• 2019-10
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  • 2021-03
  • C after coating st rd and th cycle


    C5: after coating 1st, 3rd, and 5th cycle, respectively). (b) Intensity change of different polarization peaks. (c) Transmission spectra of BP-TFG in different RI medium.
    (d) Wavelength shift against external RI.
    3.4. Label-free detection of NSE biomarkers
    The anti-NSE bound BP-TFG was employed to monitor the kinetic binding interaction between anti-NSE and target NSE biomarkers. Five consecutive binding processes for human NSE sample in 1 × PBS buffer solutions with concentration of 0.01, 0.1, 1.0, 10, and 100 ng/mL were detected by monitoring TM resonant wavelength shift in real-time. The PBS buffer (1 × PBS, Artesunate 7.4) was firstly used to prewash the device for 5 min, offering a sensing baseline where the spectrum was recorded (Fig. S4-a). Then, the NSE sample solution was applied to immerse the sensor device for 30 min (Fig. S4-b). As depicted in Fig. 6a, the first 5-min was a rapid-state bind process in which the optical signal showed a significant wavelength shift, then the reaction rate began to asymptote to a steady-state for the following 20-min, and gradually achieved sa-turation-state in the last 5-min. After that, a subsequent rinsing with PBS buffer was followed to wash away the unbound NSE molecules
    prior to the next measurement. The wavelength shift after deducting the baseline value is 210, 240, 290, 320, and 370 pm for NSE concentration of 0.01, 0.1, 1.0, 10, and 100 ng/mL, respectively. Since the evanescent field penetrating from cladding/BP boundary to the surrounding-medium with the enhanced light-matter interface, even very small local RI change due to the affinity binding would cause an observable wa-velength shift.
    The wavelength shift as a function of NSE concentration has been plotted in Fig. 6b. The red line provides the best Hill model fitting of the experimental data. Hill model can be described as (Koopal et al., 1994; Nakatsuka et al., 2018):
    where λmax is the maximum wavelength shift of the reaction, Kd is the dissociation constant of the interaction between receptor and ligand molecules, CNSE is the ligand concentration, and n is the Hill coefficient,
    Fig. 5. Biofunctionalization and bioconjugation of BP-TFG. (i) Biofunctionalization with biocompatible PLL, (ii) Immobilization of anti-NSE via EDC/NHS bio-conjugation, (iii) Bioaffinity binding between anti-NSE and target NSE biomarkers.
    Fig. 6. Label-free detection of NSE with anti-NSE immobilized BP-TFG. (a) Wavelength shift caused by binding interactions with target NSE samples with con-centration of 0.01, 0.1, 1.0, 10, and 100 pg/mL, respectively. (b) Dependence of wavelength shift against NSE concentrations. The red line is the best Hill model fit curve. (c) Specificity of BP-TFG biosensor (in PBS, IgG, PSA, and NSE). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    which is useful for determining the degree of cooperativity of the ligand binding to the receptor. The Hill equation provides a way to quantify the degree of interaction between ligand binding sites. According to Eq.
    (2), the calculated value of λmax, Kd, and n is 718.9 pm, 64.0 ng/mL and 0.10, respectively. The Hill coefficient n is less than 1, indicating the negative cooperativity with respect to ligand binding to the re-ceptor. The molecular weight of NSE is approximately 47 kDa, resulting in a dissociation constant Kd of 1.36 × 10−9 M which agrees with the previous research (Acero Sánchez et al., 2016). Based on the experi-mental results, the limit of detection (LOD, at a signal/noise ratio of 3) of NSE concentration is calculated below 1.0 pg/mL, which is 4 orders of magnitude lower than NSE cut-off value (15.2 ng/mL) of SCLC (Wang et al., 2013), demonstrating the possibility for quantitative detection of NSE in clinical samples. The enhanced biosensing sensitivity is 100-fold higher than GO- and AuNPs-based biosensors (Zhou et al., 2009; Qu et al., 2011). Due to its ultrahigh surface-to-volume ratio, wide tunable range of bandgap and superior molecular adsorption energy, BP plays an important role as bio-nano-photonic interface which increases the sensing area, number of binding sites, and affinity binding efficiency, hence amplifies the optical signal providing superior biosensing per-formance.
    For the practical biosensing application, the reusability is an im-portant function. The reusability of the proposed BP-TFG has been 
    examined by detecting the binding interaction in 10 ng/mL NSE for multiple times. Fig. S5 presents the comparison results for three cycles with the percentages of resonant wavelength shift and initial binding rate. The maximum wavelength shift as the absolute RI change after reducing the baseline signal in PBS prewashing stage retained 95.5% and 91.5% after second and third cycle, respectively. Likewise, the in-itial binding rate calculated with the data over the first 3 min of binding interaction maintained greater than 89% and 88.5% after second and third cycle, respectively. These results confirmed that the BP-TFG bio-sensor could be used to detect the NSE/anti-NSE binding for multiple times.