Facile preparation of a bacteria imprinted artificial receptor for highly

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Facile preparation of a bacteria imprinted artificial receptor for highlyselective bacterial recognition and label-free impedimetric detection Jikui Wu, Ruinan Wang, Yunfei Lu, Min Jia, Juan Yan, and Xiaojun Bian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04314 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Facile preparation of a bacteria imprinted artificial receptor for highly-selective bacterial recognition and label-free impedimetric detection Jikui Wu1, Ruinan Wang1, Yunfei Lu1, Min Jia1, Juan Yan1, Xiaojun Bian*,1,2,3 1 College 2

of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China

Laboratory of Quality and Safety Risk Assessment for Aquatic Product on Storage and Preservation

(Shanghai), Ministry of Agriculture, Shanghai 201306, China 3

Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China

*Corresponding author: Email address: [email protected]. Fax: +86-21-61900753

Abstract The effective identification and quantification of pathogenic bacteria is essential for addressing serious public health issues. Here we demonstrate a simple and universal impedimetric sensor for highly selective and sensitive detection of pathogenic bacteria based on the recognition by a bacteria-imprinted polypyrrole (BIP) film. The BIP film was facilely prepared via one-step electro-polymerization followed by in-situ removal of the bacterial template. The film structure is novel with non-cavity-like imprinted sites situated at the surface of the polypyrrole (PPy) matrix, which are more accessible for the target bacteria and should enhance the mass transfer and the binding kinetics. A limit of quantitation low to 103 CFU/mL was achieved within 1 h for the detection of E.coli O157:H7, which is comparable to the antibody-based assays. Moreover, the sensor displayed remarkable selectivity, especially regarding the specific identification of bacterial serotypes. When employed to analyze E.coli O157:H7 in real drinking water, apple juice and milk samples, the sensor showed recoveries from 96.0% to 107.9% with relative standard derivations (RSDs) less than 4%. The BIP-based sensing strategy provides a universal approach for specific, selective and rapid detection of pathogenic bacteria. Compared with 1

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conventional biosensors based on biomolecular recognition, this sensor shows clear advantages including easy-of-preparation, robustness and low cost, which may hold great potential in fields of food/public safety monitoring.

Key Words: electrochemical biosensor; molecularly imprinted polymers; pathogenic bacteria; label-free; Escherichia coli O157; bacteria imprinting

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Diseases caused by foodborne pathogenic bacteria have been a serious threat to public health and food safety for decades and remain one of the major concerns of our society. It was estimated that approximately one-third of global deaths were caused by foodborne bacteria contamination, which led to about tens of million illnesses each year in the United States.1 In developing countries, some strains of bacteria such as E.coli O157:H7, are among the most abundant and the leading cause of death.2 Traditional culture-based bacterial detection methods need time-consuming and laborious isolation, growth and biochemical tests, which often take at least a few days to get the results. More recent methodologies, especially real-time quantitative polymerase chain reaction (qPCR), have received considerable attentions in pathogens detection because of its good selectivity and ease of high throughput. The overall analysis time of qPCR method was reported to take approximately 24 h, which is much improved compared to the traditional culture method. However, qPCR method requires complicated sample preparation, strict operation conditions, expensive and large equipment, low-temperature-stored biological reagents, and well-trained personnel.3 Moreover, the accuracy of qPCR method is seriously affected by PCR inhibitors, commonly found in food and other biological samples,4 and thus leading to false negative judgment. Accordingly, development of a rapid, simple, low-cost and reliable method for detection of pathogenic bacteria is of great significance to guarantee public health and food safety. Electrochemical sensors are promising methods for pathogens detection due to their fast response, simplicity of operation, low cost and ease of miniaturization. The essential aspects in fabrication of electrochemical sensors are the selection of specific receptors and the efficient immobilization of them onto transducer surface (e.g. glass carbon, gold, etc.).5,6 Among various bacteria-specific receptors (such as antibodies,4,7,8 aptamers,3,9 phages,10,11 carbohydrate,12-14 etc.), antibodies are the most commonly used recognition elements due to their superior selectivity and binding affinity.15 Nevertheless, antibodies usually suffer from several inherent limitations including technically complexed preparation, high cost, limited pH and temperature 3



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stability, and poor reproducibility, which have extremely restricted their practical applications.16 In addition, antibodies can be subject to partially denature and lose their function, after being linked to the transducer surface via the frequently used strategies such as physical adsorption and covalent coupling.5 Molecularly imprinted polymers (MIPs) are considered as promising alternatives to natural antibodies, owing to their distinctive features of simple preparation, low cost, superior chemical/physical stability, long shelf-life, and potential reusability.17 MIPs can be prepared via chemical, photochemical or electrochemical methods in the presence of target template followed by removal of the templates.18 This process renders the formation of polymeric materials with specific recognition sites complementary in shape, size, and functional group to template molecules.19 So far, MIPs have gained broad applications in fields of sensing and separation of small molecules,20-22 biological macromolecules,23-25 and even larger species such as viruses,26,27 bacteria,17,28-30 and mammalian cells.31 In spite of the promising prospects for MIPs, we have to acknowledge that it is more challenging as the target size increases, due to the increased difficulties in template extraction inducing the failure of imprinting or the lack of imprinted sites.17 Moreover, the transport of target with huge size becomes much slower, resulting in much longer response time of the system.32

More

recently,

Turner

et

al.

have

successfully

fabricated

a

poly(3-aminophenylboronic acid) (PAPBA) based cell-imprinted polymer for the electrochemical label-free detection of Staphylococcus epidermidis.33 The boronic acid groups in the PAPBA film has high affinity for diol-containing molecules (such as sugars, bacteria, etc.), which is capable to increase the cell adhesion onto the polymer surface, and facilitate the release of the adhered cells from the polymer network by competitive displacement with fructose. But meanwhile the presence of boronic acid groups also increased the non-specific adhesion of the target and non-target bacterial cells on the non-imprinted polymer surface. Inspired by the research mentioned above, we demonstrated the facile preparation of a bacteria-imprinted polypyrrole (BIP) film on glass carbon electrode (GCE) surface via one-step electro-copolymerization of pyrrole monomer and bacterial 4


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template followed by in-situ removal of the bacteria. Electrochemical polymerization represents an advantageous method that allows depositing a uniform molecularly imprinted layer with controllable thickness of the polymer film, and good adherence to the transducer surface.34 Polypyrrole (PPy) is considered as the most promising conductive polymer for the development of electrochemical sensor because of its low non-specific adsorption, good conductivity, superior stability, and efficient polymerization at mild conditions.35,36 By optimization of the approaches for the template removal, the micrometer-scaled bacterial template was almost completely removed from the PPy matrix, producing abundant recognition sites for binding of the target bacteria. Different from the morphology of conventional MIPs, the BIP film had novel non-cavity-like imprinted sites situated at the surface of the PPy matrix, which are more accessible and could be expected to enhance the mass transfer and the binding kinetics. With the BIP film as an artificial receptor, a simple and universal impedimetric sensor for rapid and label-free detection of pathogenic bacteria was developed. As a proof of concept, E.coli O157:H7 as a common microorganism, was used for evaluation of the sensing performance. Under optimized experimental conditions, E.coli O157:H7 can be detected within 1.0 h with wide linear range, high selectivity and low limit of quantitation (LOQ). Moreover, the sensor showed high selectivity for the template bacteria, which is even capable of specific identification of E. coli group serotypes (O157:H7 and O26). The applicability of the sensor was also evaluated in real samples, including drinking water, apple juice and milk. The BIP film based impedimetric sensing strategy can serve as a universal detection platform for rapid, specific, and cost-efficient detection of pathogenic bacteria, which in turn could have great impact in the fields of food safety and public health.

EXPERIMENTAL SECTION Bacterial cultivation. Several bacterial strains were involved including E.coli O157:H7 (ATCC 43889), E.coli O6 (ATCC 25922), Listeria monocytogenes (L. monocytogenes, ATCC 19115), Staphylococcus aureus (S. aureu, ATCC 25923), Salmonella (CMCC 50094). All the bacterial strains were cultured separately in LB 5



liquid medium at 37°C overnight with continuous shaking at 200 rpm. The cultured bacterial pellets were harvested by centrifugation (5000 rpm, 10 min) and suspended in sterilized water. The accurate concentration of each bacteria was individually determined in triplicate using plate counting method. Nutrient Agar (NA) was used for the coating of E.coli O157:H7, L. monocytogenes and S. aureus. Tryptic soy agar (TSA) was employed for the enumeration of Salmonella. The 10-fold serial dilutions from 108 to 103 CFU/mL were prepared with sterilized 1×PBS (pH 7.4). Preparation of the BIP film. Prior to use, the GCE was polished with 0.3-0.05 μm of alumina aqueous slurry to a mirror-like surface followed by successively ultrasonic cleaning in water and ethanol. Electrochemical copolymerization was carried out by cyclic voltammetry (CV) in 5 mL of 0.1 M KCl containing 0.05 M pyrrole and bacterial template (105 CFU/mL). A cyclic voltage from -0.4 V to +0.7 V versus the saturated calomel reference electrode (SCE) was applied at a scanning rate of 50 mV/s under mild stirring. The fabricated modified electrode was denoted as PPy+E.coli/GCE. As a control, PPy modified electrode (PPy/GCE) was prepared in exactly the same way except for the omitting of bacterial template during the electro-polymerization. To remove the bacterial template, the fabricated modified GCEs were then treated with SDS/HAC (5% w/v) for 4.0 h under static conditions. The formed modified electrode after template removal was defined as BIP/GCE. Fluorescence microscope observation. Before capture, E.coli O157:H7 cells were stained with SYTO-9 green fluorescent dye (Life Technologies) according to the manufacturer’s instructions. Detachable GCEs (Gaoss Union, China; surface area, 0.07 cm2) were used for the formation of the BIP or PPy film. The fluorescent images of the stained E.coli O157:H7 cells captured by the BIP and PPy film were observed through an optical filter with an excitation of 465-495 nm and an emission of 510-530 nm. Optimization of experimental conditions. In order to obtain optimum sensing performance towards pathogenic E.coli O157:H7, experimental conditions including polymerization cycles, eluents and elution time used for template removal, and recognition time were optimized. The choice of proper eluents and time for template 6


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removal was based on the degree of reduction in charge transfer resistance (Rct). The more impedance dropped, the more completely template was removed. The selection of optimal polymerization cycles and recognition time were dependent on relative variation of Rct [△R/R(Ω)] after incubation with E.coli O157:H7 at 105 CFU/mL for 1.0 h. The use of relative variation of Rct as the analytical response was to minimize the variability between paralleled electrodes. 33 The value was calculated as follows: △R/R = (Rcta - Rctb)/Rctb

(1)

where Rctb and Rcta are the value of Rct before and after incubation with the target bacteria, respectively. Bacterial detection based on the BIP film. The recognition capability of the BIP film towards the template bacteria was investigated by incubation of the BIP/GCE in 250 μL of 1×PBS (pH 7.4) containing certain concentrations of bacteria for 1.0 h under constant shaking (300 rpm). Electrochemical impedance spectroscopy (EIS) was used to monitor the impedance change of the electrode surface and its interface to the buffer solution containing 1 mM K3[Fe(CN)6] and 1 mM K4[Fe(CN)6] in 0.1 M KCl. An open circuit voltage was applied to the modified GCEs in a frequency range of 0.1-100,000 Hz with an amplitude of 5 mV. The impedance data were then represented in the form of Nyquist plots. Real sample. The experiment was done with drinking water, apple juice and milk for the detection of E.coli O157:H7 to see if the system works for real samples. Drinking water was obtained from water purifier (Ozner, China). Apple juice (Huiyuan Brand, China) and milk (Yili Brand, China) were purchased from local supermarket. For both drinking water and apple juice, no sample pretreatment was needed. While for milk, centrifugation (15000 rpm, 10 min) was firstly done and the supernatant fat layer was discarded. Then the left supernatant was filtered through a sterile Millipore membrane (0.22 μm) followed by ten-fold dilution with 1×PBS (pH 7.4). The artificially contaminated real samples were prepared by spiking 9 mL of drinking water, apple juice or milk with 1 mL of E.coli O157:H7 cultures to a final concentration from 103 to 105 CFU/mL. Control experiment was done by adding 1 mL of each sample in replacement of E.coli O157:H7. Subsequently, 250 μL of each real sample with or 7



without E.coli O157:H7 were independently incubated with the BIP films followed by the EIS measurement. Each experiment was performed at least three times.

RESULTS AND DISCUSSION Construction of the BIP-based sensor for label-free detection of pathogenic bacteria. The schematic showing the fabrication of BIP-based sensor for E.coli O157:H7 detection is illustrated in Figure 1. The proposed BIP-based bacterial sensor involved three consecutive procedures: the BIP film preparation, bacterial recognition and label-free impedimetric detection. The BIP film preparation was very convenient, which included one-step electro-copolymerization of pyrrole monomer and E.coli O157:H7 template followed by in-situ removal the bacterial template. The created specific recognition sites on the BIP film could then selectively recognize E.coli O157:H7 via complementary in chemical properties with the template bacteria.18,29 The captured bacteria were subsequently detected as an increase in impedance due to the relatively lower conductivity of the bacteria. 33,37

Figure 1. Schematic illustration showing the construction of BIP film based sensor for E.coli O157:H7 detection. The CV curves recorded during the electrochemical polymerization of pyrrole in the presence (Figure S1A) and absence (Figure S1B) of E.coli O157:H7 are shown in Figure S1. In both situations, no redox peak was observed in the first cycle. As the scans increased, a pair of small redox peaks appeared due to the redox reaction of 8


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pyrrole. When the electro-polymerization was conducted in the presence of E.coli O157:H7, the redox currents and the capacitance were found to be reduced. This phenomenon could be explained that the less conductive bacteria was embedded in the PPy matrix. Electrochemical characterization for the BIP film fabrication. EIS was employed for characterization of the BIP film fabrication process. The obtained data for different modified GCEs were presented as Nyquist plots as shown in Figure 2A and Figure 2B. The Rct value of various modified GCEs as described in Figure 2D (top) was estimated according to the diameter of the observed semicircle.38 Before polymerization, the bare GCE (Figure 2B, hollow square) presented a typical mass diffusion limiting process.39 After the electro-copolymerization of pyrrole and E.coli O157:H7, the Rct value of PPy+E.coli/GCE (Figure 2A, solid square) increased into ca. 60 kΩ, which was 400 times more than that of PPy/GCE (ca. 150 Ω) without bacteria (Figure 2B, solid circle). The result indicated that E.coli O157:H7 template was successfully embedded in the PPy matrix. After treatment of the PPy+E.coli/GCE with SDS/HAC (5%, w/v), a significant decrease in the Rct value (ca. 300 Ω) of the fabricated BIP/GCE was observed (Figure 2A, hollow circle), which was 200 times lower than that of PPy+E.coli/GCE. Lower Rct indicated quicker electron transfer rate at the BIP film/electrolyte interface, owing to more thorough removal of the bacterial template from PPy.

9



Figure 2. Electrochemical characterization for the fabrication process of the BIP film. (A-B) EIS and (C) CV curves of different modified GCEs in 0.1 M KCl containing 1mM K3Fe(CN)6/K4Fe(CN)6; (D) Statistic histograms showing the value of Rct and △Ep for the various modified GCEs. CV method was simultaneously carried out for characterizations of the BIP film fabrication process. The CV curves and the corresponding numerical variation of peak potential separation (△Ep) for different modified GCEs were demonstrated in Figure 2C and Figure 2D (bottom), respectively. Obvious redox peaks could be found in the CV curves of bare GCE and PPy/GCE. After copolymerization of pyrrole and E. coli O157:H7, the △Ep of PPy+E.coli/GCE increased remarkably and the reduction peak could not even be observed in the potential range. This result further confirmed the successful embedding of the bacterial template in PPy matrix. When E.coli O157:H7 template was removed, the formed BIP/GCE exhibited an obvious reduction in △Ep and a significant rise in current response compared with PPy+E.coli/GCE. It was because there were more conductive sites in the BIP film and more Fe(CN)43−/4− penetration channels for electrochemical redox.39

10


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Figure 3. SEM images presenting the film morphology of various modified GCEs. (A) PPy/GCE, (B) PPy+E.coli/GCE, (C) magnified image of PPy+E.coli/GCE showing the bacteria on the hybrid film, and (D) BIP/GCE. Morphological characterization. The morphologies of various polymer films were characterized by scanning electron microscopy (SEM). Many rod bacteria were observed from the hybrid membrane fabricated via electrochemical copolymerization of pyrrole and E.coli O157:H7 (Figure 3B). In comparison, the PPy film formed in the absence of E.coli O157:H7 was smoother and more uniform (Figure 3A). The rod-shaped bacterial cell in the hybrid film could be more clearly seen from the magnified image as presented in Figure 3C. The dimension of the bacteria was approximately 2 μm in length by 0.6 μm in width, which was very close to that of E.coli O157:H7 found in previous report.40 The result further revealed that E.coli O157: H7 cells were successfully doped with PPy film. When the hybrid film was eluted with SDS/HAC (5%, w/v) for 4 h, no more bacteria were left on the hybrid membrane. This phenomenon suggested the E.coli O157:H7 template was almost completely removed. Recognition capability of the BIP film. In-situ EIS measurements were taken to 11



evaluate the recognition capability of the BIP film towards the template bacteria, E.coli O157:H7. After incubating the BIP/GCE with E.coli O157:H7 at a concentration of 105 CFU/mL for 1.0 h, the Rct value of BIP-E.coli/GCE reached as high as 18 kΩ (Figure 4A, solid square), which was 60 times higher than that of BIP/GCE as mentioned above (Figure 2A, hollow circle). To verify if the Rct increase was caused partly by non-specific adsorption of bacteria on PPy film, a control experiment was done by incubation of PPy/GCE with E.coli O157:H7 under otherwise the same conditions. However, the Rct value of PPy-E.coli/GCE only increased slightly by about 250 Ω (Figure 4A, solid circle), which is ＞ 70 times lower than that of BIP-E.coli/GCE. The results initially revealed that the BIP film had good recognition capability towards E.coli O157:H7. Moreover, the recognition capability of the BIP film towards the template bacteria were assessed by fluorescent microscopy (DP80, Olympus). In the experiment, BIP/GCE and PPy/GCE were separately incubated with the stained E.coli O157:H7 cells by SYTO-9 for 1.0 h under otherwise the same conditions. After incubation, a number of green rod-like forms were observed on the surface of BIP-E.coli/GCE (Figure 4B). In comparison, almost no fluorescent signals could be identified for PPy-E.coli/GCE (Figure 4C). The results further confirmed the specific recognition capability of the BIP film towards the template bacteria, E.coli O157:H7.

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Figure 4. (A) EIS showing the recognition capability of the BIP and PPy film towards the template bacteria, E.coli O157:H7; Fluorescence images of the (B) BIP/GCE and (C) PPy/GCE after incubation with E.coli O157:H7 cells stained green by SYTO-9 dye. The bacterial concentration was kept at 105 CFU/mL. Optimization

of

the

BIP-based

sensing

performance.

The

number

of

polymerization cycles was varied from 5 to 20 during the electro-copolymerization of pyrrole and E.coli O157:H7. As shown in Figure 5A, the sensing performance was improved with the increase of polymerization cycles from 5 to 15. But further increase in polymerization cycles to 20 resulted in a decreased response to template 13



bacteria. Therefore, 15 cycles of polymerization were selected as the optimal condition for the electrochemical copolymerization.

Figure 5. Optimization of experimental conditions including (A) polymerization cycles, (B) eluents for template removal, (C) template removal time, and (D) recognition time to obtain a better sensing performance towards E.coli O157:H7. The Removal of micrometer-sized bacterial template from cross-linked polymer is quite challenging but very crucial for the effective synthesis of MIPs.41 Inspired by previous studies, three different methods have been proposed for the removal of bacterial template (Figure 5B). For example, mannose instead of fructose 33 was used for competitive detachment of E.coli O157:H7 template from the polymer matrix for 4.0 h. In addition, successive treatment with lysozyme (10 mg/mL) for 2 h and 10% Triton X-100 for 80 min were employed to damage the strong interactions between polysaccharide on the bacterial cell wall and polymer surface.42 Besides, with some modifications from published literature,39 SDS/HAC (5%, w/v) was used to extract E.coli O157:H7 template for 4.0 h from PPy matrix, which was proved to be the most effective method and was finally chosen as the eluent for bacterial template removal. Also, the effect of elution time on the template removal was investigated. As shown in 14


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Figure 5C, the Rct value gradually decreased with the increase of elution time from 1.0 to 4.0 h. When extending the elution time to 5.0 h, there was almost no change in Rct. So, 4.0 h of elution with SDS/HAC (5%, w/v) was employed as the optimum condition for template removal. The incubation time or recognition time is another important parameter that influences the sensing performance. As shown in Figure 5D, the analysis response was enhanced with the incubation time varying from 0.5 to 1 h. And then the response signal changes little even with a longer incubation period of 1.5 h. Thus, an incubation time of 1.0 h was chosen for all analyses in this work. Quantitative detection of E.coli O157:H7 based on the BIP film. Under optimized experimental conditions, 10-fold serial diluted E.coli O157:H7 samples (103-108 CFU/mL) were separately incubated with the prepared BIP/GCEs. The subsequent quantitative detection was carried out using label-free EIS strategy. For each sample, at least three paralleled experiments were performed simultaneously using different BIP/GCEs. As presented in Figure 6A, the impedance increased as the bacterial concentration increases from 103 to 108 CFU/mL. This is because the bacterial cells captured by the BIP film partially block the electron transfer between the redox probe and the electrode surface, resulting in an increase in the film impedance.33 Figure 6B displayed the corresponding calibration curve of the EIS response versus log concentration of E.coli O157:H7. In a wide concentration range from 103 to 108 CFU/mL, it exhibited a linear relationship between the △R/R and the log concentration of E.coli O157:H7. The linear regression equation was expressed as △R/R (Ω)=17.99logC-56.21 with a correlation coefficient of 0.9883, where C stands for the concentration of E.coli O157:H7 in CFU/mL. When the concentration of E.coli O157:H7 was below 103 CFU/mL, it was difficult to measure a EIS response without any pretreatment procedure. Therefore, the LOQ of the BIP-based sensor for E.coli O157:H7 was 103 CFU/mL.

15



Figure 6. (A) Impedance spectra of the BIP-based biosensor for detection of E.coli O157:H7 at series of gradient concentrations from 103 to 108 CFU/mL; (B) Corresponding calibration curve of the EIS response versus log concentration of E.coli O157:H7 from 103 to 108 CFU/mL. Selectivity and universality of the BIP-based sensor. The selectivity of the BIP film with E.coli O157:H7 recognition sites was investigated by comparing the EIS responses to the template bacteria (E.coli O157:H7) and other four different interfering bacteria including both gram-positive and gram-negative bacteria (L. monocytogenes, S. aureu, Salmonella, and E.coli O6). All the bacteria were at the same concentration of 105 CFU/mL. As shown in Figure 7A and Figure 7B, the EIS response for E.coli O157:H7 was nearly 5 times higher than that for those interfering bacteria. More significantly, even closely related strains such as E.coli O157:H7 and E.coli O6 could be discriminated by the BIP film with E.coli O157:H7 imprinted sites (Figure 7B). The discoveries revealed that the BIP film with E.coli O157:H7 recognition sites had a satisfactory selectivity for detection of the template bacteria. 16


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For further confirm the selectivity, and validate the universality of the BIP-based sensor, another two BIP films with S. aureus and Salmonella recognition sites were individually prepared. For both S. aureus and Salmonella imprinted PPy films, the EIS responses to these interfering bacteria were at least 3 times lower than that to the template bacteria (Figure 7A). The above results indicated the good selectivity and universality of the proposed BIP-based sensing strategy for pathogenic bacteria detection. The high selectivity of the BIP film towards the template bacteria could be ascribed to its sensitivity to the chemical conformation of bacterial outer cell structures, as demonstrated in the previous literature.42

Figure 7. Selectivity of the BIP-based sensor. (A) EIS response of different BIPs to the corresponding template bacteria and three other kinds of interfering bacteria. (B) EIS response of E.coli O157:H7-imprinted PPy film to E.coli group serotypes (O157:H7 and O26). All the bacterial samples are at the concentration of 105 CFU/mL. The results are averages of at least three independent experiments and error bars indicate standard deviations. 17



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Detection of E.coli O157:H7 in real samples. In order to demonstrate the applicability of the proposed BIP-based sensor for E.coli O157:H7 detection in real samples, we have used it for detection of E.coli O157:H7 in artificially contaminated drinking water, apple juice and milk samples, respectively. During the experiment, we found that the drinking water and apple juice matrices had little effect on the detection, and thus no sample pretreatment was done. While for milk, the coexisting components (e.g. fats and proteins) could interfere with the bacteria in the detection. Two-step sample pretreatments including centrifugation and filtration were carried out before sample analysis, referring to previous literatures with some modifications.43,44 The obtained results were summarized in Table 1. The recoveries of E.coli O157:H7 in real samples varied between 96.0% and 107.9% with RSDs from 0.84% to 3.83%, suggesting that the proposed method could be applied for detection of E. coli O157:H7 in real samples. Table 1. Detection of E.coli O157:H7 in real samples. Sample

Added (CFU/mL)

Measured (CFU/mL)

103

1.025×103

102.5

1.42

104

1.016×104

101.6

2.69

105

1.017×105

101.7

3.83

103

9.88×102

98.8

2.72

104

1.035×104

103.5

2.96

105

1.079×105

107.9

2.50

103

1.039×103

103.9

0.84

104

1.011×104

101.1

0.90

105

9.60×104

96.0

1.77

Drinking water

Apple juice

Milk

Recovery (%)

RSD (%, n=3)

CONCLUSIONS In this study, we developed an impedimetric sensor that offers highly selective and label-free detection of pathogenic bacteria based on the recognition of bacterial cell by a facilely prepared BIP film. In contrast to conventional bacteria imprinted 18


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polymers, a significant structure novelty is the formation of a non-cavity-like imprinted sites situated at the surface of the PPy matrix, which are more accessible and could be expected to enhance the mass transfer and the binding kinetics.32 The level of detection (for example, 103 CFU/mL was achieved for E.coli O157:H7) is comparable to or even better than that afforded by conventional MIP sensors.33,42,45 Moreover, the BIP film exhibited high selectivity against the target bacteria, especially capable of the specific identification of bacterial serotypes. The sensor also demonstrated ideal recoveries for testing drinking water, apple juice and milk samples artificially spiked with E.coli O157:H7. The concept of the BIP-based sensing strategy should be expanded to the detection of other pathogenic bacteria, providing a universal platform for pathogens assay.

ACKNOWLEDGEMENTS The authors gratefully acknowledge financial support from NSFC (21405167, 21775102) and “Chenguang Program” (15CG54) supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission.

Supporting Information This section contains the cyclic voltammetry (CV) curves recorded during the electrochemical polymerization of pyrrole monomer (0.05 M) in 0.1 M KCl in the presence and absence of E.coli O157:H7 at the concentration of 105 CFU/mL.

REFERENCES (1) Chen, J.; Andler, S. M.; Goddard, J. M.; Nugen, S. R.; Rotello, V. M. Chem. Soc. Rev. 2017, 46, 1272-1283. (2) Bole, A. L.; Manesiotis, P. Adv. Mater. 2016, 28, 5349-5366. (3) Mengqun Yu, H. W., Fei Fu, Linyao Li, Jing Li, Gan Li, Yang Song, Mark T. Swihart, and Erqun Song. Anal. Chem. 2017, 89, 4085-4090. (4) Srisa-Art, M.; Boehle, K. E.; Geiss, B. J.; Henry, C. S. Anal. Chem. 2018, 90, 1035-1043. (5) Amiri, M.; Bezaatpour, A.; Jafari, H.; Boukherroub, R.; Szunerits, S. ACS Sens. 2018, 3, 1069-1086. (6) Templier, V.; Roux, A.; Roupioz, Y.; Livache, T. TrAC Trends Anal. Chem. 2016, 79, 71-79. 19



(7) Farka, Z.; Jurik, T.; Pastucha, M.; Skladal, P. Anal. Chem. 2016, 88, 11830-11836. (8) Ahmed, A.; Rushworth, J. V.; Wright, J. D.; Millner, P. A. Anal. Chem. 2013, 85, 12118-12125. (9) Wu, S.; Duan, N.; Shi, Z.; Fang, C.; Wang, Z. Anal. Chem. 2014, 86, 3100-3107. (10) Farooq, U.; Yang, Q.; Ullah, M. W.; Wang, S. Biosens. Bioelectron. 2018, 118, 204-216. (11) Rippa, M.; Castagna, R.; Pannico, M.; Musto, P.; Borriello, G.; Paradiso, R.; Galiero, G.; Bolletti Censi, S.; Zhou, J.; Zyss, J.; Petti, L. ACS Sens. 2017, 2, 947-954. (12) Yazgan, I.; Noah, N. M.; Toure, O.; Zhang, S.; Sadik, O. A. Biosens. Bioelectron. 2014, 61, 266-273. (13) Gade, M.; Paul, A.; Alex, C.; Choudhury, D.; Thulasiram, H. V.; Kikkeri, R. Chem. Commun. 2015, 51, 6346-6349. (14) Bulard, E.; Bouchet-Spinelli, A.; Chaud, P.; Roget, A.; Calemczuk, R.; Fort, S.; Livache, T. Anal. Chem. 2015, 87, 1804-1811. (15) Brosel-Oliu, S.; Uria, N.; Abramova, N.; Bratov, A. In Biosensors-Micro and Nanoscale Applications; IntechOpen, 2015. (16) Zhang, Z.; Li, M.; Ren, J.; Qu, X. Small 2015, 11, 1258-1264. (17) Zhang, Z.; Guan, Y.; Li, M.; Zhao, A.; Ren, J.; Qu, X. Chem. Sci. 2015, 6, 2822-2826. (18) Uzun, L.; Turner, A. P. Biosens. Bioelectron. 2016, 76, 131-144. (19) Wang, C.; Hu, R.; Morrissey, J. J.; Kharasch, E. D.; Singamaneni, S. Small 2017, 13. (20) Razavipanah, I.; Alipour, E.; Deiminiat, B.; Rounaghi, G. H. Biosens. Bioelectron. 2018, 119, 163-169. (21) Capoferri, D.; Alvarez-Diduk, R.; Del Carlo, M.; Compagnone, D.; Merkoci, A. Anal. Chem. 2018, 90, 5850-5856. (22) Kong, D.; Qiao, N.; Wang, N.; Wang, Z.; Wang, Q.; Zhou, Z.; Ren, Z. Phys. Chem. Chem. Phys. 2018, 20, 12870-12878. (23) Zhang, W.; Liu, W.; Li, P.; Xiao, H.; Wang, H.; Tang, B. Angew. Chem., Int. Ed. Engl. 2014, 53, 12489-12493. (24) Bie, Z.; Chen, Y.; Ye, J.; Wang, S.; Liu, Z. Angew. Chem., Int. Ed. Engl. 2015, 54, 10211-10215. (25) Cai, D.; Ren, L.; Zhao, H.; Xu, C.; Zhang, L.; Yu, Y.; Wang, H.; Lan, Y.; Roberts, M. F.; Chuang, J. H.; Naughton, M. J.; Ren, Z.; Chiles, T. C. Nat. Nanotechnol. 2010, 5, 597-601. (26) Birnbaumer, G. M.; Lieberzeit, P. A.; Richter, L.; Schirhagl, R.; Milnera, M.; Dickert, F. L.; Bailey, A.; Ertl, P. Lab Chip 2009, 9, 3549-3556. (27) Cumbo, A.; Lorber, B.; Corvini, P. F.; Meier, W.; Shahgaldian, P. Nat. Commun. 2013, 4, 1503. (28) van Grinsven, B.; Eersels, K.; Akkermans, O.; Ellermann, S.; Kordek, A.; Peeters, M.; Deschaume, O.; Bartic, C.; Diliën, H.; Steen Redeker, E.; Wagner, P.; Cleij, T. J. ACS Sens. 2016, 1, 1140-1147. (29) Zare, K. R. a. R. N. ACS nano 2012, 6, 4314–4318. (30) Shen, X.; Svensson Bonde, J.; Kamra, T.; Bulow, L.; Leo, J. C.; Linke, D.; Ye, L. Angew. Chem., Int. Ed. Engl. 2014, 53, 10687-10690. (31) Kunath, S.; Panagiotopoulou, M.; Maximilien, J.; Marchyk, N.; Sanger, J.; Haupt, K. Adv .Healthcare Mater. 2015, 4, 1322-1326. (32) Haupt, K. Anal. Chem. 2003, 75, 376A-383A. (33) Golabi, M.; Kuralay, F.; Jager, E. W.; Beni, V.; Turner, A. P. Biosens. Bioelectron. 2017, 93, 87-93. (34) Antuña-Jiménez, D.; Díaz-Díaz, G.; Blanco-López, M. C.; Lobo-Castañón, M. J.; Miranda-Ordieres, A. J.; Tuñón-Blanco, P. In Molecularly Imprinted Sensors, 2012, pp 1-34. (35) Teles, F. R. R.; Fonseca, L. P. Materials Science and Engineering: C 2008, 28, 1530-1543. (36) Zhong, M.; Teng, Y.; Pang, S.; Yan, L.; Kan, X. Biosens Bioelectron 2015, 64, 212-218. 20


Page 20 of 22

Page 21 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(37) Madiyar, F. R.; Bhana, S.; Swisher, L. Z.; Culbertson, C. T.; Huang, X.; Li, J. Nanoscale 2015, 7, 3726-3736. (38) Tavares, A. P.; Ferreira, N. S.; Truta, L. A.; Sales, M. G. Sci. Rep. 2016, 6, 26132. (39) Chen, S.; Chen, X.; Zhang, L.; Gao, J.; Ma, Q. ACS Appl. Mater. Interfaces 2017, 9, 5430-5436. (40) Shan, X.; Yamauchi, T.; Yamamoto, Y.; Niyomdecha, S.; Ishiki, K.; Le, D. Q.; Shiigi, H.; Nagaoka, T. Chem. Commun. 2017, 53, 3890-3893. (41) Pan, J.; Chen, W.; Ma, Y.; Pan, G. Chem. Soc. Rev. 2018, 47, 5574-5587. (42) Tokonami, S.; Nakadoi, Y.; Takahashi, M.; Ikemizu, M.; Kadoma, T.; Saimatsu, K.; Dung le, Q.; Shiigi, H.; Nagaoka, T. Anal. Chem. 2013, 85, 4925-4929. (43) Lian, W.; Liu, S.; Yu, J.; Li, J.; Cui, M.; Xu, W.; Huang, J. Biosens. and Bioelectron. 2013, 44, 70-76. (44) Liang, R.; Zhang, R.; Qin, W. Sensor. Actuat. B-Chem. 2009, 141, 544-550. (45) Poller, A. M.; Spieker, E.; Lieberzeit, P. A.; Preininger, C. ACS Appl. Mater. Interfaces 2017, 9, 1129-1135.

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Facile preparation of a bacteria imprinted artificial receptor for highly

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