Size-Dependent Immunochromatographic Assay with Quantum Dot

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Size-dependent Immunochromatographic Assay with Quantum Dot Nanobeads for Sensitive and Quantitative Detection of Ochratoxin A in Corn Hong Duan, Xiaolin Huang, Yanna Shao, Lingyan Zheng, Liang Guo, and Yonghua Xiong Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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Analytical Chemistry

Size-dependent Immunochromatographic Assay with Quantum Dot Nanobeads for Sensitive and Quantitative Detection of Ochratoxin A in Corn Hong Duana,b, Xiaolin Huanga,b, Yanna Shaoa,b, Lingyan Zhenga,b, Liang Guoa,b, Yonghua Xiong*a,b a

State Key Laboratory of Food Science and Technology, Nanchang University,

Nanchang 330047, P. R. China; b

Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, P. R.

China; *Correspondence to: Dr. Yonghua Xiong State Key Laboratory of Food Science and Technology, and Jiangxi-OAI Joint Research Institute, Nanchang University Address: 235 Nanjing East Road, Nanchang 330047, P.R. China Phone: +0086-791-8833-4578. Fax: +0086-791-8833-3708. E-mail: [email protected].

ACS Paragon Plus Environment


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ABSTRACT: Fluorescent microspheres are a novel luminescent nanomaterial proposed as an alternative probe to improve the detection sensitivity of competitive immunochromatographic assay (ICA). Quantum dot nanobeads (QBs) possess strong luminescence and resistance to matrix interference. Theoretically, large-sized QBs exhibit stronger luminescent intensity than small-sized QBs and are beneficial to ICA sensitivity. However, oversized QBs may reduce the sensitivity of competitive ICA. Thus, the relationship between the size and luminescent intensity of QBs and the competitive ICA sensitivity must be elucidated. In this study, QBs of different sizes (58, 124, 255, 365, and 598 nm) were synthesized. Ochratoxin A (OTA) was selected as model analyte for competitive ICA. The effects of QB size on the detection performance of competitive ICA were then evaluated. The cut-off limit of QB-ICA for naked eye detection was used for qualitative analysis, and the half maximal inhibitory concentration (IC50) and LOD were employed for quantitative analysis. Results indicated that 124 nm QBs used as labeling probes for competitive ICA showed the optimal detection performance and the lowest cut-off value of 5 ng/mL for qualitative detection and IC50 (0.39 ng/mL) for quantitative detection. Similar to commercial ELISA, QB124-ICA displayed good accuracy, specificity, reproducibility, and practicability. In summary, 124-nm QBs can be used as a new labeling probe for competitive ICA. Keywords: different-sized quantum dot beads, immunochromatographic assay, ochratoxin A


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INTRODUCTION Immunochromatography assay (ICA) is an important platform widely used in clinical diagnosis, food safety, animal health, and environment monitoring.1,2 ICA exhibits outstanding characteristics, such as easy to use, rapidity, low cost, and user friendliness, particularly for on-site screening.3-5 ICA with competitive format has been extensively applied to detect small chemical molecules with one epitope. Traditional competitive ICA commonly uses gold nanoparticles (GNPs) as colorimetric labels for signal output.3,6 However, low bright intensity of conventional 20–30 nm GNPs results in a poor sensitivity of competitive ICA,7 which constraints its further application in some cases required with high sensitivity. Various fluorescent microspheres, such as quantum dot (QD) or dye-doped beads, with diameter ranging from 10 nm to 1.0 µm,8-12 are proposed as alternative to GNP probes to improve the analytical performance of competitive ICA because of their strong luminescence. Theoretically, large-sized luminescent beads are beneficial for enhancing the detection sensitivity of competitive ICA because they can contain higher amounts of luminescent materials compared with small-sized nanobeads. However, using oversized nanobeads as probes would reduce the sensitivity of competitive ICA. For example, Laitinen et al. explored the size of GNPs in range of 20 nm to 40 nm on the sensitivity of competitive ICA, and found that the larger size of GNPs (39 nm) increased both maximal signals and sensitivity of the assay than smaller particles for determination of progesterone in milk.13 Our previous study further compared four kinds of AuNPs with sizes of 20, 60, 100, and 180 nm on the



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sensitivity of competitive ICA, and demonstrated that large-sized GNPs (100 nm) used as probe exhibited potential to improve the detection performance of competing ICA method because of their high molar extinction coefficients; however, oversized GNPs (180 nm) could reduce the sensitivity of strip assay.7 Therefore, the effect of the size and fluorescent intensity of fluorescent microspheres on the detection performance of competitive ICA must be investigated for further applications. QDs are optimum fluorescent labels because of their broad excitation, narrow fluorescent emission spectra, high quantum yield, and large molar extinction coefficient.14 QD-embedded luminescent beads (QBs) are used to improve the sensitivity of ICA because it encapsulates thousands of QDs into a polymer or silica matrix, leading to more intense fluorescence signals than the original QDs.15,16 For example, Li et al. used QBs (60 nm) as signal probe in a sandwich ICA for highly sensitive detection of prostate specific antigen.17 Hu et al. developed QBs (272 nm)-based sandwich ICA for sensitive detection of C-reaction protein.18 Our previous study demonstrated the feasibility of using QBs (247 nm) as label to construct highly sensitive competitive ICA.9 However, to our best knowledge, the effect of the size and fluorescent intensity of luminescent beads on the analytical performance of competitive ICA has not yet been comprehensively evaluated. In this study, we synthesized five kinds of strong luminescent QBs, with sizes of 58, 124, 255, 365, and 598 nm (named QB58, QB124, QB255, QB365, and QB598, respectively). The resultant QB58, QB124, QB255, QB365, and QB598 exhibited approximately 3.2×102, 1.8×103, 1.5×104, 3.9×104, and 1.3×105 times higher


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luminescence intensity than the corresponding QDs. Ochratoxin A (OTA) was selected as model analyte in the assay. OTA is a common mycotoxin contaminant in agricultural products and categorized as a potential human group 2B carcinogen.19,20 Anti-OTA ascetics were covalently conjugated to the surface of QBs as labeling probes in competitive ICA. The effects of size and fluorescent intensity of the resultant QBs on the detection performance of competitive ICA were evaluated using the cut-off limit of QB-ICA for naked eye detection and the half maximal inhibitory concentration (IC50) by strip reader for quantitative analysis. Results demonstrated that oversized QBs inversely showed lower sensitivity in competitive ICA although presented stronger luminescence. Therefore, this work can serve as a reference for selecting the appropriate size of fluorescent microsphere in competitive ICA for detection of small chemical molecules.



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EXPERIMENTAL SECTION Synthesis and Modification of QBs. Highly luminescent QBs with five

different sizes of 58, 124, 255, 365, and 598 nm were synthesized according to our previously reported method with some modifications.21 Briefly, 5 mg of octadecylamine-coated CdSe/ZnS QDs were fully dissolved in 25 µL of cyclohexane. Then, 500 µL of 3.5 mM SDS aqueous solution was added into the organic phase. The mixture of organic and aqueous phases was emulsified with an ultrasonicator for 2 min (working: 5 s, pausing: 10 s) at power of 27, 72, 90, 135, and 270 W, respectively. Afterward, the mini-emulsion was heated at 70 °C for 2 h to evaporate the cyclohexane and to obtain the QBs stabilized by SDS in the solution. The resulting QBs with sizes of 58, 124, 255, 365, and 598 nm were collected by centrifugation at 13500, 12000, 10000, 8000, and 6000 rpm for 15 min, respectively. The precipitates were resuspended in 1 mL of PB solution (0.01 M, pH 7.4) and then mixed with 100 µL of 10% BSA solution (w/v) under magnetic stirring for 2 h. After purification by centrifugation,

the

resultant

QB@BSA

was

added

into

the

excess

EDC/NHSS-activated Carboxyl-PEG2K-Carboxyl solution. After reaction for 1 h, the carboxyl-modified QB@BSA (QB@COOH) was obtained through centrifugation. Preparation of QB@mAbs. The unpurified ascetic fluid containing 4% anti-OTA mAbs was conjugated to QBs directly, as previously described with some modifications.22 Briefly, 100 µL of 10 mg/mL EDC, 0.2 mg of QB@COOH with size of 58, 124, 255, 365, and 598 nm, and desired anti-OTA mAbs were added to 1 mL of 0.01 M PB buffer (pH 6.0), respectively. After reacting at room temperature for 1 h


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under magnetic stirring, 100 µL of 10% BSA (w/v) and 100 µL of 10 mg/mL EDC were added to the above mixture for another 1 h reaction. Finally, the mixtures were centrifuged, and the precipitates were resuspended with 1 mL of phosphate buffered saline (PBS) (0.01 M, pH 7.4) containing 2% fructose, 1% polyethylene glycol (PEG20000), 5% sucrose, 1% BSA, and 0.4% Tween-20. The resuspension solution was stored at 4 °C for further use. Fabrication of QB-ICA. The preparation of the strip was conducted according to some previous reports.21 As shown in Scheme 1B, the QB-ICA was composed of three parts: sample pad, nitrocellulose (NC) membrane, and absorbent pad. The BSA@OTA conjugates and goat anti-mouse IgG were spotted onto NC membranes as the test (T) and control (C) lines, respectively, and then dried at 37 °C for 12 h. The sample pads were treated with 20 mmol/L sodium borate buffer (pH 8.0) containing 1.0% (w/v) BSA, 0.1% (w/v) NaN3, and 0.25% Tween-20. The distance between the T and C lines was approximately 5 mm. After drying at 60 °C for 2 h, the sample pad, NC membrane, and absorption pad were attached to a plastic backing plate, and then cut into 4 mm-wide strips and packaged in a plastic casing for subsequent storage in a drying cylinder at room temperature. Performance Comparison with Differently Sized QB-ICA. The desired amounts of QB probes with five different sizes were premixed with 70 µL of PBS for 5 min, and then added into the well of the sample pad. After a 5-min reaction, the fluorescence intensity in the T and C lines (FIT and FIC) were recorded by using a homemade fluorescent reader, which was provided by Shanghai Huguo Science



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Instrument Co., Ltd. (Shanghai, China). The competitive inhibition curves for five differently sized QB probes were established by detecting a series of standard OTA solutions with final concentrations of 0 (as negative control) to 30 ng/mL. The FIT/FIC values of negative control and positive standard samples were designated B0 (FIT/FIC of negative control) and B (FIT/FIC of positive samples), respectively. The standard curves were constructed by plotting the inhibition rate (B/B0) against the logarithm of the OTA concentrations. Comparison of ICA detection performance with five-sized QB was performed based on the cut-off limit by the naked eye detection for qualitative analysis and the IC50 by strip reader for quantitative analysis. Preparation of OTA-Spiked Corn Samples. OTA-free corn samples (determined by LC–MS/MS9) were collected from the grain procurement agencies (Shandong, China). The above real samples were fortified with OTA concentrations ranging from 4.8 µg/kg to 36 µg/kg for the proposed QB-ICA assay. For comparison, a commercial OTA ELISA Kit was simultaneously used to detect these spiked samples. The detailed sample extraction procedure followed the manufacturer’s instructions. In brief, 5.0 g of the pulverized sample was extracted with 10 mL of methanol–water (60:40, v/v) for 20 min on a vortex shaker. After centrifugation at 12,000 rpm for 10 min, the supernatant solutions were stored at −20 °C and further diluted 12-fold with PB (0.01 M, pH 7.4) prior to analysis.


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RESULTS AND DISCUSSIONS Synthesis and Characterization of QBs. Although various commercial

fluorescent microspheres have been widely proposed as labeling probes to enhance the detection sensitivity in immunoassays, no definite good correlation exists between the luminescence intensity and size of various commercial fluorescent microspheres. This observation may be attributed to the difference in the synthetic methods used for the preparation of commercial fluorescent microspheres. Thus, using differently sized commercial fluorescent microspheres directly to investigate the effects of luminescence intensity and size of fluorescent microspheres on the detection performance of competitive ICA method is not appropriate. To circumvent the difference from different synthetic methods, in this present study, differently sized QD-embedded fluorescent microspheres (QBs) were prepared according to our previously described method with slight modification. Scheme 1A exhibits the procedure of QB synthesis. First, the hydrophobic octadecyl amine-coated CdSe/ZnS QDs with an average size of 8.5 ± 1 nm (Figure S1) were completely dissolved in cyclohexane to form an oil phase. The oil phase was mixed with a water phase containing SDS to obtain oil droplets as a mini-emulsion under the ultrasound. After evaporation of the cyclohexane, the hydrophobic QDs tended to form compact aggregates stabilized by the SDS in the solution. After centrifugation, the resultant aggregates were separated and added into BSA solution to allow the strong electrostatic adsorption of BSA on their surface, where BSA was introduced to reduce unwanted nonspecific binding and provide the amino group for the conjugation of



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Carboxyl-PEG2K-Carboxyl. After being introduced into double carboxyl reagent to perform the carboxyl coating for good biocompatibility, the proposed QB@COOH complex particle was finally obtained. Previous studies proved that the size of the complex particle can be adjusted by changing the soluble solid content in oil phase and with the increase of ultrasonic power.23 Moreover, the more soluble solid content, the bigger the complex particle. In this present study, we synthesized five kinds of different sizes of QBs by successively increasing the ultrasonic power. Transmission electron microscopy (TEM) images shown in Figures 1A1-E1 indicate that the five kinds of QBs present relatively uniform and regular quasi-spherical with average sizes of 58, 124, 255, 365, and 598 nm, respectively. These results are in accordance with the dynamic light scattering (DLS) data (Figures 1A2–E2). Meanwhile, DLS analysis revealed that the polydispersity indices (PDIs) of QB58, QB124, QB255, QB365, and QB598 were 0.109, 0.114, 0.187, 0.060, and 0.152, indicating that the as-prepared QBs exhibit good mono-dispersity. The fluorescent spectrum of the obtained QBs in Figure 2A displayed that a characteristic fluorescence emission peak at 620 nm was observed, and no significant shifts appeared in the wavelength of the five QBs compared with that of the original QDs, indicating that the surface ligands of the original QDs were not damaged during QB preparation. Although the emission peaks of five differently sized QBs were in the same position as the single QDs, the FIs of QB58, QB124, QB255, QB365, and QB598 were approximately 3.2×102, 1.8×103,1.5×104, 3.9×104, and 1.3×105 times stronger than the corresponding QDs, respectively. The detailed calculation can be found in Supporting Information (Figures S1–S2, Tables S1–S3).


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Moreover, Figure 2B shows the photograph and fluorescence image of five sized QBs under the same molar concentration. The above-mentioned results suggested that the five sized QBs with an excellent luminescent intensity were successfully synthesized, which could be used as labels for improving the analytical sensitivity of ICA. Optimization of the QB Probes and QB–ICA. The QB@mAb conjugates were prepared by coupling the carboxyl group of the QB@COOH with the amino group of anti-OTA mAbs using the EDC method as previously described. Here, the unpurified anti-OTA ascitic fluids containing 4% anti-OTA mAbs were directly used to conjugate with QB@COOH, while the miscellaneous protein in the ascitics was used to block the excess carboxyl groups of the QBs to reduce the nonspecific binding of the QBs with NC membrane and the antigen on the test line. The saturated labeled amounts of anti-OTA ascitic fluids of differently sized QB were evaluated by recording the FIT value using fluorescent reader. Figure 3A indicates that the FIT values of five sized QB-based strips exhibit a similar trend, where the FIT values increase sharply with increasing ascitic fluid content, and then gradually decline with further increase of ascitic fluid amounts. This observation is attributed to the excessive mAbs on the surface of QBs, which may result in steric hindrance of the mAbs, as a result, QB@mAbs present low biological activities that is bad for the interaction of QB@mAbs and OTA@BSA pre-immobilized on the T line, thereby leading to a low FIT value. Therefore, the highest FIT values of 1584 ±33, 1557 ± 39, 1514 ± 42, 1551 ± 23, and 1506 ± 78 were obtained when the saturated labeled amounts of anti-OTA ascitic fluids were 400, 265, 200, 132.5, and 66.25 µg/mg of QB58, QB124, QB255,



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QB365, and QB598, respectively. To obtain the best sensitivity and an appropriate FI signal on both T and C lines for QB158, QB124, QB255, QB365, and QB598, five orthogonal experiments of L9 (3)3 were designed to optimize the amounts of QB@mAbs, the conjugation ratio between OTA and BSA, and the concentration of OTA@BSA on T line. The FIT, FIC, and competitive inhibition rate ((1-B/B0) ×100%) were recorded to determine the optimal parameters, where B0 corresponds to the FIT/FIC of the negative control, and B corresponds to FIT/FIC of the OTA standard solution. QB124-ICA shows the best sensitivity than other four QB-ICA, results shown in Table S5 indicate that the optimal combinations were 2.0 mg/mL of OTA@BSA with a mole ratio of 10:1 and 1.0 mg/mL of goat anti-mouse IgG sprayed on the T and C lines, respectively; and 0.5 µL of QB124@mAb (0.2 mg/mL) pre-incubated with 70 µL of PBS for running the QB124-ICA strip. Under the optimal conditions, the means of the FIT and FIC were 661 ± 33 and 220 ± 40, respectively. The competitive inhibition rate for 1 ng/mL OTA-spiked corn sample was 80.14% ± 2.95%. The results for QB124-ICA, QB255-ICA, QB365-ICA, and QB598-ICA were shown in Table S5, Table S6, Table S7, and Table S8, respectively. To better estimate the effect of QB size on the analytical sensitivity, the FIT values were adjusted to the same level of 650 for five different-sized QB to avoid the interference from fluorescence variation of the T line on the ICA sensitivity. Immunological Kinetics Analysis of Five-sized QB Probes on the Strip. Large-sized QBs exhibit a slower migration rate than small-sized QBs on the NC


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membrane, which can affect the immunological reaction of QB@mAbs and BSA@OTA on the T line, as well as QB@mAbs and goat anti-mouse IgG on the C line, further influencing the readout time of QB-ICA. Thus, to acquire the optimized readout time for five-sized QB-ICA, the immunological kinetics analysis was performed by running PBS solution containing the desired QB probes. After 1 min reaction, both the FIT and the FIC were recorded every 1 min for a total of 25 min. The reaction kinetics of QB@mAbs and BSA@OTA on the T line, as well as QB@mAbs and goat anti-mouse IgG on the C line, was determined by plotting the curve of FIT and FIT/FIC against time. As shown in Figure 3B, all five FIT values displayed a parallel change tendency, where the FIT increase continuously without a balance during the 25-min observation period. However, the FIT/FIC values was quickly reached after a 15-min reaction for the five QB-ICA (Figure 3C). These findings indicated no significant difference in the immunoreaction time regardless of the QB size. Thus, 15 min of immunoreaction time was enough for five-sized QB-ICA through using FIT/FIC values as signal readout, which can effectively avoid the strip-to-strip effects and shorten the strip detection time.24 Analytical Performance Comparison of Five-sized QB-ICA. To evaluate the effect of five-sized QB on the ICA detection performance, qualitative assays using the naked eye and quantitative analysis by fluorescence reader for the five kinds of QB-ICA were conducted. For qualitative assay, the cut-off values representing the concentrations of OTA that cause no fluorescence on the T line observed by the naked eye under the excitation of UV light were 7.5 ng/mL for QB58-ICA, 5 ng/mL for



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QB124-ICA, 7.5 ng/mL for QB255-ICA, 15 ng/mL for QB365-ICA, and 30 ng/mL for QB598-ICA (Figure S3). For quantitative analysis, the standard curves of five different-sized QB-ICA in Figure 3D were constructed by plotting the B/B0×100% against the logarithm of various concentrations of the OTA standard (0–30 ng/mL), respectively. According to their respective standard curves, the IC50 for QB58-ICA, QB124-ICA, QB255-ICA, QB365-ICA, and QB598-ICA were calculated to be 0.46 ng/mL, 0.43 ng/mL, 0.51 ng/mL, 0.90 ng/mL, and 1.44 ng/mL, respectively. Given all this, we found that the QB124-ICA exhibits the best detection performance with the lowest cut-off value (5 ng/mL) for naked-eye detection, as well as IC50 (0.43 ng/mL) for quantitative detection. Meanwhile, we found that with the increase of QB size from 255 nm to 569 nm, the luminescent intensity improved 8.67 times, whereas the IC50 increased from 0.51 ng/mL to 1.44 ng/mL, indicating that large-sized QB will give rise to the decrease in analytical sensitivity of QB-ICA. Presumably, oversized QBs could result in poor signal on the test zone because of significant steric hindrance that could deteriorate the detection sensitivity of competitive ICA. In other words, although the oversized QBs have higher luminescence intensity that is beneficial for improving the detection sensitivity, they are still not suitable as labels for developing the competitive ICA method due to the presence of steric hindrance. Therefore, we selected the QB124-ICA for all succeeding evaluation. Effects of pH and Methanol for QB124-ICA. In general, pH and methanol content of the sample are two key factors that could immensely influence the sensitivity and reproducibility of the QB124-ICA. Previous study demonstrated that


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both of them could influence the detection sensitivity of ICA by disturbing the antigen–antibody interaction25. To evaluate the effects of pH on FIT, FIC, and FIT/FIC, the pH values of the sample solutions were adjusted to final pH values of 4.5, 5.5, 6.5, 7.5, 8.5, and 9.5, respectively. Figure 4A showed that FIT improved sharply from 121 ± 10 to 468 ± 6 with the pH shift from 4.5 to 6.5, and then remained relatively constant at a pH ranging from 6.5 to 7.5 (468 ± 6 to 472 ± 22). Eventually, with the pH ranging from 7.5 to 9.5, FIT showed a significant decline from 472 ± 22 to 221 ± 17. Meanwhile, the FIT/FIC ratio variation maintained a similar trend, which improved sharply from 0.38 ± 0.02 to 3.19 ± 0.22 as the pH changed from 4.5 to 6.5, and then declined to 1.19 ± 0.07 when the pH shifted to 9.5. The competitive inhibition rates for 0.5 ng/mL OTA solution changed from 14.7% ± 1.7% to 51.3% ± 1.7%, with a pH shift from 4.5 to 7.5. Although the competitive inhibition rates reached to 50.6 ± 0.4% at a pH of 9.5, we yet selected the sample solution with a pH of 7.5 as the optimal pH condition for all succeeding experiments because of the strong fluorescence signal on the test line. The OTA extraction from the corn sample requires high concentration of methanol (60%, v/v) because of the strong hydrophobicity of OTA. However, the immunoreaction was vulnerable to the interference of methanol at high concentration. Therefore, optimization of methanol content in sample solution is necessary. In this work, we used sample solution with a pH of 7.4 containing different methanol contents ranging from 5% to 30% (v/v) according to our previous research. Figure 4B showed that both FIT and FIC values significantly ascended as the methanol



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concentration increased to 5%, and then decreased (especially for FIC) with increasing methanol concentration from 5% to 30%. These results indicated that a certain concentration of methanol (here is 5%) is beneficial for the interaction of OTA and QBs probes. Similarly, the competitive inhibition rate maintained a relatively constant value at a methanol concentration ranging from 0% to 5%, whereas it showed a significant decline from 50.7% ± 2% (methanol concentration at 5%) to 10.9% ± 2% (methanol concentration at 30%). Thus, considering the ultrasensitivity of the QB124-ICA, the actual sample extract should be diluted with a 0.01 M PBS buffer (pH 7.5) to obtain a final methanol concentration of 5%. Evaluation of QB124-ICA. The calibration curves of QB124-ICA were constructed by plotting the B/B0×100% against the logarithm of various concentrations of the OTA standard (0–5 ng/mL) under the optimal experimental conditions, respectively. Figure 4C shows that the regression equation of QB124-ICA is y = −0.232ln(x) + 0.2798 (R² = 0.9856), where y is the ratio of B/B0, and x is the OTA concentrations. Moreover, a good linear detection range from 0.1 ng/mL to 2 ng/mL was obtained. The IC50 of this proposed QB124-ICA was calculated to be 0.39 ng/mL (n = 3), and the LOD was as low as 0.085 ng/mL, according to 10% OTA competitive inhibition concentration.21 To evaluate the specificity of this developed ICA, the QB124-ICA was tested with OTA at a concentration of 4 ng/mL, and other eight mycotoxins, including CIT, AFB1, AFB2, AFG1, ZEN, T-2, FB1, and DON at concentrations of 1 µg/mL, respectively. In addition, negative control was prepared with a 0.01 M PB buffer at pH 7.4 containing


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5% methanol. Figure 4D shows the FIT/FIC ratios of QB124-ICA in detecting CIT, AFB1, AFB2, ZEN, AFG1, T-2, FB1, and DON, which exhibited negligible difference with the negative control, whereas a significant decrease of FIT/FIC ratio was only observed in detecting OTA. These results indicated that the QB124-ICA lacks immune response to CIT, AFB1, AFB2, ZEN, AFG1, T-2, FB1, and DON even at high concentrations. In other words, the designed QB124-ICA in this study can specifically detect OTA. We used intra- and inter-assay to investigate the accuracy and precision of the proposed method by analyzing three spiked corn extracts with low (0.2 ng/mL), medium (0.5 ng/mL), and high (1.5 ng/mL) levels of OTA concentrations. Intra-assay was carried out within one day with five replicates at each spiked level, whereas inter-assay was conducted for three consecutive days with five replicates at each spiked level. Table 1 showed that the average recoveries for the intra-assay varied from 98.80% to 109.88%, with a CV ranging from 1.64% to 9.72%. Moreover, the results for the inter-assay ranged from 97.40% to 108.09%, with a CV ranging from 2.80% to 9.02%, respectively. All these intra- and inter-assay variations indicated that the proposed QB124-ICA showed acceptable levels of precision for OTA quantification.26 The reliability of the as-prepared QB124-ICA was further compared with a widely accepted ELISA Kit method by blindly detecting 25 OTA-spiked real corn samples. Figure 5 shows that the two methods exhibited good agreement with a highly significant correlation (R2 = 0.91), and the slope of the resultant linear regression



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curve was equal to 0.9524. These results suggested that the two methods are not significantly different in the quantitative determination of OTA. However, the proposed QB124-ICA requires only 15 min to complete an analysis, whereas the traditional ELISA method takes 90 min. Therefore, these findings suggested that the newly developed QB124-ICA scan can serve as an alternative of conventional ELISA for rapid screening of OTA in real samples.


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CONCLUSIONS We successfully demonstrated that the size and luminescent intensity of QBs can

significantly affect the analytical performance of QB-ICA both in qualitative and quantitative analysis. Although large-sized QBs displayed stronger luminescence relative to small-sized QBs, lower detection sensitivity in competitive ICA was obtained owing to their significant steric hindrance that hinders the immunological recognition of OTA and corresponding antibody on the strip, further resulting in poor signal intensity. In addition, the best detection performance was acquired by using QBs with a size of 124 nm as labeling probes in competitive ICA assay. Nevertheless, this work mainly focused on studying the effects of the luminescence intensity and size of fluorescence microspheres on the detection performances of competitive ICA. For sandwich ICA assay, whether the same trend with competitive ICA assay should be followed or not is still unclear. Further study must be conducted to explore the relationship between the luminescence intensity and size of fluorescence microspheres and the analytical performance using sandwich ICA format.



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Acknowledgments This work was supported in part by the National Basic Research Program of China (2013CB127804), Major projects of Natural Science Foundation of Jiangxi province (20161ACB20002), and Training Plan for the Main Subject of Academic Leaders of Jiangxi Province (20142BCB22004). The Innovation Fund Designated for Graduate Students of Jiangxi Province (YC2016-B012).


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Supporting Information Details about the following are available: Materials, TEM image of CdSe/ZnS QDs, Signal intensity tested by ICP–OES, Calibration curve for cadmium (Cd), Molar concentration of the resultant QBs, Fluorescence-enhanced fold of the QBs compared with the QDs. Orthogonal test for parameter optimization of the QB58-ICA, QB124-ICA, QB255-ICA, QB365-ICA, and QB598-ICA, respectively, Digital photo of the fluorescent strip of the five kinds of QB-ICA for OTA detection.



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REFERENCES

(1) Sajid, M.; Kawde, A.-N.; Daud, M. J. Saudi. Chem. Soc. 2015, 19, 689-705. (2) Anfossi, L.; Baggiani, C.; Giovannoli, C.; D'Arco, G.; Giraudi, G. Anal. Bioanal. Chem. 2013, 405, 467-480. (3) Ngom, B.; Guo, Y.; Wang, X.; Bi, D. Anal. Bioanal. Chem. 2010, 397, 1113-1135. (4) Huang, X.; Aguilar, Z. P.; Xu, H.; Lai, W.; Xiong, Y. Biosens. Bioelectron. 2016, 75, 166-180. (5) Michael G. W.; Fresenius J. Anal. Chem. 2000, 366, 635-645 (6) Singh, J.; Sharma, S.; Nara, S. Food Chem. 2015, 170, 470-483. (7) Li, J.; Duan, H.; Xu, P.; Huang, X.; Xiong, Y. RSC Adv. 2016, 6, 26178-26185. (8) Majdinasab, M.; Sheikh-Zeinoddin, M.; Soleimanian-Zad, S.; Li, P.; Zhang, Q.; Li, X.; Tang, X.; Li, J. Food Control 2015, 47, 126-134. (9) Ren, M.; Xu, H.; Huang, X.; Kuang, M.; Xiong, Y.; Xu, H.; Xu, Y.; Chen, H.; Wang, A. ACS Appl. Mater. Interfaces 2014, 6, 14215-14222. (10) Wang, D.; Zhang, Z.; Li, P.; Zhang, Q.; Zhang, W. Sensors 2016, 16, 1094. (11) Wang, Z.; Li, H.; Li, C.; Yu, Q.; Shen, J.; De Saeger, S. J. Agric. Food Chem. 2014, 62, 6294-6298. (12) Hu, L. M.; Luo, K.; Xia, J.; Xu, G. M.; Wu, C. H.; Han, J. J.; Zhang, G. G.; Liu, M.; Lai, W. H. Biosens. Bioelectron. 2017, 91, 95-103. (13) Laitinen M. P. A.; Vuento M. Biosens. Bioelectron.1996,12,1207-1294. (14) Jun, H. K.; Careem, M. A.; Arof, A. K. Renew. Sust. Energ. Rev. 2013, 22, 148-167. (15) Jun, B.-H.; Hwang, D. W.; Jung, H. S.; Jang, J.; Kim, H.; Kang, H.; Kang, T.; Kyeong, S.; Lee, H.; Jeong, D. H.; Kang, K. W.; Youn, H.; Lee, D. S.; Lee, Y.-S. Adv.Funct. Mater. 2012, 22, 1843-1849. (16) Ranzoni, A.; den Hamer, A.; Karoli, T.; Buechler, J.; Cooper, M. A. Anal. Chem. 2015, 87, 6150-6157. (17) Li, X.; Li, W.; Yang, Q.; Gong, X.; Guo, W.; Dong, C.; Liu, J.; Xuan, L.; Chang, J. ACS Appl. Mater. Interfaces 2014, 6, 6406-6414. (18) Hu, J.; Zhang, Z. L.; Wen, C. Y.; Tang, M.; Wu, L. L.; Liu, C.; Zhu, L.; Pang, D. W. Anal. Chem. 2016, 88, 6577-6584. (19) el Khoury, A.; Atoui, A. Toxins 2010, 2, 461-493. (20) Meulenberg, E. P. Toxins 2012, 4, 244-266. (21) Duan, H.; Chen, X.; Xu, W.; Fu, J.; Xiong, Y.; Wang, A. Talanta 2015, 132, 126-131. (22) Shen, J.; Zhou, Y.; Fu, F.; Xu, H.; Lv, J.; Xiong, Y.; Wang, A. Talanta 2015, 142, 145-149. (23) Fu, R.; Jin, X.; Liang, J.; Zheng, W.; Zhuang, J.; Yang, W. J. Mater. Chem. 2011, 21, 15352. (24) Li, C.; Luo, W.; Xu, H.; Zhang, Q.; Xu, H.; Aguilar, Z. P.; Lai, W.; Wei, H.; Xiong, Y. Food Control 2013, 34, 725-732. (25) Ji, Y.; Ren, M.; Li, Y.; Huang, Z.; Shu, M.; Yang, H.; Xiong, Y.; Xu, Y. Talanta 2015, 142, 206-212. (26) Bai, Y.; Liu, Z.; Bi, Y.; Wang, X.; Jin, Y.; Sun, L.; Wang, H.; Zhang, C.; Xu, S. J. Agric. Food Chem. 2012, 60, 11618-11624.


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Tables Table 1. Precision and accuracy of the QB124-ICA inter-assay precisiona

intra-assay precision

spiked OTA (ng/mL)

meanb

0.2

0.22

recovery (%) 109.88

meanb

0.004

CV (%) 1.64

0.5

0.52

104.55

0.01

1.5

1.48

98.80

0.04

SD

0.21

recovery (%) 107.09

0.006

CV (%) 9.04

2.14

0.52

103.47

0.03

6.35

9.72

1.46

97.40

0.08

5.34

a

Assay was completed every 1 d for 3 d continuously.

b

Mean value of 5 replicates at each spiked concentration.


SD


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Figure captions Scheme 1. A. Schematic representation of the procedure to prepare different-sized QBs by emulsion droplet solvent evaporation method. B. Relationship between size of QB and performance for QB-ICA. Figure 1. Characterization of different-sized QBs. A1-E1, high-resolution transmission electron microscope image of QB58, QB124, QB1255, QB365, and QB598, respectively; A2-E2, hydration diameter of QB58, QB124, QB1255, QB365, and QB598, respectively. Figure 2. Fluorescence spectral Characterization of different-sized QBs. A. Fluorescence comparison between different-sized QBs and corresponding QDs; B. Luminant photos of different-sized QBs and corresponding QDs. (excited by 365 nm). Figure 3. A. Optimization of the content of anti-OTA ascites on the surface of QBs. B. Immunoreaction dynamic curve of the test line (FIT) against immunoreaction time with different-sized QBs probes; C. Immunoreaction dynamic curve of FIT/FIC against immunoreaction time with different-sized QBs probes; D. Inhibitory curves of five kinds of QB-ICA for OTA detection. Figure 4. Parameter optimization of the QB124-ICA. A. Effect of pH value of samples on FIT, FIC, and FIT/FIC ratio. Competitive inhibition rate was defined as (1 - B/B0) × 100%, where B0 and B represent FIT/FIC of the negative sample and an OTA spiked sample solution (0.5 ng/mL), respectively; B. Effect of methanol in samples on FIT, FIC, FIT/FIC ratio, and Competitive inhibition rate ratio; C. Calibration curves of QB124-ICA for OTA detection; D. Specificity experiment for 4 ng/mL of OTA, and 1µg/mL of CIT, AFB1, AFB2, AFG1, T-2, FB1, DON, and ZEN;


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Figure 5. Methodology comparison between the QB124-ICA and ELISA methods (n=25).



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Scheme 1


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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for TOC only


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Size-Dependent Immunochromatographic Assay with Quantum Dot

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