2.3.1. PCT Detection in Human Serum Samples.
PCT was selected as the serum target, which is a biomarker of sepsis and pyemia caused by bacterial infection. Accurate monitoring of PCT at low concentration ranges is of great importance for indicating the degree of inflammation and guiding antibiotic therapy.[43,44] To begin with, the coupling conditions of the MANP6:6@PCT-mAbs were adjusted to optimize the final performance. This optimization included adjusting the coupling pH of PCT-mAbs, the amount of EDC used, and the saturated labeling amount of anti-PCT mAbs on MANP6:6 (Figures S 9a–c). As shown in Figure S9 d, the as-obtained MANP6:6@PCT-mAbs nanoprobes exhibited good monodispersity with a slight size increase of 20 nm compared with that of MANP6:6. Additionally, the zeta potential of MANP6:6@PCT-mAbs was decreased by 23.7 mV, due to the presence of the PCT-mAbs with negative charge (FigureS9 e). These observations demonstrate the successful modification of antibodies on the MANP6:6. Moreover, the as-fabricated MANP6:6@PCT-mAbs probes maintained the fluorescent profile and brightness (Figure S9 f), which remarkably ensured the detection sensitivity of the LFIA strip. Besides, maximum responses were further obtained by optimizing the strip and running parameters, such as the sprayed concentration of PCT-mAbs on the T line, the added amount of MANP6:6@PCT-mAbs probes for each strip, the immunoreaction time, and the magnetic separation volume (Figures S10 a–d). Furthermore, the serum dilution volume was investigated to achieve the best immunoreaction and magnetic capture efficiency in practical samples. As revealed in Figure S10 e, the fluorescent intensity increases with the dilution degree, and a signal plateau is reached with a twofold serum dilution, effectively eliminating serum matrix interference on the immunoreaction and LFIA fluorescent reading.
Under the developed conditions mentioned above, PCT standards in serum solutions with various concentrations were magnetically enriched and detected using MANP6:6@PCT-mAbs probes-immunolabeled LFIA test strips. As shown in Figure 4 a, the fluorescent signal on the T line gradually increases as the concentration of PCT increases, followed by a decrease in the fluorescent signal on the C line. The logarithm of the ratiometric FIT/FIC linearly increases with the logarithm of the antigen concentration in the range from 0.0018 ng mL−1 to 62.5 ng mL−1, as shown inFigure 4b. The linear regression equation for PCT in serum isY = 0.9992 X 0.7798, where Y represents the logarithm of FIT/FIC, and X is the logarithm of PCT concentration, with a linear regression coefficient of 0.9977. The LOD of this method for PCT was determined to be 0.0012 ng mL−1 (defined as the concentration corresponding to 20 negative means plus triple standard deviation). In comparison, direct detection of PCT in serum without magnetic separation resulted in a liner responding range (0.0075–62.5 ng mL−1, R 2 = 0.9947) with a higher LOD of 0.0068 ng mL−1(Figures 4 b, S11 ). Moreover, a traditional AuNPs-based colorimetric LFIA (AuNPs-LFIA) was constructed, which showed a much narrower liner responding range (0.06–15.6 ng mL−1,R 2 = 0.9826) and a higher LOD of 0.06 ng mL−1 (Figures 4 b, S12 ). This indicates a 5.6-fold and 50-fold higher sensitivity of the magnetic-assisted MANP-LFIA compared with MANP-LFIA without magnetic operation and AuNPs-LFIA, demonstrating the remarkable superiority of the developed sensor in detecting trace targets. Furthermore, the upper limit of PCT detection by magnetic-assisted MANP-LFIA is lifted by four times compared with that of AuNPs-LFIA, which is highly important to severe inflammation diagnosis. Such excellent analytical performance is attributable to the magnetic operation, which effectively enriches the PCT protein from a larger sample volume to obtain a higher target amount and extracts the PCT from the serum to mitigate matrix interference, and high fluorescent brightness of MANP6:6 with weak IFE.Figure 4 c shows the typical strip prototypes after the detection of various concentration (0–250 ng mL−1) of PCT-spiked serum samples. Clearly, the brightness of the T lines remarkably increases with an increasing concentration of the target. The particle location was also confirmed by SEM. As shown in Figure4 d, masses of MANP6:6@PCT-mAbs are immobilized tightly on the NC fiber in the T line region in the case of a PCT concentration of 100 ng mL−1, whereas negligible nonspecific adsorption is observed on the NC membrane in the PCT-negative sample, which matches well with the visual result. These results demonstrate that the proposed MANP-FLIA provided good sensitivity for POCT detection, and the paper strip platform provides visual analysis of biomarker monitoring. The detection specificity is also a key issue in immunoassays. Here, hepatitis B surface antigen (HBsAg), prostate-specific antigen (PSA), human chorionic gonadotropin (HCG), HIV p24 antigen, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), N-terminal prohormone of brain natriuretic peptide (NT-ProBNP), and C-reactive protein (CRP) were selected as the interfering proteins to evaluate the selectivity of the immunoassay platform. The FIT/FIC of PCT at 1 ng mL−1 was compared with those of interference species at the 1000 ng mL−1 concentration level. As shown in the spider diagram in Figure 4 e and photograph inFigure S13 , a negligible signal was detectable in the interference species, whereas a strong fluorescent signal was observed in the PCT sample. This result demonstrates that the proposed MANP-FLIA platform exhibits good specificity to the target protein and has the ability to differentiate it from complex samples. In addition, the accuracy and precision of the MANP-LFIA method were investigated by determining the intra- and inter-assay recoveries and percent coefficients of variation (%CVs) of seven PCT-spiked serum samples with concentrations of 0.05, 0.1, 0.5, 1, 5, 10, and 50 ng mL−1. As revealed in Table S1 , the average recoveries of the intra-assay ranged from 84.7% to 105.6% with CVs ranging from 3.49% to 9.66%, and the average recoveries of the inter-assay ranged from 85.2% to 96.3% with CVs ranging from 7.3% to 14.4%. These results indicate that the proposed MANP-LIFA is suitable for accurate PCT quantification with satisfactory precision.
Furthermore, we demonstrated the reliability and practicability of the proposed method by measuring 34 human blood plasma samples from patients. As shown in Figure 4 f and Table S2 , the detection results showed a high level of agreement compared with those measured by commercial chemiluminescence immunoassay (CLIA) kits, without any false-positive or false-negative signals. On this basis, a regression analysis was conducted between the proposed method and the standard CLIA by the hospital. Figure 4 g demonstrates a strong positive correlation between the MANP-FLIA method and the standard CLIA, with correlation coefficients of 0.966. These results confirm that the proposed method is accurate and valid for clinical diagnosis.