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.