Label-Free 3D Ag Nanoflower-Based Electrochemical Immunosensor for the Detection of Escherichia coli O157:H7 Pathogens
- He Huang†1,
- Minghuan Liu†2,
- Xiangsheng Wang1,
- Wenjie Zhang1,
- Da-Peng Yang2Email author,
- Lianhua Cui3 and
- Xiansong Wang1Email authorView ORCID ID profile
© The Author(s). 2016
Received: 19 July 2016
Accepted: 1 November 2016
Published: 17 November 2016
It is highly desirable to develop a rapid and simple method to detect pathogens. Combining nanomaterials with electrochemical techniques is an efficient way for pathogen detection. Herein, a novel 3D Ag nanoflower was prepared via a biomineralization method by using bovine serum albumin (BSA) as a template. It was adopted as a sensing interface to construct an electrochemical bacteria immunosensor for the rapid detection of foodborne pathogens Escherichia coli (E. coli) O157:H7. Bacterial antibody was immobilized onto the surface of Ag nanoflowers through covalent conjugation. Electrochemical impedance spectroscopy (EIS) was used to detect and validate the resistance changes, where [Fe(CN)6]3−/4− acted as the redox probe. A linear relation between R et and E. coli concentration was obtained in the E. coli concentration range of 3.0 × 102–3.0 × 108 cfu mL−1. The as-prepared biosensor gave rise to an obvious response to E. coli but had no distinct response to Cronobacter sakazakii, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus albus, Lactobacillus easei, and Shigella flexneri, revealing a high selectivity for the detection of the pathogens down to 100 cfu mL−1 in a short time. We believe that this BSA-conjugated 3D Ag nanoflowers could be used as a powerful interface material with good conductivity and biocompatibility for improving pathogen detection and treatment in the field of medicine, environment, and food safety.
The risk being infected by pathogenic bacteria in food and drinking water is one of the major concerns on human health. Detection and identification of harmful organisms, for example, Salmonella typhimurium and Escherichia coli O157:H7, in a cost-effective, rapid, and selective manner remains a challenging task. Conventional methods including bacterial culture, counts of colonies, and use of the polymerase chain reaction and immunological techniques such as ELISA are time-consuming, labor intensive, and often produce nonspecific results. Thus, these methods no longer meet the requirements of pathogenic bacteria diagnosis in food, in the clinic and the general environment . Therefore, it is important to develop effective techniques for disease prevention, for medical diagnosis, and to ensure food safety.
The introduction of nanotechnology has provided important new insights into the problems involved in pathogen detection and identification . Up to now, a great number of nanomaterials including noble metal nanoparticles, quantum dots, and carbon nanomaterials as well as metal oxide nanoparticles have been actively explored for the detection of pathogenic bacteria [2–4]. Taking advantage of their unusual attributes, such as optical, electrical, magnetic, and acoustic properties, various biosensors including surface-enhanced Raman scattering (SERS), fluorescence, and surface plasmon resonance, as well as electrochemical (amperometric, impedance, and luminescence) biosensors based on nanomaterials and recognition elements, have been developed [5–9]. Of all the biosensors, electrochemical impedance spectroscopy (EIS) biosensors have emerged as extremely useful tools for pathogen detection by antigen-antibody interactions on nanomaterial-modified electrode surfaces [3, 10]. EIS works by detecting alterations in an electrochemical system over a wide range of sine wave frequencies. The output signal is electrical current, whose intensity is a function of frequency , with a Nyquist impedance graph commonly used to determine the electron transfer resistance, R et. When bacteria affix to an electrode surface, they reduce the output current and thus increase the impedance of the interface. R et increases with increasing bacterial cell concentration . Nanomaterials on the electrode increase the number of biomolecules that become immobilized (due to the large surface-volume ratio) and then amplify the biomolecular recognition events, resulting in a greatly enhanced signal. By constructing a recognition interface on the electrode that is biocompatible (using biofunctionalized nanomaterials), label-free electrochemical cytosensing can be achieved . On the one hand, the interface material should possess good conductivity, which could accelerate the electron transfer; on the other hand, it also should afford a highly stable and biocompatible matrix, which is fit for the attachment and growth of cells .
In an earlier study, we generated noble metal microspheres (Au, Ag, and Pt) to investigate their potential electrochemical sensing applications [13–16]. The preparation method was based on a green pathway, where bovine serum albumin (BSA) was used as a template and stabilizing agent. In the present study, we utilized a modified method to obtain BSA-conjugated Ag nanoflower architectures with attractive features (widely open porosity, large surface area, intrinsic conductivity, and unique platform for functionalization) to act as an electrochemical sensing interface for E. coli detection. The first aim of the present study was to develop an electrochemical impedance system capable of detecting various types of bacteria, with BSA-conjugated Ag nanoflowers employed as the working electrode. The second aim was to evaluate the effectiveness of Ag@BSA nanoflowers compared with previously reported nanomaterials.
Chemicals and Materials
Ascorbic acid and AgNO3 were sourced from Sinopharm Chemical Reagent Co. (Shanghai, China). Bovine serum albumin (68 kDa) was bought from Xiamen Sanland Chemicals Company Limited. The anti-E. coli O157 antibody was purchased from Abcam (Hong Kong) Ltd. A [Fe(CN)6]3−/4− solution containing 10 mM K3Fe(CN)6, 10 mM K4Fe(CN)6, and 0.1 M KCl (the supporting electrolyte) was used as the redox probe. Phosphate-buffered solution (PBS, 10 mM, pH 7.4) containing 14 mM KH2PO4, 87 mM Na2HPO4, 2.7 mM KCl, and 137 mM NaCl was employed to dilute the anti-E. coli antibody and also as the washing solution. A Millipore Milli-Q system was used to produce ultrapure water. Analytical grade reagents were used in all experiments; it was deemed unnecessary to purify them further.
Synthesis of 3D Ag Nanoflowers
The synthesis of Ag nanoflowers was as previously described . Briefly, a 10 mL BSA solution (5 mg/mL) and a 10 mL AgNO3 solution (10 mM) were added to a 50-mL beaker and magnetically stirred for 10 min at room temperature and then placed in a water bath for 5 min at 25 °C. Subsequently, 50 mg of ascorbic acid was rapidly added and the mixture maintained at 55 °C for 30 min, before being allowed to cool to room temperature. The Ag nanoflowers were harvested and washed three times with water and then three times with ethanol. Finally, the sample was stored in a refrigerator (4 °C) for subsequent use as required.
Experimental Apparatus and Measurement Equipment
A field emission scanning electron microscope (FESEM, ZEISS ULTRA 55) and a transmission electron microscopy (TEM, JEOL 2011) were used to examine the morphology of Ag@BSA nanoflowers. A CHI 660D electrochemical workstation (Shanghai CH Instruments Co., China) was used to carry out EIS and differential pulse voltammetric (DPV) experiments. The electrochemical cell consisted of three compartments, namely, a modified Au electrode served as the working electrode, a platinum wire served as the auxiliary electrode, and the reference electrode was a saturated calomel electrode (SCE). Electrochemical measurements were carried out in sterile PBS containing K3Fe(CN)6/K4Fe(CN)6 (10 mM, 1:1) and 0.1 M KCl (10 mM, pH 7.4). A frequency range of 10−2–105 Hz was used to record the impedance spectra (signal amplitude 5 mV). Nyquist plots were generated using ZSimpWin software (ver. 3.10). DPV experiments were performed with a CHI 660B electrochemical workstation which utilized a conventional three-electrode system (vide supra). CV measurements were carried out in PBS containing 5 mM K3Fe(CN)6 and 0.1 M KCl (10 mM, pH 7.4). A scan rate of 100 mV s−1 was employed over a −0.2-V and +0.8-V range. Fourier transform infrared (FTIR) spectrophotometer measurements were made using a Bruker EQUINOX 55 FTIR spectrometer (range 4000–400 cm−1). X-ray diffraction measurements were performed using a Bruker AXS D8 instrument at 40 kV and 40 mA with Cu-Kα radiation (λ = 1.5406 A).
All bacterial strains (including E. coli, Cronobacter sakazakii, and MRSA) used in this study were purchased from the Institute of Microbiology, Chinese Academy of Sciences. Prior to use, they were incubated twice at 37 °C for 24 h in tryptic soy broth. The initial concentrations were determined using plate counting and serial dilution techniques. Experiments involving the subculture of pathogenic bacteria, their maintenance, and various treatments were performed in a level II biosafety cabinet.
Human skin fibroblast cells were obtained from the cell bank of the Chinese Academy of Sciences and cultured in RPMI 1640 medium supplemented with 10% FBS. Cells were incubated for 2–3 days at 37 °C in a humidified chamber containing 5% CO2. Subsequently, 100 μL volumes containing 1 × 104 cells were added to well plates and incubated for 24 or 48 h, respectively. The cell medium was then replenished and various concentrations of Ag@BSA nanoflowers added. The control groups consisted of human skin fibroblast (HSF) cells alone. After removal of the medium containing the matrix, 20 μL of a solution of 5 mg/mL of MTT was added and incubation was initiated for 4 h. Then, 150 μL of DMSO was added and the solution agitated for 15 min.
Assembly and Recognition of Bacteria Biosensors Based on Ag@BSA Nanoflowers
Results and Discussion
Characterization of Immunosensors Based on Ag@BSA Nanoflowers
XRD was further used to analyze the crystalline structure of the Ag nanoflowers. As shown in Fig. 3b, the diffraction peaks corresponded to the (111), (200), (220), and (311) planes. It was possible to index the planes to the face-centered cubic (fcc) structure of Ag. No peaks of any impurities were observed, showing that the products were composed of pure crystalline Ag.
In addition, the MTT cell viability assay (Additional file 3: Figure S3) was conducted to determine the cytotoxicity of Ag@BSA nanoflowers at different concentrations at 37 °C for 24 and 48 h. HSF (1 × 104 cells) were seeded wells in a 96-well plate and then exposed to Ag@BSA nanoflowers. Control experiments were carried out under the same conditions but without the addition of Ag@BSA. When the Ag@BSA concentration was increased from 0.375 to 5 mM at 24 h, no significant viability decreases of HSF were detected in the MTT assay. MTT assays at 48 h (5-mM Ag nanoflowers) showed slightly lower absorbance, but more than 75% of cells were viable, revealing the excellent biocompatibility of Ag@BSA nanoflowers.
Optimization of Biosensing Conditions
The binding time of antibody was also an important parameter, which affected the measurement of R et. Figure 5b shows the influence of antibody binding time on R et differences prior to E. coli recognition. It was apparent that the R et values exhibited a tendency to increase as a function of an increased antibody binding time, reaching a plateau after 8 h. Thus, 8 h was used as the optimal binding time for anti-E. coli to obtain maximal electrochemical signals from the sensor.
EIS Detection of E. coli Based on Anti-E. coli Antibody/Ag@BSA Nanoflowers
Detection Performance of the Immunosensor Stability, Reproducibility, and Specificity
Comparison of bacterial detection performance with other reported EIS immunosensors
Detection range, cfu mL−1
E. coli O157:H7
1.0 × 102–1.0 × 107
1.8 × 102–1.8 × 108
1.0 × 103–1.0 × 105
1.8 × 101–1.8 × 107
E. coli O157:H7
3.0 × 101–3.0 × 104
Nanoporous Al membrane
E. coli O157:H7
1.0 × 100–1.0 × 104
E. coli K12
1.0 × 103–1.0 × 108
E. coli O157:H7
3.0 × 102–3.0 × 108
In summary, we have constructed a Ag@BSA nanoflower electrochemical impedimetric biosensor functionalized with anti-E. coli antibody for the rapid, label-free, and specific detection of E. coli. The biosensor was capable of detecting very low concentrations of E. coli strains with the limit of detection circa 102 cfu mL−1 and a broad detection range of 3.0 × 102–3.0 × 108 cfu mL−1. We believe that the Ag@BSA electrochemical sensing interfaces will be useful for detecting a wide range of analytes of practical importance including disease-related proteins, cancer cells, and heavy metal ions.
The work is supported by the National Natural Science Foundation of China (Grant No. 31400851 and 81472001), the Initial Foundation for Distinguished Scholars of Quanzhou Normal University, Natural Science Foundation of Shanghai (Grant No. 15ZR1425300 and 15JC1490600) and Funding of SJTU (Grant No. YG2014MS01).
YDP* and WXS*, designed experiments. HH, LMH, WXS carried out the experiments. YDP* drafted the manuscript, YDP* WXS* and HH created the figures. ZWJ, HH, and CLH performed the statistical analysis. All authors discussed the results and commend on the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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