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State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. ChinaJiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R. China
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. ChinaJiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R. China
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. ChinaJiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R. China
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R. ChinaJiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R. China
The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase–hydrogen peroxide (Cat–H2O2) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H2O2 through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 105 cfu/mL. This method also revealed a good linear range (3 × 102 to 3 × 107 cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 102 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 104 cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.
Plasmonic enzyme-linked immunosorbent assay (pELISA) has emerged as a powerful platform for environment monitoring, food safety control, and clinical diagnosis owing to its ultra-high sensitivity and multiple color response to noble nanoparticles (
). Nonetheless, most nanoparticle aggregation-based pELISA usually face potential problems, including unstable auto-aggregation and narrow linear detection ranges (
). Nanoparticle growth-based pELISA are inclined to obtain high sensitivity owing to the small-to-large nanoparticle morphology change that results in low-to-high extinction coefficient of noble nanoparticles (
). The formation and growth kinetics of noble nanoparticles strictly depend on reducing reagents for nanoparticle growth-based pELISA. At high concentration of reducing reagents, the growth kinetics of noble nanoparticles are fast, resulting in a uniform morphology. On the contrary, the kinetics of crystal growth proceed slowly at low concentrations of reducing reagents, thereby contributing to aggregated nanoparticles with ill-defined morphology (
reported a sandwich pELISA based on hydrogen peroxide (H2O2)-mediated gold nanoparticle (AuNP) growth for ultra-low biomarker detection. In this work, altering H2O2 by approximately 0.05 µM (from 119.5 to 120 µM) can produce significant changes in local surface plasmon resonance of AuNP and color of solution (
). Controlling the growth kinetics of noble nanoparticles by reducing reagents can achieve a high sensitivity. However, growth sensing mode directly derived by reducing reagent is more likely obtain nonreproducible results because most reducing reagents are easily oxidized and influenced by surrounding pH, salt, and temperature at low concentration (
). This shortcoming considerably impedes practical applications of nanoparticle growth-based pELISA. Therefore, developing sensitive and robust pELISA remains a prominent challenge.
DNA is a well-known biopolymer and has been used as template to position nanoparticles through DNA metallization or to control the sizes and photoluminescent property of metal fluorescent nanoclusters (
Fluorescence immunoassay through histone-ds-poly (AT)-templated copper nanoparticles as signal transductors for the sensitive detection of Salmonella choleraesuis in milk.
). Moreover, single-strand DNA (ss-DNA) can adsorb on noble nanoparticles due to the strong affinity between the nitrogen of DNA bases and noble atoms (
). During the synthesis of noble nanoparticles, the ss-DNA attached on the surface of noble nanoparticles guides the anisotropic crystal growth and affects the outcome morphology of individual nanoparticles and final color of colloidal solution (
). In the DNA-directed AuNP growth system, excess NH2OH (>20 mM) should be introduced to ensure that Au3+ completely reduces to Au atom and then AuNP anisotropic growth is only relevant to DNA types and amounts adsorbed on the particle surface (
). Furthermore, the high concentration of NH2OH makes the crystal grow much faster to uniform size and subtle changes in NH2OH concentration cannot make a big difference to the final morphology of AuNP because the NH2OH concentration largely exceeds the Au3+ concentration (
). Therefore, DNA-directed AuNP growth is a desirable tool to regulate the local surface plasmon resonance of AuNP in fabricating robust plasmonic sensors. To the best of our knowledge, a pELISA performed through mediation of DNA in the growth kinetic of AuNP has not been reported.
In this work, we report the pELISA performed through mediation of DNA in the growth kinetics of AuNP to control the morphology of resulting AuNP. The ss-DNA is easily sheared by trace amounts of hydroxyl radical (·OH), which generates damaged DNA bases, such as 8-oxoguanine, 8-oxoadenine, thymine glycol, and 8-hydroxycytosine. The damaged DNA cannot attach on the surface of the gold nanoseeds (AuNS); therefore, no DNA can guide the crystal growth of the gold, and ·OH could be produced by Fenton reagent (Fe2+) catalyzed H2O2 (
). Therefore, to integrate DNA-mediated growth kinetic of AuNP into conventional ELISA, a catalase (Cat)–H2O2 system was used to bridge the pELISA and DNA-mediating AuNP growth. Cronobacter was selected as the model target given its association with the disease outbreak causing premature and immunocompromised infants by contaminating infant formula (
). Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (C. muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 105 cfu/mL. This method also revealed a good linear range from 3 × 102 to 3 × 107 cfu/mL for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection (LOD) of 1.6 × 102 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase (HRP)-based conventional ELISA (2.6 × 104 cfu/mL). The proposed pELISA showed good performance in powdered infant formula (PIF) samples spiked with C. muytjensii ATCC 51329. Recoveries ranged from 90.79 to 119.09% with coefficients of variation (CV) below 10%. These results demonstrated that our proposed pELISA can be applied for sensitive detection of Cronobacter in real food samples and exhibits potential for detection of other targets.
MATERIALS AND METHODS
Materials
Luria–Bertani (LB) broth and Baird–Parker agar base were purchased from Land Bridge Technology Co. Ltd. (Beijing, China). Biotin-3-sulfo-N-hydroxysuccinimide ester sodium salt, streptavidin (SA), hydroxylamine hydrochloride (NH2OH·HCl), Cat, HRP, iron (II) chloride tetrahydrate, hydrogen peroxide (H2O2, 35 wt%), chloroauric acid (HAuCl4·3H2O), trisodium citrate (Na3C6H5O7·2H2O), and BSA were purchased from Sigma-Aldrich (St. Louis, MO). The ss-DNA (5′-TAGCTATGG-3′) was commercially synthesized by Sangon Biological Engineering Technology and Service Co. Ltd. (Shanghai, China). Our laboratory prepared monoclonal antibody against C. muytjensii ATCC 51329 and minimal cross-reacted with Cronobacter dublinensis China Center of Industrial Culture Collection (CICC) 24179, Cronobacter turicensis CICC 24178, and C. muytjensii SR 1074B. This antibody labeled as 5H-6 is utilized as capture (cAb) and detection antibodies (dAb) at the same time. The 96-well polystyrene plates were obtained from Corning Inc. (New York, NY). The 180 nm magnetic beads (MB) were obtained from Allrunnano Technology Co. Ltd. (Shanghai, China). The deionized water used throughout the experiments was purified and prepared by using a Milli-Q system (Millipore, Milford, MA). All other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Corp. (Shanghai, China). All reagents were used without further purifications.
Bacteria Strains and Culture Conditions
Supplemental Table S1 (https://doi.org/10.3168/jds.2019-17067) shows the bacterial strains used in this study. All bacterial strains were grown overnight in LB broth medium in an orbital shaker incubator at 180 rpm at a temperature of 37°C. The collected bacterial pellets were resuspended in PBS and stored at 4°C for further utilization. The viable cell count of bacterial strains was determined by surface plating with 100 µL of suspension liquid through a conventional plate counting method. All counting results were counted as colony-forming units. The bacterial samples were sterilized before use for safety.
Synthesis of Citrated-Capped AuNS and Procedure of DNA-Mediating AuNP Growth
A hydrothermal citrate-induced reduction method was used for the synthesis of citrate-capped AuNS with an average diameter of 13 nm (
). In brief, 250 mL of 1 mM HAuCl4·3H2O solution was boiled under vigorous stirring in a conical flask. After 5 min of reflux, 25 mL of 38.8 mM Na3C6H5O7 was rapidly added. The reaction was continually heated for 15 min. During this process, the hue of the mixture changed from earthy yellow to dark red, indicating the formation of citrate-capped AuNS. The resultant solution was immediately kept in an ice bath for another 15 min to terminate the reaction and then filtrated by a 0.22 µM filter membrane. Figure 3C(a) shows the transmission electron microscopy images of the obtained AuNS. The concentration of obtained AuNS was approximately 16.3 nM according to Beer's law.
The DNA-mediating AuNP growth was based on a modified previous method (
). A total of 40 µL of AuNS (16.3 nM) was first incubated with 40 µL of DNA (10 µM) for 2 min to allow DNA adsorption onto the AuNS surface. A total of 20 µL of NH2OH·HCl (80 mM) was then added to the AuNS solution. After vortexing, 20 µL of HAuCl4·3H2O (1 mM) was introduced to the AuNS mixture solution to initiate reduction reaction. A change in color (depending on the amounts of intact DNA absorbed on AuNS surface) was observed in seconds.
pELISA for Cronobacter Detection
The 96-well plates were coated overnight with 80 µL of cAb (10 µg/mL) in bicarbonate buffer (0.01 M, pH 8.6) at 4°C and then blocked with 1 mg/mL BSA solution at 37°C for 1 h. After washing thrice with PBS-Tween (PBST), 80 µL of the desired final concentrations of Cronobacter was added and incubated at 37°C for 1 h. After washing thrice with PBST, 80 µL of dAb@Bio (10 µg/mL) was added for 45 min incubation at 37°C. After washing the plate thrice with PBST, 80 µL of SA (1.25 µg/mL) was added. Following the 30 min reaction at 37°C and after washing the plates thrice with PBST, 80 µL of Cat@Bio (2.5 µg/mL) was added into each well for 45 min incubation at 37°C. Then, the plate was washed 4 times with PBST and twice with ultrapure water, followed by addition of 80 µL of H2O2 (100 µM) for 30 min incubation at 37°C. Thereafter, 30 µL of Fe2+ (2 mM) and 40 µL of DNA (10 µM) were added into each plate well for another 30 min reaction. Eventually, 40 µL of each plate well with resultant solution containing intact ss-DNA and DNA fragments was used for routine DNA-capped AuNP synthesis similar to that described previously. Colorimetric signal was detected by the naked eye and microplate reader. The HRP-based conventional ELISA was developed for comparison with detailed procedures provided in the Supplemental Information (https://doi.org/10.3168/jds.2019-17067).
Sample Pretreatment
Cronobacter muytjensii ATCC 51329 was pretreated by using our previously developed 2-step large-volume immunomagnetic separation method (
). Different concentrations of C. muytjensii ATCC 51329 were separately added into 10 g of PIF. The samples spiked with C. muytjensii ATCC 51329 were mixed with 90 mL of sterilized PBS to achieve a 10% infant formula solution. All samples were prepared in triplicate. A total of 3 µg of dAb@Bio was added to 10 mL of infant formula solution spiked with C. muytjensii ATCC 51329. After incubation at 37°C for 1 h with gentle shaking, 700 µg of SA-modified MB (MB@SA) was subsequently added into the mixture solution at room temperature for 90 min with gentle shaking. Finally, MB@SA@dAb@Bio@C. muytjensii ATCC 51329 complex was separated from the homogenate solution using an external magnetic field and then resuspended in 1 mL of PBS solution. Cronobacter muytjensii ATCC 51329 was dissociated from the resuspension solution by heating at 90°C for 15 min and then separated using an external magnetic field. The supernatants containing C. muytjensii ATCC 51329 were analyzed using our proposed pELISA.
RESULTS AND DISCUSSION
Principle of pELISA for Cronobacter Detection
A novel pELISA with sandwich format based on DNA-directed AuNP growth as signal output was fabricated for Cronobacter detection, and the detailed principle is shown in Figure 1. In this case, the SA–Bio system was introduced as a bridge for the linking of dAb with Cat. When Cronobacter was present in the PIF sample, the microorganism was captured by cAb. The dAb@Bio was captured by Cronobacter in the plate well. The SA acted as a scaffold for linking dAb@Bio and Cat@Bio. Thereafter, H2O2 was consumed by Cat, and a minimal amount of ·OH was generated. Then, a small amount of ss-DNA was damaged and cleaved into fragments by ·OH, contributing a large number of intact ss-DNA adsorbed on AuNS. Under high intact ss-DNA coverage, the AuNP developed flower-like morphology by adding NH2OH·HCl to reduce Au3+ into Au, with a resultant blue solution. Conversely, high H2O2 retention in the plate well led to better ·OH generation, and spherical AuNP were obtained, featuring resultant red solutions. Such morphological and color changes can be further characterized by UV–visible spectrum. Therefore, the concentration of Cronobacter can be qualitatively detected by the naked eye and quantitatively detected by plate reader.
Figure 1The principle of the proposed plasmonic ELISA. SA = streptavidin; cAb = capture antibody; dAb = detection antibody; Cat@bio = biotin-labeled catalase; dAb@bio = biotin-labeled detection antibody.
To verify the possible application of AuNP growth through DNA mediation in pELISA, we first investigated the influence of H2O2 on AuNP growth with NH2OH·HCl as a reducing reagent. Normally, special buffer, such as 2-(N-morpholino)ethanesulfonic acid, and exact pH are required for AuNP kinetic growth tuned by H2O2 (
). In our designed DNA-directed AuNP growth system, NH2OH.HCl is used to completely consume the Au3+, resulting in an extremely acidic environment, whereas H2O2 cannot play its reduction activation. As shown in Supplemental Figure S1A, in the absence of NH2OH·HCl and at H2O2 concentration ranging from 0 to 400 µM in the AuNP growth system, the color of AuNP solution remained unchanged with a low and stable optical density (OD)520 value. However, when 40 mM of NH2OH·HCl was added, the color of AuNP solution notably changed from pale to brick red, and the OD520 value increased from 0.32 to 0.75. These results proved that H2O2 was irrelevant for AuNP growth, and NH2OH·HCl was a driving factor for AuNP growth in our designed system. In addition, the effect of NH2OH·HCl concentration on AuNP growth response was investigated over a range of 0 nm to 160 mM as excessive amounts of NH2OH·HCl can cause the aggregation of AuNP (
). The results in Supplemental Figure S1B (https://doi.org/10.3168/jds.2019-17067) show that when NH2OH·HCl concentration was above 80 mM, the solution color changed from red to blue, and absorbance presented a significant red shift. Thus, 80 mM NH2OH·HCl was selected for the following experiment. Moreover, excess Fe2+ can adsorb on the surface of citrated AuNP screening the negative charge of AuNP and result in aggregation of AuNP (
). The effect of Fe2+ on AuNP growth was evaluated by adding 0 to 16 mM Fe2+ into AuNP growth solution containing 80 mM NH2OH·HCl. Results showed no remarkable color or absorbance change in the resultant solution when Fe2+ concentration was below 4 mM (Supplemental Figure S1C; https://doi.org/10.3168/jds.2019-17067). Finally, 2 mM Fe2+ was used for the following experiments because this concentration is sufficient to convert H2O2 into ·OH.
A previous study demonstrated that the morphology of AuNP growth is independent of the length of DNA sequences (
). Moreover, the synthesis of a short DNA sequence (e.g., less than 5 bases) is technically difficult. Thus, a ss-DNA of 9 bases with a random sequence was designed to mediate the anisotropic crystal growth of AuNP. The effect of the extent of ss-DNA adsorption on AuNP growth reaction was investigated. As evident in Figure 2A, the color of AuNP solution changed from red to purple and blue as ss-DNA amounts increased in a typical AuNP growth run. The corresponding UV–visible absorption spectra showed that the red AuNP solution exhibited a single surface plasmon resonance peak centered at 520 nm, whereas the purple and blue AuNP solutions displayed 2 peaks at 520 and 620 nm, respectively (Figure 2B). For convenience of characterizing surface plasmon resonance changes, the absorbance ratio (OD620/OD520) was used to evaluate the optimal parameters for the following experiments. A large OD620/OD520 indicates the flower-like appearance of resulting AuNP due to high amounts of ss-DNA adsorbed on AuNP. On the contrary, adsorption of low amounts of ss-DNA on AuNP led to spherical shapes in the outcome AuNP. As shown in Figure 2C, the value of OD620/OD520 increased as ss-DNA concentration increased from 0 to 10 µM. The value started to plateau when ss-DNA concentration further increased. The AuNP solution turned blue with 10 µM ss-DNA mediating the AuNP growth. Such change in color is easily distinguished from that of AuNP solution without DNA mediation. Therefore, 10 µM ss-DNA was applied for mediating AuNP growth.
Figure 2(A) Photographs of resultant gold nanoparticle (AuNP) solution with the addition of different DNA concentrations. (B) Ultraviolet–visible absorption spectra of resultant AuNP solution with the addition of different DNA concentrations. (C) Corresponding optical density at 620 nm (OD620)/optical density at 520 nm (OD520) of the resultant AuNP solution with the addition of different DNA concentrations. Each value represents the mean of 3 independent experiments (n = 3). Error bars represent SD of 3 measurements. a.u. = arbitrary units.
In addition, H2O2 is an important component in determining the sensitivity of pELISA because it can be converted into ·OH with the aim of regulating the amounts of intact ss-DNA by Fe2+. Thus, AuNP growth through DNA mediation in the presence of H2O2 was investigated by incubating 10 µM ss-DNA with different concentrations of H2O2 ranging from 0 to 800 µM in the presence of 2 mM Fe2+ for 30 min. Then, a typical DNA-mediating growth reaction was carried out. Figure 3A shows that the color of AuNP solution significantly changed from blue to purple and then to red with increasing H2O2 concentration, whereas the corresponding OD620/OD520 decreased from 0.56 to 0.23 (Figure 3B). The OD620/OD520 value of the AuNP solution also displayed a good linear trend with H2O2 concentration ranging from 6.25 to 100 µM, yielding a reliable correlation coefficient of 0.9807 (Figure 3B insert). The LOD of H2O2 toward the change of OD620/OD520 value was calculated to be 8.03 µM. Notably, when H2O2 concentration was 100 µM, a vivid color change from red to blue was observed by the naked eye. The AuNP growth through DNA-mediating in the presence of H2O2 was further confirmed by transmission electron microscopy. With increasing H2O2 levels, flower-like AuNP gradually diminished and transformed into a spherical shape similar to the AuNP without DNA adsorption (Figure 3C, a–e). Moreover, good dispersion and physically uniform nanoparticles demonstrated the high stability of our designed AuNP growth system. This finding was also validated by monitoring of OD620/OD520 values for 2 h. As shown in Supplemental Figure S2 (https://doi.org/10.3168/jds.2019-17067), the OD620/OD520 reached the maximum value within 2 min and remained relatively stable for the next 2 h. The above results indicate that the DNA-directed AuNP growth system features the potential to fabricate sensitive and robust pELISA. Finally, to obtain a stable colorimetric signal and spectral signal, we designated 100 µM H2O2 as the optimal concentration in the subsequent experiments.
Figure 3(A) Photograph of resultant gold nanoparticle (AuNP) solution under different concentrations of H2O2-induced DNA-mediating AuNP growth. (B) Optical density at 620 nm (OD620)/optical density at 520 nm (OD520) value of resultant AuNP solution under different H2O2 concentrations. (C) Transmission electron microscopy images of resultant AuNP under different concentrations of H2O2-induced DNA-mediating AuNP growth. (a) Gold nanoseeds (AuNS), (b) 100 µM, (c) 50 µM, (d) 25 µM, and (e) 0 µM H2O2. Each value represents the mean of 3 independent experiments (n = 3). Error bars represent SD of 3 measurements. LOD = limit of detection.
Optimization of Experimental Parameters and Analytical Performance
Before establishing the AuNP growth-based pELISA through DNA mediating for Cronobacter detection, several parameters that affect analytical sensitivity, including the concentrations of cAb, dAb@Bio, SA, and Cat@Bio, were investigated. The preparation and characterization of dAb@Bio and Cat@Bio are shown in Supplemental Figures S3A–S3B (https://doi.org/10.3168/jds.2019-17067). The concentrations of cAb and dAb@Bio were optimized by using checkboard titration with different of dAb@Bio contents under a series of cAb on the plate well. In this case, the concentration of C. muytjensii ATCC 51329 was set to 106 cfu/mL. The results in Supplemental Table S2 and Figure S4 (https://doi.org/10.3168/jds.2019-17067) indicate that both 10 µg/mL of cAb and 10 µg/mL of dAb@Bio were the optimized concentrations for the following experiments. These values were obtained because the plate well of C. muytjensii ATCC 51329 positive sample appeared purple, whereas that of OD620/OD520 slightly changed when dAb@Bio and cAb continually increased. In addition, the concentrations of SA and Cat@Bio were optimized by determining C. muytjensii ATCC 51329 at 106 cfu/mL. The results in Supplemental Figure S5A (https://doi.org/10.3168/jds.2019-17067) suggest that the OD620/OD520 increased as SA concentration increased from 0.31 to 1.25 µg/mL. The OD620/OD520 reached a plateau when the SA concentration was further increased. Thus, 1.25 µg/mL was determined as the optimal concentration of SA in subsequent experiments. Supplemental Figure S5B (https://doi.org/10.3168/jds.2019-17067) indicates that the OD620/OD520 significantly increased from 0.44 to 0.72 as the Cat@Bio concentration increased from 0.31 to 1.25 µg/mL and then stabilized when Cat@Bio concentration further increased. An excess amount of Cat@Bio benefits the achievement of a stable signal. Thus, 2.5 µg/mL of Cat@Bio was selected as the optimal concentration for the following experiments.
Under optimal conditions, the sensitivity of the proposed pELISA was evaluated by detecting different concentrations of C. muytjensii ATCC 51329 from 3 × 100 to 3 × 108 cfu/mL. The calibration curve was constructed by plotting the OD620/OD520 values versus the logarithmic of C. muytjensii ATCC 51329 concentration. The linear relationship for quantitative C. muytjensii ATCC 51329 detection ranged from 3 × 102 to 3 × 107 cfu/mL, whereas the regression for quantitative C. muytjensii ATCC 51329 detection can be described as y = 0.0217 ln(x) + 0.3418 (R2 = 0.9932), as shown in Figure 4b. The LOD, defined as the blank signal minus 3 standard deviations, of the proposed pELISA for C. muytjensii ATCC 51329 detection measured 1.6 × 102 cfu/mL. This value is approximately 162.5 times lower than that of HRP-based conventional ELISA (Supplemental Figure S6; https://doi.org/10.3168/jds.2019-17067). Stereographs in Figure 4a indicate that the AuNP solution produced contrasting colors from red to purple, and no false results were observed in 5 duplications when C. muytjensii ATCC 51329 concentrations exceeded 3 × 105 cfu/mL. This color change was easily distinguished by the naked eye, and the robust plasmonic signal output was attributed to the DNA-directed AuNP growth. Eventually, 3 × 105 cfu/mL was defined as the cut-off limit for the qualitative detection of C. muytjensii ATCC 51329 by the naked eye. In addition, we further compared the detection performance of our proposed method with that of other reported works. Results in Table 1 reveal that the sensitivity of our method is comparable with that of other methods, and our proposed pELISA possesses advantage in terms of high throughput and stable data collection for the detection of Cronobacter.
Figure 4Qualitative or quantitative detection of Cronobacter muytjensii ATCC 51329 with the proposed plasmonic enzyme-linked immunosorbent assay (pELISA). (a) Qualitative detection (naked eye) of C. muytjensii ATCC 51329 by using our proposed pELISA with 5 duplications. (b) Calibration curve of C. muytjensii ATCC 51329 detected by the proposed pELISA. Each value represents the mean of 5 independent experiments (n = 5). Error bars represent SD of 3 measurements. OD = optical density; LOD = limit of detection.
A disposable electrochemical immunosensor arrays using 4-channel screen-printed carbon electrode for simultaneous detection of Escherichia coli O157: H7 and Enterobacter sakazakii..
Electrochemical coupled immunosensing platform based on graphene oxide/gold nanocomposite for sensitive detection of Cronobacter sakazakii in powdered infant formula.
Rapid and sensitive detection of Cronobacter spp.(previously Enterobacter sakazakii) in food by duplex PCR combined with capillary electrophoresis–laser-induced fluorescence detector.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2013; 921–922 (23416290): 15-20
Immunochromatographic strip for rapid detection of Cronobacter in powdered infant formula in combination with silica-coated magnetic nanoparticles separation and 16S rRNA probe.
To determine the specificity of the proposed method, we performed the proposed pELISA to detect C. muytjensii ATCC 51329, Cronobacter sakazakii ATCC 29544, C. turicensis CICC 24178, C. condimenti CICC 24184, Cronobacter malonatics National Center for Medical Culture Collections (CMCC) 45402, C. dublinensis CICC 24179, C. sakazakii YC 633B, C. muytjensii SR 1074B, and 11 common pathogenic bacterial strains, including Salmonella choleraesuis, Salmonella enteritidis, Salmonella typhimurium, Shigella flexneri, Listeria monocytogenes, Proteus vulgaris, Escherichia coli O157:H7, Micrococcus lutes, Bacillus subtilis, and 2 Staphylococcus aureus strains. Figure 5 reveals that significant increases in OD620/OD520 only occurred in the presence of C. muytjensii ATCC 51329 (107 cfu/mL), whereas weak signals for other Cronobacter species and negligible signal changes for nontarget bacteria (107 cfu/mL) were observed. These findings demonstrate the high selectivity of the proposed pELISA for Cronobacter species.
Figure 5Selectivity of the proposed plasmonic enzyme-linked immunosorbent assay (pELISA). A negative control test was performed by adding sterile PBS solution. Each value represents the mean of 3 independent experiments (n = 3). Error bars represent SD of 3 measurements. OD = optical density; ATCC = American Type Culture Collection; CICC = China Center of Industrial Culture Collection; CMCC = National Center for Medical Culture Collections.
The precision of intra- and interassay was used to evaluate the accuracy of the proposed pELISA by analyzing 4 different concentrations (3 × 103, 3 × 104, 3 × 105, and 3 × 106 cfu/mL) of PIF samples spiked with C. muytjensii ATCC 51329. The intra-assay was carried out in quadruplicate on the same day, whereas inter-assay was executed on 3 sequential days. Interferences from the infant formula solution matrix were assessed by using 2-step large-volume immunomagnetic separation for the isolation and enrichment of C. muytjensii ATCC 51329 from samples before detection. Table 2 shows the average recovery for intra-assay ranging from 90.79 to 115.90%, with CV ranging from 4.24 to 9.55%. On the other hand, recovery for inter-assay ranged from 92.48 to 119.09%, whereas CV ranged from 5.88 to 9.13%. These results reveal that our developed pELISA exhibits an acceptable level of accuracy for detection of C. muytjensii ATCC 51329.
Table 2Recovery and precision of the proposed plasmonic enzyme-linked immunosorbent assay (pELISA) in Cronobacter muytjensii 51329-spiked milk samples
In this study, AuNP growth through DNA mediation was successfully integrated with a conventional ELISA platform to detect Cronobacter in PIF sample by the naked eye and using a plate reader. Compared with reducing reagent-regulated AuNP growth, the DNA-directed AuNP growth is tolerant to the surrounding environment. Therefore, the signal readout is considerably stable. The proposed sandwich pELISA also exhibited an excellent sensitivity for quantitative detection of Cronobacter and good accuracy in PIF samples. The vivid color change from red to blue can be successfully used to screen Cronobacter, and DNA was directly applied to pELISA without complex modification. Consequently, the proposed method is easy and convenient to operate and suitable for use in resource-constrained regions. Therefore, the developed sandwich pELISA shows potential for naked eye and quantitative detection of other analytes in food safety, clinical diagnostics, and environmental monitoring.
ACKNOWLEDGMENTS
This work was supported by a grant from the National Key Research and Development Program of China (2018YFC1602202 and 2018YFC1602203), National Natural Science Foundation of China (31760485), and “5511” superior science and technology innovation team project of Jiangxi province (China). Ying Xiong acknowledges financial support from the Chinese Scholarship Council.
Immunochromatographic strip for rapid detection of Cronobacter in powdered infant formula in combination with silica-coated magnetic nanoparticles separation and 16S rRNA probe.
A disposable electrochemical immunosensor arrays using 4-channel screen-printed carbon electrode for simultaneous detection of Escherichia coli O157: H7 and Enterobacter sakazakii..
Rapid and sensitive detection of Cronobacter spp.(previously Enterobacter sakazakii) in food by duplex PCR combined with capillary electrophoresis–laser-induced fluorescence detector.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2013; 921–922 (23416290): 15-20
Electrochemical coupled immunosensing platform based on graphene oxide/gold nanocomposite for sensitive detection of Cronobacter sakazakii in powdered infant formula.
Fluorescence immunoassay through histone-ds-poly (AT)-templated copper nanoparticles as signal transductors for the sensitive detection of Salmonella choleraesuis in milk.
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