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Escherichia coli O157:H7 is an extremely serious foodborne pathogen accounting for a vast number of hospitalizations. In this system, a simple, rapid, and safe compound method was developed based on carbonyl iron powder (CIP) and multiwalled carbon nanotubes (MWCNT). Then, the CIP@MWCNT-based aptasensor was constructed by strong π-stacking between nanocomposite and aptamer, single-strand DNA, causing fluorescent quenching of the dye-labeled aptamer. The restoration of dye fluorescence could be achieved when aptamer came off the surface of the CIP@MWCNT nanocomposite due to the presence of target bacteria. To the best of our knowledge, this fabrication of magnetic carbon nanotubes without irritating and corrosive reagents is described for the first time. The sensing platform was also an improvement on the conventional formation of the aptasensor between carbon materials and DNA aptamer. The nanocomposite was verified by diverse characterization of zeta potential, Fourier-transform infrared spectroscopy, transmission electron microscopy, and energy dispersive x-ray analysis. The CIP@MWCNT-based aptasensor was an effective nanoplatform for quantitative detection of E. coli O157:H7, and was measured to have high specificity, good reproducibility, and strong stability. The aptasensor's capacity to quantify E. coli O157:H7 was as low as 7.15 × 103 cfu/mL in pure culture. The detection limit of E. coli O157:H7 was 3.15 × 102 cfu/mL in contaminated milk after 1 h of pre-incubation. Hence, the developed assay is a new possibility for effective synthesis of nanocomposites and sensitive tests of foodborne pathogens in the dairy industry.
Foodborne pathogens are a public issue and account for many hospitalizations and deaths each year (https://www.cdc.gov/foodborneburden/index.html). Of all the foodborne pathogens, Escherichia coli O157:H7 is regarded as the most concerning. Certain efforts for controlling E. coli O157:H7 in food processes have been made, such as abiding by good manufacturing practices, but the effect of prevention is still not obvious (
). To monitor food safety, microbial culture and molecular-based measurements are used frequently. However, these methods are time consuming, multistep, and require complicated equipment and specialized personnel (
). Thus, establishing a rapid, sensitive, and reliable detection assay is particularly important for reducing the infection of E. coli O157:H7 in the dairy industry.
Nanotechnology-based detecting assays are a rapidly extending field that covers various nanomaterials and biomolecules (e.g., aptamer, a low-cost substitute for antibodies targeting antigens specifically). In recent years, these assays have had explosive growth for food safety applications. They have a great capacity for offering superiority over conventional laboratory methods (
). Due to their large surface area, carbon nanotubes have been employed as a carrier to load plentiful biomolecules (such as aptamers) by noncovalent interaction, achieving ultrasensitive detection of targets (
). A nanoplatform was constructed by multiwalled carbon nanotubes (MWCNT) and DNA aptamer according to the π–π stacking interaction, which was a safe functional strategy that passed cellular toxicity assays (
). However, the creation of this nanoplatform required several centrifugations to remove excess aptamers, which were cumbersome and time consuming. The operation can be simplified when magnetizing MWCNT is washed in aqueous solution using only a magnetic separator and this sort of treatment has been described several times. The MWCNT nanocomposites with magnetism were generally obtained by a solvothermal or coprecipitation method (
Removal of reactive red 198 from aqueous solution by combined method multi-walled carbon nanotubes and zero–valent iron: Equilibrium, kinetics, and thermodynamic.
). The unique adsorption property of magnetic carbon nanomaterials was widely applied to multiple fields of prevention and control, such as organic acids (
). An aptasensor was constructed by graphene oxide/iron (GO/Fe3O4) nanocomposites and DNA aptamer for quantifying E. coli O157:H7 in milk, avoiding tedious chemical modifications by combining aptamer self-assembly with GO and magnetic separation (
A novel universal colorimetric sensor for simultaneous dual targets detection through DNA-directed self-assembly of graphene oxide and magnetic separation.
). These Fe3O4/GO nanoparticles were also prepared by an intricate chemical coprecipitation method, and the sensing results could be inaccurate due to incomplete adsorption of Fe3O4/GO nanoparticles for unbound aptamer from solution because nanocomposites were added in the mixture after incubation of aptamer and target bacteria. The surface area of GO is not as large as MWCNT for a nanoparticle, which could adsorb fewer single-stranded DNA aptamers. Thus, the fabrication of carbon nanomaterials with magnetism must be created through a novel approach, and the accuracy of the aptasensor needs to be improved.
In the present study, we introduced a novel, simple, and safe synthetic method for magnetic carbon nanomaterials without irritating and corrosive reagents. Afterward, the CIP@MWCNT-based aptasensor was modified by noncovalent functionalization concatenation due to π–π stacking of CIP@MWCNT nanocomplex with a dye-labeled aptamer targeting E. coli O157:H7. This caused fluorescent quenching and the restoration of fluorescence could be achieved when aptamer combined with E. coli O157:H7. The detailed construction of nanocomposite and multiple performance analysis of the aptasensor were evaluated to verify the effectiveness. The neo-aptasensor in this research provides a simple, rapid, and sensitive system for detecting pathogens for dairy safety.
MATERIALS AND METHODS
Materials and Reagents
Carboxylated MWCNT with an outer diameter <8 nm were purchased from Nanjing XFNANO Materials Tech Co. Ltd. (Nanjing, China). Carbonyl iron powder (CIP) was obtained from Jilin Zhuochuang New Materials Co. Ltd. (Jilin, China). Ethanesulfonic acid and N-hydroxysuccinimide (NHS) were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). Carbodiimide hydrochloride (EDC), anti-E. coli O157:H7 aptamer (
), conjugated fluorescein amidite (FAM), as shown below, were purchased from Sangon Biotech Co. Ltd. (Shanghai, China) and purified using HPLC. All other reagents used in this work were of analytical grade and solutions were prepared with ultrapure water.
The chosen aptamer could specifically bind to LPS of E. coli O157:H7 according to the report by
Escherichia coli O157:H7 ATCC 25922 and other common pathogenic bacteria associated with dairy products and human health used in this research were cultured in Luria-Bertani broth under gentle shaking (150 rpm) at 37°C and remained overnight. Information about these strains is shown in Table 1. Cell concentrations were determined by plating on Luria-Bertani agar and the cultivated bacteria were washed 3 times with an aseptic phosphate buffer solution (PBS, 0.1 M, pH = 7.4).
Table 1The specificity of CIP@MWCNT-aptasensor assay with selected strains
ATCC = American Type Culture Collection (Manassas, VA); CMCC = China Medical Culture Collection (Beijing, China); CICC = China Center of Industrial Culture Collection (Beijing, China); DSM = DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany); CECT = Colección Española de Cultivos Tipo (Valencia, Spain).
2 ATCC = American Type Culture Collection (Manassas, VA); CMCC = China Medical Culture Collection (Beijing, China); CICC = China Center of Industrial Culture Collection (Beijing, China); DSM = DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany); CECT = Colección Española de Cultivos Tipo (Valencia, Spain).
Fabrication and Characterization of the CIP@MWCNT Nanocomposites
The carboxylated MWCNT (1 mg/mL) were activated and stabilized with EDC (40 mM, 100 µL) and NHS (40 mM, 100 µL) into a clean glass bottle that contained ethanesulfonic acid buffer (0.1 M, pH 5.0) with shaking (180 rpm) for 2 h at room temperature (RT). Afterward, CIP and MWCNT were blended at a mass ratio of 3:1, and the mixture was conducted by shaking (180 rpm) for 60 min at RT. The nanocomposites were magnetically separated and washed repeatedly with deionized water to remove excess reagents. The complex was dried for several hours and stored at RT following application. All above reagents used in this work were filtered through Millipore Express Membrane Filters (0.22 μm, Merck Millipore Ltd., Billerica, MA). The zeta potential of nanomaterials was measured utilizing dynamic laser light scattering (Zetasizer Nano ZS90, Malvern Instruments Ltd., Malvern, UK). The surface functional groups from nanocomposites were probed by Fourier-transform infrared spectroscopy (FT-IR, Thermo Fisher, Waltham, MA). The morphology of nanomaterials was observed using transmission electron microscopy (H-7650, Hitachi High-Technologies Corporation, Tokyo, Japan). The element of nanocomplex was measured by energy dispersive x-ray analysis (EDAX, S-3400N, Hitachi High-Technologies Corporation).
Preparation of the CIP@MWCNT-Based Aptasensor
The CIP@MWCNT-based aptasensor was prepared according to
with modifications. The CIP@MWCNT nanocomplex was dispersed in deionized water by an ultrasound bath (model KQ-500DE, 500 W, 40 kHz, Kunshan Ultrasonic Instrument Co. Ltd., Kunshan, Jiangsu, China) at a concentration of 1 mg/mL for 60 min at 20°C. Then, the FAM-aptamer (100 nM, 100 µL) was mixed with dispersed CIP@MWCNT complex, and the dispersion was treated in deionized water by sonication (500 W, 40 kHz) for 60 min at 20°C. The compounds were maintained overnight at 4°C to attain equilibrium after sonication. Subsequently, the nanocomplex was magnetically separated and washed with deionized water to remove excess aptamer. The washing steps were repeated 4 times and the efficiency of removal free aptamer was measured by a fluorescence microplate reader (model Infinite 200 PRO, Tecan, Grödig, Austria; λexcitation = 492 nm, λemission = 532 nm). After washing, the CIP@MWCNT-based aptasensor pellet was resuspended in autoclaved deionized water.
Principle of the Sensing Strategy
Figure 1 illustrates the analytical process for E. coli O157:H7 on the CIP@MWCNT-based aptasensor. The magnetic nanocompounds based on CIP and MWCNT were formed by redox reaction. The CIP reacted with H+ in acidic solution to form positively charged Fe2+ that adsorbed on the surface of nanoparticles. Under the attraction of the opposite charge, MWCNT with negative charge would bind with CIP particles, which were tightly adsorbed to form magnetic nanocomplexes. The chemical reaction formulas that took place are shown below. Then, the FAM-labeled aptamer wrapped randomly around the sidewalls of CIP@MWCNT due to π–π stacking interactions, and the FAM molecules were in close proximity to the CIP@MWCNT, effectively quenching the fluorescence of the dye. In the presence of E. coli O157:H7, the aptamer left the sidewalls of CIP@MWCNT, causing augmented fluorescence because the aptamer had higher affinity and specificity against E. coli O157:H7 than CIP@MWCNT. Conversely, the fluorescence was turned off because the aptamer was still adsorbed onto the CIP@MWCNT.
Figure 1Schematic illustration of the CIP@MWCNT-based aptasensor for the detection of Escherichia coli O157:H7. CIP = carbonyl iron powder; MWCNT = multiwalled carbon nanotubes; FAM = conjugated fluorescein amidite.
The system containing 500 µL of E. coli O157:H7 and 50 µL of CIP@MWCNT-based aptasensor was vortexed for 20 s in a black microcentrifuge tube. Then, the mixing solution was incubated for 10 min at 37°C in an incubator. Then 200 µL of mixture was taken in a black 96-well polystyrene plate. The fluorescence intensity (FI) was tested by a microplate reader at 532 nm with an excitation wavelength of 492 nm. The whole process applied black vessels and avoided light treatment to protect the fluorophore. Each test was repeated in triplicate. Line charts were recorded as digital documents for subsequent reset and analysis.
Analytical Specificity, Sensitivity, Reproducibility, and Stability
Each solution (500 µL) containing target bacteria was vortexed with a CIP@MWCNT-based aptasensor (50 µL) in microcentrifuge tubes. The FI was measured by fluorescence microplate reader (λexcitation = 492 nm, λemission = 532 nm) after the mixing solutions were incubated at 37°C for 10 min. The specificity of the CIP@MWCNT-based aptasensor was proven against a cluster of pathogenic bacteria (Table 1) considered to be hazardous in food under the same cell concentrations as target detection for E. coli O157:H7. The E. coli O157:H7 was 10-fold serially diluted to obtain different cell concentrations prepared in PBS buffer ranging from 107 to 101 cfu/mL and each dilution was used to analyze sensitivity. In the reproducibility analysis, 8 of the same batch and different batches of CIP@MWCNT-based aptasensor were randomly selected to test the intragroup differences and intergroup differences. Each sample (500 µL) containing E. coli O157:H7 was vortexed with a CIP@MWCNT-based aptasensor (50 µL) from different groups in microcentrifuge tubes. The bacterial concentration of 107 cfu/mL was used for each test. After the mixing solutions were incubated for 10 min at 37°C, the FI was measured by fluorescence microplate reader (λexcitation = 492 nm, λemission = 532 nm). The relative standard deviation (RSD) values were calculated and compared with 5%. In general, a good reproducibility is indicated by RSD values of less than 5% (
). Furthermore, we evaluated the stability of this system for washing E. coli O157:H7 (estimated 107 cfu/mL) by utilizing the CIP@MWCNT-based aptasensor stored at 4°C, −20°C, and RT. Other conditions were maintained equally and the CIP@MWCNT-based aptasensor was tested for 7 wk, and the interval of measurement was 1 wk. For all the above project analyses, a negative control (NC) was designed with PBS and the FI of the assay was tested at 532 nm with an excitation wavelength of 492 nm. These indexes were evaluated using the CIP@MWCNT-based aptasensor in 3 replications. Line charts were recorded as digital documents for subsequent reset and analysis.
Artificially Contaminated Milk with E. coli O157:H7
Commercially available UHT sterilized milk was used to verify the suitability of the sensing assay of the CIP@MWCNT-based aptasensor. The UHT sterilized milk was purchased from supermarkets in Harbin, China, and verified by the conventional culturing method for the absence of E. coli O157:H7 (
). The UHT sterilized milk containing several different concentrations (e.g., 100 to 106 cfu/mL) of E. coli O157:H7 was prepared in PBS (0.1 M, pH = 7.4). The NC group was prepared with UHT sterilized milk, which had no artificial contamination of E. coli O157:H7. The artificially contaminated milk samples and NC group were simultaneously incubated by shaking (180 rpm) for 1 h at 37°C. Each sample (500 µL) was vortexed with CIP@MWCNT-based aptasensor (50 µL) in microcentrifuge tubes. The FI was tested at 532 nm with an excitation wavelength of 492 nm after the mixture was incubated at 37°C for 10 min. These procedures were conducted using the CIP@MWCNT-based aptasensor in 3 replications. Line charts were recorded as digital documents for subsequent reset and analysis.
RESULTS
Characterization of the CIP@MWCNT Nanocomposite
Figure 2A and B show pure MWCNT and CIP@MWCNT nanocomposite in H2O. The CIP@MWCNT nanocomposite was more easily aggregated, which could be relative with the result of transform in charge after the redox reaction. As shown in Figure 2C and D, CIP@MWCNT were attracted by the magnetic stand in aqueous solution, but MWCNT were not. Furthermore, 3 kinds of characterization from different aspects were performed to verify the formation of nanocomposite. Zeta potential and FT-IR spectra are common characterizations of surface charge and functional groups from nanoparticles. As shown in Figure 3A, the zeta potential of MWCNT and CIP was −21.7 and 3.4 mV, respectively, whereas the zeta potential of CIP@MWCNT was −1.4 mv. The reduction of the negative charge might be redox reaction in liquor, and decrease of the same charge could lead to more aggregation. Figure 3B shows the FT-IR spectra of pristine MWCNT, CIP, and CIP@MWCNT. The absorption peaks of O-H and C = O appeared at 3,425 and 1,682 cm−1, respectively, which was due to the carboxyl functional groups on MWCNT and CIP@MWCNT. The new peak at 610 cm−1 was ascribed to the Fe-O bond. The consumption of O-H and generation of Fe-O about signal intensity were attributed to the formation of CIP@MWCNT complexes. In addition, transmission electron microscopy images in Figure 4 revealed that CIP@MWCNT was synthesized successfully. In the present experiment, morphology, texture, and diameter were well preserved comparing MWCNT with CIP@MWCNT in Figure 4A and B. The pristine MWCNT appeared to be reuniting, twisting, and having a large length distribution; nevertheless, some of the MWCNT were slightly curled and exfoliated after redox reaction. Furthermore, the dispersity of CIP@MWCNT was enhanced after the composite reaction. According to the results in Figure 4C, certain CIP were distinctly observed on the outer walls of the MWCNT. The EDAX result showed that the amount of Fe doped into the MWCNT was about 93.25 wt% (Figure 4D). This was verified to determine the CIP in the nanocomposite materials. All of these results implied that the CIP and MWCNT were successfully connected, even in tiny diameters (<8 nm) of MWCNT that were purchased as minimum sizes from Nanjing XFNANO Materials Tech Co. Ltd.
Figure 2(A) Multiwalled carbon nanotubes (MWCNT) and (B) CIP@MWCNT nanomaterials in H2O. (C) MWCNT and (D) CIP@MWCNT nanomaterials in H2O using a magnetic stand. CIP = carbonyl iron powder.
Figure 4Transmission electron microscopy images of (A) pristine multiwalled carbon nanotubes (MWCNT) and (B and C) CIP@MWCNT. (D) Energy dispersive x-ray analysis results of CIP@MWCNT. CIP = carbonyl iron powder.
Determination and Evaluation of the CIP@MWCNT-Based Aptasensor
The washing times of the preparation process needed to be determined to achieve higher accuracy. Figure 5 illustrates the consequence of the wash steps to remove excess aptamer by fluorescent detection. A gradual decline in fluorescence signal values was examined and the FI changes of supernatant were no longer monitored after 4 successive washes, which were consistent with the decreased amount of aptamer in these residues. Thus, the 4 washing times were handled following subsequent operation after the anti-E. coli O157:H7 aptamer and CIP@MWCNT complex were connected.
Figure 5Fluorescence values for residues in the washing. FI = fluorescence intensity. Error bars = mean ± SD (n = 3).
To verify the advantage of the CIP@MWCNT-based aptasensor, the specificity, sensitivity, reproducibility, and stability analyses were examined. From the results of Table 1, the CIP@MWCNT-based aptasensor had excellent specificity toward E. coli O157:H7 over the other pathogenic bacteria. The analytical sensitivity of the CIP@MWCNT-based aptasensor assay was determined by diluting cell concentrations of E. coli O157:H7 from 107 to 101 cfu/mL. As shown in Figure 6, the resulting calibration curve hinted that the FI was proportional to the concentration of the target in the dynamic range from 103 to 107 cfu/mL (Figure 6A) and a good linear response to E. coli O157:H7 was acquired in the range from 104 to 107 cfu/mL (Figure 6B). The linear regression equation is FI = 342.6 log [E. coli O157:H7 (cfu/mL)] – 1,200.8 with a correlation coefficient of 0.9902. Limit of detection of the CIP@MWCNT-based aptasensor was as low as 7.15 × 103 cfu/mL in pure culture. It was calculated as background +3 s, which is explained as the concentration of sample generating signal intensity in accordance with the average of background plus 3 times its standard deviation (
A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.
Validity of a single antibody-based lateral flow immunoassay depending on graphene oxide for highly sensitive determination of E. coli O157:H7 in minced beef and river water.
), and reproducibility was executed with CIP@MWCNT-based aptasensor by comparing identical and different batches (8 parallel for each group). We found that the RSD values of intragroup and intergroup were 3.57% and 4.43%, respectively (Figure 6C), and the measured values of latter were more dispersed. Hence the RSD values of both showed that the CIP@MWCNT-based aptasensor had good reproducibility (<5%). Meanwhile, the stability of the nanomaterial-based biosensor, known as a significant element (
), was also considered in this work. From the results shown in Figure 6D, the fluorescent signals of the CIP@MWCNT-based aptasensor had no significant decline over 4 wk, and were always above the initial value of 94.1%. Of note, the fluorescent signal started to decrease after 4 wk. Although the fluorescent values were still above 91.2% of the initial value at 4°C and −20°C, the fluorescent intensity had decreased to 79.9% stored at RT. These implied that low temperature could maintain the properties of the fluorophore. Thus, the CIP@MWCNT-based aptasensor had good fluorescence stability at 3 usual temperatures for almost a month.
Figure 6(A and B) Sensitivity, (C) reproducibility, and (D) stability of the CIP@MWCNT-based aptasensor for Escherichia coli O157:H7 in pure culture. a.u. = arbitrary units; NC = negative control; FINC = fluorescence intensity (FI) value of negative control FIn = FI value of the nth sample, n = 1, 2, 3, 4, 5, 6, 7, 8; FI1 = FI value of the first sample; RT = room temperature. CIP = carbonyl iron powder; MWCNT = multiwalled carbon nanotubes. Error bars = mean ± SD (n = 3).
Quantification of CIP@MWCNT-Based Aptasensor in Milk
We further explored the performance of the CIP@MWCNT-based aptasensor with spiked E. coli O157:H7 in UHT sterilized milk. The aptasensor had a higher level for directly detecting concentration of E. coli O157:H7 in milk (104 cfu/mL) without incubation than pure culture (103 cfu/mL), which was attributed to the matrix interference of milk. Similar interference of milk on other technologies for detecting E. coli O157:H7 was previously reported (
Rapid and sensitive detection of Escherichia coli O157: H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification.
) and incubation was required. In Figure 7, the results of the calibration curve hinted that the FI was proportional to the concentration of target in the dynamic range from 102 to 106 cfu/mL (Figure 7A) after incubation at 37°C for 1 h. A good linear response to E. coli O157:H7 was acquired in the range from 103 to 106 cfu/mL (Figure 7B). The linear regression equation is FI = 421.9 log [E. coli O157:H7 (cfu/mL)] – 995.13 with a correlation coefficient of 0.9916. Limit of detection of the CIP@MWCNT-based aptasensor was as low as 3.15 × 102 cfu/mL in UHT sterilized milk after 1 h of pre-incubation. It was calculated as background +3 s, which is explained as the concentration of sample generating signal intensity in accordance with the average of background plus 3 times its standard deviation (
A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.
Validity of a single antibody-based lateral flow immunoassay depending on graphene oxide for highly sensitive determination of E. coli O157:H7 in minced beef and river water.
The utilization of carbon materials with nanoscale for preparation of biosensors has had increasing attention following the discovery of carbon nanotubes by Sumio Ijima in 1991 (
). Some studies reported that solvothermal and deposition in situ synthesis of magnetic carbon nanomaterials were widely used, but corrosive and irritating agents were needed in these methods, which could be harmful to the body in case of maloperation. In the present experiment, we synthesized the CIP@MWCNT nanocomposite by a safe and effective one-step method for the first time. The carboxylated MWCNT were activated with EDC and stabilized with NHS, which conquered agminate restriction of MWCNT and improved the solubility in aqueous medium (
Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNT chromatography.
Enhanced detection of infectious pancreatic necrosis virus via lateral flow chip and fluorometric biosensors based on self-assembled protein nanoprobes.
). Then, the compound was obtained by a one-step method based on redox reaction. This would be a promising approach for the synthesis of magnetic carbon nanocomposites due to its rapidity and safety.
Otherwise, the aptasensor was formed from the aptamer carried by CIP@MWCNT, rather than the free aptamer absorbed by nanocomposite after incubation of bacteria and aptamer. This ensured that the entire aptamer of the system came from the CIP@MWCNT-based aptasensor, which was a more accurate detecting strategy that differed from previous assays (
Aptamer biosensing platform based on carbon nanotube long-range energy transfer for sensitive, selective and multicolor fluorescent heavy metal ion analysis.
). In preparation, the application of ultrasound helped to enhance the dissolution and diffusion capacities of the CIP@MWCNT nanocomposite, which facilitated higher reaction rates, more uniform polymer formation, and higher yields (
). The CIP@MWCNT-based aptasensor was effective for detection of E. coli O157:H7 via thoughtful evaluation of specificity, sensitivity, reproducibility, and stability. Meanwhile, the aptasensor was valid and sensitive for detection of E. coli O157:H7 in milk. The detection limit of the present report was acceptable, and was also compared with other detection methods toward E. coli O157:H7 (Table 2). The chemiluminescence-Fe3O4/GO (
Rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium in foods using an electrochemical immunosensor based on screen-printed interdigitated microelectrode and immunomagnetic separation.
) detected E. coli O157:H7 of 4.5 × 103, 3.05 × 103, and 2.05 × 103 cfu/mL in skim milk, milk, and ground beef, respectively. Additionally, some research reported E. coli O157:H7 of 1 × 103 and 1.46 × 103 cfu/mL in meat and PBS solution using a fiber optic biosensor (
Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples.
A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.
Validity of a single antibody-based lateral flow immunoassay depending on graphene oxide for highly sensitive determination of E. coli O157:H7 in minced beef and river water.
), as well as this work. In terms of detecting assays with nanotechnology, the CIP@MWCNT-based aptasensor has several advantages: (1) CIP@MWCNT nanocomposite can be obtained without irritating and corrosive reagents, thus avoiding dangerous and intricate procedures; (2) CIP@MWCNT shows a certain degree of stabilization to single-stranded aptamer by π–π stacking, which can react satisfactorily at RT for up to a month; and (3) the CIP@MWCNT-based aptasensor can tolerate complex dairy matrices and detect pathogens with sensitivity. These distinguishing features showed that this assay could be a prospective way of inspecting pathogens in the dairy industry.
Table 2Comparison of different detection methods toward Escherichia coli O157:H7 in food
Validity of a single antibody-based lateral flow immunoassay depending on graphene oxide for highly sensitive determination of E. coli O157:H7 in minced beef and river water.
A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.
Rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium in foods using an electrochemical immunosensor based on screen-printed interdigitated microelectrode and immunomagnetic separation.
Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples.
In summary, our newly developed CIP@MWCNT-based aptasensor with fluorescence detection could be successfully applied for sensitive diagnosis of E. coli O157:H7 in a contaminated sample matrix. Four kinds of characterization of surface charge, functional groups, and morphology revealed the successful synthesis of nanocomposites through CIP and MWCNT. Under the prepared system of aptasensor, E. coli O157:H7 could be detected at a concentration as low as 7.15 × 103 cfu/mL in pure culture. The detection limit of E. coli O157:H7 was 3.15 × 102 cfu/mL in contaminated milk after 1 h of pre-incubation. In addition, this new type of magnetic nanocomposite modified aptasensor is reliable and stable with good targeting ability according to the performance analyses. All statistical information verified that the CIP@MWCNT-based aptasensor has potential for rapid, sensitive, and reliable detection of E. coli O157:H7 in a practical sample. In the future, this type of developed aptasensor will be used to simultaneously monitor multiple types of foodborne pathogens in real-world samples.
ACKNOWLEDGMENTS
This work was supported by the National Key Research and Development Program of China (No. 2018YFE0120500). The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
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Rapid and sensitive detection of Escherichia coli O157: H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification.
Enhanced detection of infectious pancreatic necrosis virus via lateral flow chip and fluorometric biosensors based on self-assembled protein nanoprobes.
A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.
Validity of a single antibody-based lateral flow immunoassay depending on graphene oxide for highly sensitive determination of E. coli O157:H7 in minced beef and river water.
Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNT chromatography.
Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples.
Removal of reactive red 198 from aqueous solution by combined method multi-walled carbon nanotubes and zero–valent iron: Equilibrium, kinetics, and thermodynamic.
Aptamer biosensing platform based on carbon nanotube long-range energy transfer for sensitive, selective and multicolor fluorescent heavy metal ion analysis.
Rapid detection of Escherichia coli O157:H7 and Salmonella typhimurium in foods using an electrochemical immunosensor based on screen-printed interdigitated microelectrode and immunomagnetic separation.
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