposed biosensor. Three parallel
This study was supported in part by National Key R&D Program of China (No. 2016YFD0500706), and Science and Technology Support Plan of Hebei Province (No. 16275508D). The authors would like to thank Aibit Biotech LLC for providing the screenprinted interdigitated electrodes. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodcont.2016.09.003. References Chen, Q., Lin, J., Gan, C., Wang, Y., Wang, D., Xiong, Y., et al. (2015). A sensitive impedance biosensor based on immunomagnetic separation and urease catalysis for rapid detection of Listeria monocytogenes using an immobilization-free interdigitated array microelectrode. Biosensors and Bioelectronics, 74, 504e511. Daniels, J. S., & Pourmand, N. (2007). Label-free impedance biosensors: Opportunities and challenges. Electroanalysis, 19(12), 1239e1257. Etayash, H., Jiang, K., Thundat, T., & Kaur, K. (2014). Impedimetric detection of pathogenic gram-positive bacteria using an antimicrobial peptide from class IIa bacteriocins. Analytical Chemistry, 86(3), 1693e1700. Huang, X. L., Xu, Z. D., Mao, Y., Ji, Y. W., Xu, H. Y., Xiong, Y. H., et al. (2015). Gold nanoparticle-based dynamic light scattering immunoassay for ultrasensitive detection of Listeria monocytogenes in lettuces. Biosensors and Bioelectronics, 66, 184e190. Kanayeva, D. A., Wang, R., Rhoads, D., Erf, G. F., Slavik, M. F., Tung, S., et al. (2012). Efficient separation and sensitive detection of Listeria monocytogenes using an impedance immunosensor based on magnetic nanoparticles, a microfluidic chip, and an interdigitated microelectrode. Journal of Food Protection, 75(11), 1951e1959. Law, J. W., Ab Mutalib, N. S., Chan, K. G., & Lee, L. H. (2014). Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Frontiers in Microbiology, 5, 770. Lin, J. H., Li, M., Li, Y. B., & Chen, Q. (2015). A high gradient and strength bioseparator with nano-sized immunomagnetic particles for specific separation and efficient concentration of E-coli O157:H7. Journal of Magnetism and Magnetic Materials,
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Efficient separation and quantitative detection of Listeria monocytogenes based on screen-printed interdigitated electrode, urease and magnetic nanoparticles Dan Wang a, Qi Chen a, Huiling Huo b, Shanshan Bai a, Gaozhe Cai a, Weihua Lai c, Jianhan Lin d, * a MOA Key Laboratory of Agricultural Information Acquisition Technology (Beijing), China Agricultural University, 17 East Qinghua Road, Beijing, 100083 China b Hebei Province Institute of Veterinary Drug Control, 19 Yangtze River Avenue, Shijiazhuang, 050035 China c State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047 China d Modern Precision Agriculture System Integration Research Key Laboratory of Ministry of Education, China Agricultural University, 17 East Qinghua Road, Beijing, 100083 China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2016 Received in revised form 1 September 2016 Accepted 2 September 2016 Available online xxx
Rapid screening of pathogenic bacteria contaminated foods is the key to prevent and control the outbreaks of foodborne illness. In this study, an impedance biosensor was developed using immunomagnetic nanoparticles for efficient separation and concentration of the Listeria monocytogenes cells, urease for amplifying the weak signal, and screen-printed interdigitated electrode for quantitative measurement of the impedance change of the catalysate. The magnetic nanoparticles (MNPs) coated by the monoclonal antibodies (MAbs) were used to separate the Listeria cells from the background and concentrate them in small volume of PBS. Then, the gold nanoparticles (GNPs) modified with the urease and the polyclonal antibodies (PAbs) were used to react with Listeria to form the MNP-Listeria-GNP sandwich complexes. The complexes were re-suspended with the urea to catalyze the hydrolysis of the urea into ammonium ions and carbonate ions, which were measured by the electrode. A new equivalent circuit was designed for simulation of the biosensor with a good fitting result. Under the optimized conditions, a linear relationship between the impedance changes and the concentrations of Listeria from 1.9 103 to 1.9 106 CFU/mL was obtained. The limit of detection of this biosensor was 1.6 103 CFU/mL and the recovery of the spiked lettuce sample ranges from 94.7% to 103.8%. This proposed biosensor was developed at much lower cost than our previous studies and could be more applicable for in-field detection of foodborne pathogens. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Impedance biosensor Screen-printed interdigitated electrode Immuno-magnetic nanoparticles Urease Listeria monocytogenes
1. Introduction In the recent two decades, foodborne diseases caused by pathogens have become a global public health issue due to their significantly increasing occurrences (Law, Ab Mutalib, Chan, & Lee, 2014). It was reported by WHO that an estimated 600 million, i.e., almost 1 in 10 people in the world, got ill after eating contaminated food and 420,000 people died every year, resulting in a loss of 33 million healthy life years. Bacteria are the most common foodborne pathogens, accounting for 91% of the total outbreaks of foodborne
* Corresponding author. E-mail address: [email protected] (J. Lin).
illness in the US (Yang & Bashir, 2008). According to the statistics of National Health and Family Planning Commission of China, bacteria were responsible for over 50% of the total number of food poisoning in the past decade. Listeria monocytogenes is one of the most common and dangerous foodborne pathogenic bacteria, which exists in many kinds of food, such as meat, dairy products, aquatic products, vegetables, and so on. Infection of Listeria can lead to gastroenteritis, septicemia, meningitis, abortion or other symptoms, and the mortality rate of people at high risk is 30% (Rocourt, Jacquet, & Reilly, 2000). Many countries conduct “zero-tolerance” policy for the presence of Listeria monocytogenes in ready-to-eat foods (Kanayeva et al., 2012). The current methods for detecting foodborne pathogenic
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Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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bacteria mainly include the culture method based on the physical and chemical differences of bacteria (biochemical reaction, selective medium, etc.) (Zhao, Lin, Wang, & Oh, 2014), the PCR methods based on nucleic acid amplification (conventional PCR, quantitative PCR, LAMP, etc.) (Mori, Kanda, & Notomi, 2013; Xu et al., 2016), and the immunological methods based on the reaction of antigen and antibody (ELISA, immune-chromatographic test strip, etc.) (Tu et al., 2016). Culture is the gold standard method with the highest sensitivity and accuracy, but it is time-consuming (2e4 d) and laborious due to multiple steps of separation, cultivation and identification of the bacteria (Xu, Wang, & Li, 2016). The PCR methods are fast (3e6 h) and sensitive, but they require complicated nucleic acid extraction procedures and expensive equipment. The immunological methods are fast (2e4 h) and relative easier, but their detection sensitivity is often not high. For instance, the detection limit of ELISA ranges from 103 to 105 CFU/mL (Kanayeva et al., 2012), and to obtain a detectable signal, it requires 16e24 h to enrich the bacteria (Xu et al., 2016). Therefore, it is very important to develop sensitive and low-cost methods for rapid screening of the bacteria in contaminated foods. As an alternative, various biosensors have drawn increasing attention due to their simplicity, short time and high sensitivity in the detection of foodborne pathogenic bacteria. Impedance biosensor is a type of electrochemical biosensor, relying on the measurement of the electrochemical impedance change on the interface of an electrode under an alternating perturbation with a constant bias, and has shown the advantages over the conventional biodetection methods, such as compact design, rapid detection, relatively low cost and ease to integration (Daniels & Pourmand, 2007; Pejcic, De Marco, & Parkinson, 2006). Interdigitated array microelectrodes are often used in the development of impedance biosensors and have demonstrated for sensitive detection of bacteria (Etayash, Jiang, Thundat, & Kaur, 2014; Liu, Settu, Tsai, & Chen, 2015; Pal, Sharma, & Gupta, 2016). The commonly used strategy of an impedance biosensor is to immobilize the specific antibodies onto the surface of the electrode for capturing the target bacteria, thus resulting in an increase in the impedance of the electrode measured by an impedance meter or electrochemical workstation (Lum et al., 2012; Wang et al., 2009). However, this strategy has three main drawbacks: (1) The immunoreaction between the target and the antibodies is a solid-liquid reaction, so the capture efficiency is often as low as 35% (Wang et al., 2015); (2) The procedure
of immobilizing the antibodies onto the electrode is complex, so it requires well-trained technicians; (3) The electrode is often difficult to clean thoroughly, so it can hardly be reused. These greatly limit the practical applications of the impedance biosensors. Therefore, simple and low-cost impedance biosensors should be promising for bacterial detection in the food industry. In this study, a low-cost and simple impedance biosensor combining immune magnetic separation, urease catalysis with electrochemical impedance analysis was developed based on our previous research (Chen et al., 2015). As shown in Fig. 1, the streptavidin modified magnetic nanoparticles (MNPs) were conjugated with the biotinylated monoclonal antibodies (MAbs) against Listeria, and then used for the specific separation of the Listeria cells in the sample, resulting in the forming of the MNPListeria complexes. The complexes were then incubated with the gold nanoparticles (GNPs), which were modified with the urease and the polyclonal antibodies (PAbs) against Listeria, to form the MNP-ListeriaeGNP-Urease sandwich complexes. The urease in the complexes were used to catalyze the hydrolysis of the urea into carbonate ions and ammonium ions, thus resulting in an increase in the ion strength of the solution and a decrease in its impedance. The impedance was measured using a low-cost screen-printed interdigitated electrode (SPIE) instead of the expensive interdigitated array microelectrodes in our previous studies for quantitative determination of the amount of the Listeria cells in the sample. 2. Materials and methods 2.1. Materials and reagents Phosphate buffer saline (PBS, 10 mmol L1, pH 7.4), gold chloride acid (HAuCl4$3H2O), sodium citrate, streptavidin, urease (E.C.3.5.1.5, Type III, 15,000e50,000 units/g solid), urea (NH2CONH2), ammonium carbonate ((NH4)2CO3), and 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC$HCl) were purchased from Sigma-Aldrich (St. Louis, MO). The biotinylated monoclonal antibodies and the polyclonal antibodies against Listeria monocytogenes were developed by Nanchang University and obtained from Zodolabs Biotech (Nanchang, China) for specific binding with the Listeria cells. Bovine serum albumin (BSA) from EM Science (Gibbstown, NJ) was prepared in PBS for blocking. Tween-20 from Amresco (Solon, OH) was prepared in PBS for lubricating. The magnetic nanoparticles
Fig. 1. The principle of the impedance biosensor for rapid detection of foodborne pathogenic bacteria.
Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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were purchased from Allrun Nano (PM3-020, Shanghai, China) and modified with the monoclonal antibodies for immunomagnetic separation of Listeria. The deionized water was produced by Millipore Advantage 10 (18.2 MU cm, Billerica, MA) for preparation of all the solutions. 2.2. Fabrication of the screen-printed interdigitated electrode The screen-printed interdigitated electrode was designed as shown in Fig. 2, and fabricated by DropSens (Asturias, Spain). The electrode is consisted of three pairs of concentric circle-shaped interdigitated gold fingers on a ceramic substrate with the length of 34 mm, the width of 10 mm and the height of 1 mm. The width of each finger and the gap between two adjacent fingers are 200 mm. The outer diameter of the electrode is 5.4 mm and the effective working area is about 12 mm2. The holder for housing the electrode was fabricated using a 3D printer (Objet 24, Stratasys, Eden Prairie, MN) with an inverted truncated cone-shaped well for holding the aqueous solution and a USB port for connecting with the impedance analyzer to measure its impedance. The depth, the base circle's diameter, the top circle's diameter of the well are 6 mm, 7 mm and 18 mm, respectively, and the volume of the well is 783 mL. 2.3. Bacteria culturing and counting Prior to culture, Liseria monocytogenes (ATCC13932) was frozen stored at 20 C with 20% glycerol, and revived by streaking on Luria-Bertani (LB) agar plates. After a colony was cultured in LB medium (Aoboxing Biotech, Beijing, China) at 37 C for 12e16 h with shaking at 180 rpm, the bacteria culture generally had the concentration of ~109 CFU/mL and were serially diluted with PBST (PBS with 0.05% Tween20) to ~103 CFU/mL. For bacteria counting, the bacteria samples were serially diluted with sterile PBS to ~103 CFU/mL and triplicate diluents (100 mL) were surface plated on the LB agar plates. After incubating at 37 C for 22e24 h, the bacterial colonies were counted to calculate the concentration of bacteria culture. 2.4. Preparation of immuno-MNPs and immuno-GNPs The anti-Listeria monoclonal antibodies were conjugated onto the surface of the MNPs based on streptavidin-biotin binding. 1.5 mg of the carboxyl monodisperse MNPs with the diameter of 180 nm and the Fe content of 10 mg/mL were covalently bound with 100 mg of streptavidin by the activation reaction with EDC, which is the same as previously reported protocol (Mao et al.,
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2016). Then, the streptavidin-coated MNPs were mixed with 100 mg of biotinylated MAbs and incubated for 45 min at 15 rpm. After washing with PBS twice to remove the surplus MAbs, the MAbs modified MNPs were finally suspended in PBS containing 1% (w/v) BSA and 0.02% (w/v) NaN3 at a final concentration of 1.5 mg/ mL (Fe content) or 100 mg/mL (monoclonal antibodies). The immuno-MNPs were stored at 4 C and magnetically separated to remove the storing solution prior to use. The synthesis of GNPs and the modification of the polyclonal antibodies and the urease onto the GNPs had been reported in our previous paper (Chen et al., 2015). Briefly, the GNPs were obtained by adding 0.1% sodium citrate into boiling 0.01% HAuCl4$3H2O and heating for 10 min. Then, the PAbs (3.6 mg/mL, prepared by DI water) and the urease (10 mg/mL, in 5 mM MES, prepared by DI water) were successively added into the GNPs with the incubation time of 5 min and 1 h, respectively. Finally, the GNPs were blocked by 1% (w/v) PEG 20,000 for 30 min and 10% (w/v) BSA for 30 min, respectively, and centrifuged at 10,000 rpm/min for 15 min to remove the surplus PAbs and urease and dissolved in the DI water. The immune-GNPs were stored at 4 C and ready for use.
2.5. Bacteria separation and detection The Listeria cells were first conjugated with the immune-MNPs for separation from the sample (pure culture or spiked lettuce sample), then conjugated with the immune-GNPs for the form of MNP-Listeria-GNP complexes, and finally detected using the SPIE to measure the impedance of the catalysate of urea catalyzed by urease. First, 50 mL of the immuno-MNPs was incubated with 450 mL of the sample containing Listeria at different concentrations (PBS as negative control sample) at 15 rpm for 45 min to form the MNP-Listeria complexes (magnetic Listeria) and magnetically separated for 2 min using a super strong magnetic separator with a maximum strength of 1.65 T and a mean gradient of 90 T/m, which has been reported in our previous study (Lin, Li, Li, & Chen, 2015). After the magnetic Listeria cells were re-suspended with 190 mL of the PBST, they were incubated with 10 mL of the immuno-GNPs at 15 rpm for 30 min to form the MNP-Listeria-GNP complexes (urease-Listeria). The urease-Listeria cells were magnetically separated and washed with 200 mL of the deionized water containing 0.05% Tween-20 one time and the deionized water three times, respectively, to remove the unbound GNPs and conductive ions. Finally, the urease-Listeria cells were re-suspended and incubated with 300 mL of the urea at the concentration of 100 mM at 15 rpm, and magnetically captured against the wall of the tube to pipette 100 mL of the catalysate for impedance measurement.
Fig. 2. (a) The screen-printed interdigitated electrode and its 3D printed holder with USB port; (b) the structure diagram of the electrode (in mm).
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2.6. Impedance measurement and analysis The electrochemical impedance spectra (EIS) of the catalysate were measured using the impedance analyzer (E4980A, Agilent, Santa Clara, CA) and the screen-printed interdigitated electrode. For all the impedance measurements, a sinusoidal alternating potential with an amplitude of 5 mV, a direct-current bias of 0 V and a frequency range of 100 Hze1 MHz was applied on the SPIE. The impedance changes were calculated at the characteristic frequency of 7 kHz. Besides, the bode plots and an equivalent circuit were used to interpret the EIS data for better understanding. 3. Results and discussions 3.1. Optimization of the enzymatic catalysis time The enzymatic catalysis time for the urease to catalyze the hydrolysis of the urea into the catalysate (carbonate ion and ammonium ion) (Vial, Prevot, & Forano, 2006) is a key factor to the sensitivity of this proposed biosensor since it has a great impact on the amount of the catalysate. Different enzymatic catalysis times ranging from 5 min to 60 min were applied for the incubation of the urease-Listeria cells at the concentration of 106 CFU/mL with the urea at the fixed concentration of 100 mM and the impedance of the catalysate was measured using the SPIE. As shown in Fig. 3, the impedance decreases since more non-conductive urea is catalyzed into the conductive ammonium ions and carbonate ions, while the enzymatic catalysis time increases. The increasing rate of the catalysate, i.e., the decreasing rate of the impedance, in the period of 5e30 min is obviously faster than that in the period of 30e60 min. Thus, the enzymatic catalysis time was optimized to be 30 min after trading off the time and the sensitivity. 3.2. Comparison of the sensitivity of the SPIE and the microelectrode The sensitivity of the impedance measurement of the catalysate is another key factor to the sensitivity of the proposed biosensor. In our previous study, an interdigitated array microelectrode (IDAM) with the finger width and space of 15 mm was used for the impedance measurement, but it was fabricated using photolithography and wet etching at a much higher cost than the proposed SPIE. The sensitivity of these two electrode for the impedance measurement were compared using different concentrations (1e100 mM) of ammonium carbonate (the simulation of ammonium ions and carbonate ions). As shown in Fig. 4, both electrodes
Fig. 3. The impedance measured at the characteristic frequency of 7 kHz for different enzymatic catalysis time ranging from 5 min to 60 min in the detection of 106 CFU/mL of Listeria monocytogenes.
Fig. 4. Comparison of the sensitivity of the proposed screen-printed interdigitated electrode and the previously-used interdigitated array microelectrode for the impedance measurement of ammonium carbonate.
have shown a good linear relationship between the impedance of the electrodes and the concentration of ammonium carbonate, and the slope of the linear fitted curve for the IDAM is a smaller than that of the SPIE, indicating that the IDAM is less sensitive than SPIE. Besides, the cost of the IDAM is around 10 times more than that of the SPIE. However, the relative errors of the IDAM are obviously less than those of the SPIE, indicating the stability of the IDAM is better than that of the SPIE. Considering the sensitivity, the cost and the stability, the SPIE is probably more suitable for practical applications of bacteria contaminated food screening. 3.3. Characterization of electrochemical impedance spectra The electrochemical impedance spectra of the proposed impedance biosensor were collected for determining the characteristic frequency, building the calibration model between the impedance measured at the characteristic frequency and the concentration of the catalysate, and analyzing the equivalent circuit for better understanding the electrochemical responses. Fig. 5 (a) shows the impedance of the proposed biosensor for the detection of Listeria at the concentrations ranging from 103 CFU/mL to 106 CFU/mL and negative control. Comparing to the impedance spectrum of the negative control, the impedance spectra of the Listeria sample at a higher concentration have a larger change. The maximum impedance changes of each sample occurs almost at the same frequency range of 200 Hz to 20 kHz, and the impedances measured at the low frequency range from 200 Hz to 2 kHz has a relative deviation of 5.5% and is larger than those at the middle frequency range from 2 kHz to 20 kHz with a deviation of 3.2%. Thus, the characteristic frequency of the biosensor was selected as 7 kHz. Fig. 5 (b) shows the phase angle of the proposed biosensor for the detection of Listeria at the concentrations ranging from 103 CFU/ mL to 106 CFU/mL and negative control. At the low frequencies (100 Hze5 kHz), the phase angles range from 0 to 10 , indicating that the biosensor is like a resistor; at the middle frequencies (5 kHze500 kHz), the phase angles decreases from 10 to 90 ; and at the high frequencies (500 kHze1 MHz), the phase angles are close to 90 , indicating that the biosensor is like a capacitor. Besides, it can also been seen that the phase angle shifts towards the high frequency direction, while the concentration of Listeria increases. To interpret the electrochemical impedance spectra, a new equivalent circuit was used to simulate the biosensor. As shown in Fig. 5c, the equivalent circuit is consisted of two double layer capacitors (Cdl), two electron transfer resistors (Ret), and a solution
Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003
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(Cs) was used to represent the impedance of the solution since the resistance of the solution is much larger due to the long distance of electron transfer between the fingers. Cdl stands for the effect of the ions on the capacitance at the electrode/solution interface; Ret stands for the resistance of the electron transfer from the interface to the solution; and Cs stands for the dielectric characteristics of the solution. To verify the fitting of the equivalent circuit to the biosensor, the measured impedance data (magnitude and phase angle) obtained from the impedance spectrum for the detection of Listeria at the concentration of 104 CFU/mL and the fitted impedance data calculated by equation (1) are compared in Fig. 5d.
Z ¼ Cdl ==Ret þ Cdl þ Cdl ==Ret ¼ 2Ret =ð1 þ juRet Cdl Þ þ 1=ðjuCs Þ (1) where, u is the angular frequency. The mean relative errors between the measured data and the calculated data are 2.12% for impedance and 2.62% for phase angle, respectively, indicating that the equivalent circuit has a good fitting to the proposed biosensor. To further understand the change of the equivalent circuit at the detection of Listeria at different concentrations, the impedance simulation software, ZMAN, was used to calculate the values of the elements in the equivalent circuit and shown in Table 1. When the concentration of Listeria increases, Cdl remains almost unchanged or less changes; Ret has a significant decrease from 96.1 kU to 52.8 kU; and Cs also has an obvious increase from 35 nF to 87 nF. The increase of Cs from 35 nF to 87 nF can only cause an impedance decrease of 389 U at the characteristic frequency of 7 kHz based on the calculation of 1/(juCs), however, the decrease of Ret from 96.1 kU to 52.8 kU can cause a much larger impedance decrease of 28.2 kU based on the calculation of 2Ret/(1þjuRetCdl). Thus, the impedance changes of the biosensor for different concentrations of Listeria were mainly attributed to Ret, resulting from the ionic strength change of the catalysate. 3.4. Detection of Listeria monocytogenes in pure culture and spiked lettuce sample The separation efficiency of the Listeria cells have been demonstrated in our previously report to be over 95% for the pure cultures and over 85% for the spiked lettuce samples, respectively (Chen et al., 2015). In this study, different concentrations of Listeria monocytogenes were serially diluted with PBS and detected using this proposed biosensor. TEM imaging was used to check the forming of the MNP-Listeria-GNP complexes (see Fig. 6a). As shown in Fig. 6b, a good linear relationship between the impedance change (DZ) measured at the characteristic frequency of 7 kHz and the concentration (C) of Listeria from 1.9 103 to 1.9 106 CFU/mL are obtained and can be expressed by DZ ¼ 11631*log (C) e 82,274 (R2 ¼ 0.97). DZ is calculated by subtracting the impedance of the bacterial sample from that of the negative control. To evaluate the applicability of this proposed biosensor for the detection of Listeria in real samples, Listeria at different concentrations of 2.0 103 to 2.0 106 CFU/mL were prepared and added into the Listeria-free Fig. 5. (a) The electrochemical impedance spectra for the detection of Listeria at the concentrations from 103 to 106 CFU/mL and negative sample; (b) The phase angle spectra for the detection of Listeria at the concentrations from 103 to 106 CFU/mL and negative sample; (c) The equivalent circuit for simulation of the proposed biosensor; (d) Comparison of the measured impedance and phase angle data with the calculated ones.
capacitor (Cs). In the interface of the finger and the solution, the double layer capacitor (Cdl) in parallel with the electron transfer resistor (Ret) was used to represent the impedance of the interface, and in between the two adjacent fingers, only a solution capacitor
Table 1 Simulated value of the elements in equivalent circuit for impedance measurement of the control and the samples at different concentrations. Listeria Conc.(CFU/ml)
Cdl(pF)
Control 1.9 103 1.9 104 1.9 105 1.9 106
50.0 52.4 55.2 52.2 52.4
± ± ± ± ±
Ret(kU) 3.3 4.6 2.3 3.5 4.4
95.4 96.1 83.7 64.9 52.8
± ± ± ± ±
Cs(nF) 2.5 3.4 1.4 1.7 1.7
35.1 35.4 52.2 64.8 87.7
± ± ± ± ±
4.0 5.5 4.9 6.9 1.8
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tests on the mixture of Listeria and E. coli O157:H7 at the concentration of 104 CFU/mL, only E. coli O157:H7 at the concentration of 104 CFU/mL, and only Listeria at the concentration of 104 CFU/mL were conducted, and the results showed that the impedance change of the mixture of Listeria and E. coli O157:H7 (29.1 kU) was close to that of only Listeria (28.9 kU) and the impedance change of only E. coli O157:H7 (2.5 kU) was close to that of the negative control (1.3 kU), indicated that this proposed biosensor had a good specificity. This might be attributed to the following reasons: (1) most of the E. coli O157:H7 cells in the mixture were not captured and separated by the MNPs and only few of them (<3%) (Chen et al., 2015) were non-specifically bound onto the MNPs; (2) The few E. coli cells did not react with the GNPs modified by the PAbs against Listeria, and no or trace urease were captured for catalyzing the hydrolysis of urea, resulting in very small change in the impedance of the SPIE. 4. Conclusion In this study, we have developed and demonstrated an efficient separation and quantitative detection method for rapid detection of Listeria using the screen-printed interdigitated electrode, the urease and the polyclonal antibody modified gold nanoparticles, and the monoclonal antibody coated magnetic nanoparticles. This proposed impedance biosensor has a good linear relationship between the impedance and the concentration from 103 to 106 CFU/ mL and can detect Listeria as low as 1.6 103 CFU/mL within 3 h. The recovery of the spiked lettuce samples ranges from 94.7% to 103.8%. The use of the low-cost screen-printed electrode makes it more promising in in-field screening. Acknowledgments Fig. 6. (a) The TEM image of a Listeria monocytogenes cell conjugated with the monoclonal antibodies coated magnetic nanoparticles and the polyclonal antibodies and the urease modified gold nanoparticles; (2) The linear relationship between the impedance change measured at the characteristic frequency of 7 kHz and the concentration of Listeria (N ¼ 3).
lettuce samples, followed by three parallel tests of the spiked lettuce samples using the proposed biosensor. The mean relative error for the parallel detection is 10.16%, indicating that the biosensor has a good repeatability. The recoveries for the Listeria with the concentrations of 2.0 103 CFU/mL, 2.0 104 CFU/mL, 2.0 105 CFU/ mL, and 2.0 106 CFU/mL were 94.7%, 103.8%, 100.6%, 93.7%, respectively, and the mean recovery was 98.2%, indicating the feasibility of the proposed biosensor for practical applications of foodborne bacteria detection. To obtain the detection limit of this proposed biosensor, ten negative control samples were tested using this biosensor with an average impedance value of 186.4 ± 1.3 kU. The detection limit was determined by three times of signal to noise ratio and was calculated as 1.6 103 CFU/mL. Also, this proposed biosensor was compared with some recently-reported Listeria detection methods and shown in Table S1 in the supplemental material. It can be seen that the LOD for this work is close to or lower than the previously reported methods and the detection time for this work is less than these reported methods. The specificity of this proposed biosensor depends on the monoclonal antibodies and the polyclonal antibodies against Listeria. Since these anti-Listeria antibodies have been tested with good specificity to more than 15 common foodborne pathogenic bacteria by Mao and Huang (Huang et al., 2015; Mao et al., 2016), only E. coli O157:H7 was used in this study as the non-target bacteria to test the specificity of this proposed biosensor. Three parallel
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Please cite this article in press as: Wang, D., et al., Efficient separation and quantitative detection of Listeria monocytogenes based on screenprinted interdigitated electrode, urease and magnetic nanoparticles, Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.09.003