Immunoassay-Amplified Responses Using a Functionalized MoS₂-Based SPR Biosensor to Detect PAPP-A2 in Maternal Serum Samples to Screen for Fetal Down’s Syndrome

Figures & data

Figure 1 Carboxyl-MoS₂ nanocomposite synthesis and the sensing mechanism of the carboxyl-MoS₂-based SPR biosensor to detect PAPP-A2 protein in maternal serum samples. (A) Schematic diagram of sonication-assisted liquid-phase exfoliation (LPE) for the preparation of MoS₂ sheets. (B) MoS₂ exhibits a layered structure with planar Mo–S bonds and sulfur vacancies on the MoS₂ surface. (C) The carboxyl-MoS₂ nanocomposites were successfully modified by chloroacetic acid. Carboxyl-MoS₂-based SPR chip immobilization steps and containing. (D) Au film substrate. (E) Cys linker containing a thiol-group to allow for assembly on the Au film substrate. (F) Covalent binding of carboxyl-MoS₂ on the Cys-linker. (G) EDC/NHS activated carboxyl groups resulting in a higher density of immobilized antibodies. (H) To detect PAPP-A2 protein to screen for Down’s syndrome in maternal serum samples.

Abbreviations: MoS₂, molybdenum disulfide; Carboxyl-MoS₂, carboxyl-molybdenum disulfide; SPR, surface plasmon resonance; PAPP-A2, pregnancy-associated plasma protein-A2; Au, gold; Cys, cystamine; NH₂, amine groups; EDC/NHS, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide.

Figure 2 (A) SEM image of carboxyl-MoS₂ sheets. (B) Cross-sectional SEM image of lateral flake thickness of carboxyl-MoS₂ sheets. (C) TEM image of the carboxyl-MoS₂ sheets. (D) TEM image of the MoS₂ sheets. (E) EDS analysis of the carboxyl-MoS₂ sheets (insert shows the carboxyl-MoS₂ sheet for the EDS analysis).

Abbreviations: MoS₂, molybdenum disulfide; Carboxyl-MoS₂, carboxyl-molybdenum disulfide; SEM, scanning electron microscopy; TEM, transmission electron microscopy; EDS, energy dispersive X-ray spectroscopy.

Figure 3 The XPS survey spectra of (A) MoS₂ sheets and (B) carboxyl-MoS₂ sheets. The high-resolution XPS spectra of (C) C1 2p, (D) Mo 3d, (E) S 2p for MoS₂ and carboxyl-MoS₂ sheets. (F) Analysis of XPS surface atomic intensity ratios of C1s/Mo3d and O1s/Mo3d on MoS₂ and carboxyl-MoS₂ sheets.

Abbreviations: MoS₂, molybdenum disulfide; Carboxyl-MoS₂, carboxyl-molybdenum disulfide; XPS, X-ray photoelectron spectroscopy.

Figure 4 High energy resolution XPS spectra of (A) C1s and (B) O1s regions on carboxyl-MoS₂ sheets. (C) UPS spectra of different interfaces of Au, Au/MoS₂ and Au/carboxyl-MoS₂ film. Band diagram of (D) Au/MoS₂ and (E) Au/carboxyl-MoS₂ heterojunctions obtained from UPS measurements. The band-gap energies used in the diagram are optical gaps.

Abbreviations: MoS₂, molybdenum disulfide; Carboxyl-MoS₂, carboxyl-molybdenum disulfide; Au, gold; XPS, X-ray photoelectron spectroscopy; UPS, ultraviolet photon spectroscopy.

Figure 5 (A) Optimization of the immobilization process of antibodies in carboxyl-MoS₂-based SPR chips. (B) Non-specific molecular dissociation reactions were tested using PBS and PBS_BNT buffer, respectively.

Abbreviations: Carboxyl-MoS₂, carboxyl-molybdenum disulfide; SPR, surface plasmon resonance; PBS, phosphate-buffered saline; PBS_BNT, phosphate-buffered saline_{(BSA, NaCl, Tween-20)}.

Figure 6 Sensorgrams showing the SPR responses generated with a flow rate of 60 μL/min and different dilution factors of 10 FDS maternal serum samples. The 10 FDS samples were derived from blood drawn at a gestational age of (A) 19, (B) 20, (C) 14, (D) 14, (E) 22, (F) 19, (G) 21, (H) 18, (I) 19, and (J) 18 weeks, respectively.

Abbreviations: SPR, surface plasmon resonance; FDS, fetal Down’s syndrome.

Figure 7 Sensorgrams showing the SPR responses generated with a flow rate of 60 μL/min and different dilution factors of maternal serum samples from four healthy women (NPW group). The NPW samples were derived from blood drawn at a gestational age of (A) 14, (B) 12, (C) 14, and (D) 16 weeks, respectively.

Abbreviations: SPR, surface plasmon resonance; NPW, normal pregnant women.

Figure 8 Calibration curves obtained with (A) ELISA kit assays for PAPP-A2 quantification ranging from 2.5 to 108 ng/mL. (B) Calibration curve of the average SPR response to various PAPP-A2 concentrations ranging from 0.1 to 1100 pg/mL with the carboxyl-MoS₂-based SPR chip. Error bars indicate standard deviations of means obtained from three replicates using SPR and three replicates using ELISA. Correlation between the concentration of PAPP-A2 in serum obtained by ELISA and signal responses by SPR. (C) NPW group (n = 24, R² = 0.97) and (D) FDS group (n = 60, R² = 0.91) samples.

Abbreviations: ELISA, enzyme-linked immunosorbent assay; PAPP-A2, pregnancy-associated plasma protein-A2; SPR, surface plasmon resonance; carboxyl-MoS₂, carboxyl-molybdenum disulfide; NPW, normal pregnant women; FDS, fetal Down’s syndrome.

Figure 9 SPR responses of serum samples of the FDS and NPW groups. (A) Kinetic analysis of the FDS and NPW groups on the carboxyl-MoS₂-based SPR biosensor for affinity binding analysis of the PAPP-A2 interaction. (B) Correlation between two variables of the FDS and NPW groups to detect PAPP-A2 in serum samples. Data expressed as mean ± SD for triplicate tests. Asterisks indicate statistical significance using a generalized mixed effects model (highly significant recognition (***) (p < 0.001); very significant recognition (**) (0.001 < p < 0.01); significant recognition (*) (0.01 < p < 0.05). p: probability value). (C) The dot plot shows the distribution of SPR responses at the dilution rate (100-, 500-, and 1k-fold) of different serum samples and the difference between the FDS and NPW groups at the cut-off limit for screening. (D) Recovery rates of PAPP-A2 protein assay in the FDS group using the carboxyl-MoS₂-based SPR chip.

Abbreviations: SPR, surface plasmon resonance; FDS, fetal Down’s syndrome; NPW, normal pregnant women; carboxyl-MoS₂, carboxyl-molybdenum disulfide; PAPP-A2, pregnancy-associated plasma protein-A2; SD, standard deviation.

Table 1 Comparison Between the FDS and NPW Clinical Samples

	FDS	NPW	p value
Maternal age (years)	37.20 ± 4.52	30.25 ± 2.87	0.015
Miscarriage (times)			0.105
0	3 (30%)	4 (100%)
1	3 (30%)	0 (0%)
2	4 (40%)	0 (0%)
SPR angle (millidegrees)	102.88 ± 15.71	43.52 ± 2.34	< 0.001

Notes: Continuous variables are presented as mean ± standard deviation and categorical variables as n (%). p < 0.05 was considered statistically significant.

Abbreviations: FDS, fetal Down’s syndrome; NPW, normal pregnant women; SPR, surface plasmon resonance.