The OTA standard solutions were prepared by spiking OTA stock solution to OTA-free solutions of grape juice and wine. acid (C76H52O46), and silver nitrate (AgNO3) solution. The as-prepared AgNPs were well distributed and exhibited relatively uniform spheres with an average diameter of 63.6 3.7 nm (= 50), as shown in Figure 2b. The UVCvis spectrum demonstrates that the maximum SPR peak of AgNPs is 450 nm (Figure 2c). The SPR absorption peak of AgNP totally R1487 Hydrochloride overlaps with the RuNP excitation peak. Open in a separate window Figure 2 Characterization of Ru(phen)= 3); (b) Ultraviolet absorption spectra of AgNPs before and after labeling anti-OTA ascites. 2.3. Parameter Optimization of AgNPCRuNPCcLFIA Sensor The background FIs on both lines, the amounts of AgNP probes, and the BSACOTA concentration on the T line were considered the most important factors that affected the sensitivity of the cLFIA sensor. First, the amounts of BSA-RuNP on both lines were optimized by spraying a different concentration of BSA-RuNP solution into the NC membrane as the dispensing density of T and C lines was 0.75 L/cm. The NC membrane was then dried with a vacuum dryer at 37 C for 2 h. The FI value on the T line increased from 124 to 1786 as the BSA-RuNP concentration increased from 20 g/mL to 320 g/mL, as shown in Figure S1. A low fluorescent signal on the T line indicates a low signal-to-noise ratio, which would cause a narrow linear range and a large error. Meanwhile, a strong fluorescence signal could lead to a low sensitivity because of more probe consumption. The fluorescent signals on both lines were approximately 480 when the BSA-RuNP concentration was 80 g/mL, and two significant red fluorescence bands were observed under 450 nm LED arrays (the stereogram displayed in Supplementary Material Figure S1). Thus, 80 g/mL of BSA-RuNP solution was NOX1 selected as the optimal concentration to produce the background fluorescent signals on both lines. A similar checkerboard titration was performed with different AgNP probe contents under a series of BSA-OTA concentrations on the T line for various combinations to further achieve the best sensitivity. R1487 Hydrochloride The FI values of the T line for OTA-free and OTA-positive samples (0.5 g/L) were used to confirm the optimal parameters. For the OTA-free sample, the fluorescent signals of the T line from seven of the different combinations were almost totally quenched by the AgNP probe. The No. 13 combination showed the maximum FI under a 0.5 g/L OTA concentration among the seven combinations (Table S1). Therefore, the optimal parameters for the AgNPCRuNPCcLFIA sensor were 5 L AgNP probes (OD450 = 1.5) and 1.2 mg/mL BSACOTA. In addition, we also optimized the donkey antimouse IgG amounts because excessive donkey antimouse IgG amounts can cause the nonappearance of the fluorescent transmission within the C collection. Therefore, a relatively low donkey antimouse IgG concentration of 0.2 mg/mL was suggested to be sprayed within the C collection to achieve an essential fluorescence value for OTA quantitative analysis. The T/C percentage was utilized for OTA quantitative assay because it can efficiently offset the effects R1487 Hydrochloride of the inherent heterogeneity of the LFIA sensor in the present study. The accurate readout time of the T/C percentage was from an immunological kinetic curve by operating OTA-spiked phosphate buffer remedy (PBS, 0.05 g/L OTA). The percentage of T/C was constant after a 20 min operating time, as demonstrated in Number S2a. We further ran the immunological kinetic curves at a 0.5 g/L OTA concentration in grape juice and wine samples to confirm the above effects. Similar results are demonstrated in Number S2b. Therefore, the 20 min immunoreaction time was necessary to guarantee the reproducibility of the quantitative analysis using cLFIA sensor. In summary, the optimal experimental parameters of the AgNPCRuNPCcLFIA sensor were as follows: 80 g/mL BSA-RuNP remedy comprising either 1.2 mg/mL BSACOTA antigen (the mole percentage of BSA and OTA is 1:15) or 0.2 mg/mL donkey antimouse IgG was dispensed within the NC membrane as the T or C collection at a spraying density of 0.75 L/cm. A total of 70 L of sample remedy with 5.0 L AgNP probes (OD450 = 1.5) was used to run the cLFIA sensor. The value of the T/C percentage of the cLFIA sensor was recorded with a commercial strip reader for OTA quantitative analysis after 20 min incubation. 2.4. Overall performance Evaluation of AgNPCRuNPCcLFIA Sensor The calibration curve was performed by operating 18 different OTA concentrations from 0 g/L to 40 g/L in PBS. Number 4a.
