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

Abstract

Background: Due to educational, social and economic reasons, more and more women are delaying childbirth. However, advanced maternal age is associated with several adverse pregnancy outcomes, and in particular a high risk of Down's syndrome (DS). Hence, it is increasingly important to be able to detect fetal Down's syndrome (FDS).

Methods: We developed an effective, highly sensitive, surface plasmon resonance (SPR) biosensor with biochemically amplified responses using carboxyl-molybdenum disulfide (MoS₂) film. The use of carboxylic acid as a surface modifier of MoS₂ promoted dispersion and formed specific three-dimensional coordination sites. The carboxylic acid immobilized unmodified antibodies in a way that enhanced the bioaffinity of MoS₂ and preserved biorecognition properties of the SPR sensor surface. Complete antigen pregnancy-associated plasma protein-A2 (PAPP-A2) conjugated with the carboxyl-MoS₂-modified gold chip to amplify the signal and improve detection sensitivity. This heterostructure interface had a high work function, and thus improved the efficiency of the electric field energy of the surface plasmon. These results provide evidence that the interface electric field improved performance of the SPR biosensor.

Results: The carboxyl-MoS₂-based SPR biosensor was used successfully to evaluate PAPP-A2 level for fetal Down's syndrome screening in maternal serum samples. The detection limit was 0.05 pg/mL, and the linear working range was 0.1 to 1100 pg/mL. The women with an SPR angle >46.57 m° were more closely associated with fetal Down's syndrome. Once optimized for serum Down's syndrome screening, an average recovery of 95.2% and relative standard deviation of 8.5% were obtained. Our findings suggest that carboxyl-MoS₂-based SPR technology may have advantages over conventional ELISA in certain situations.

Conclusion: Carboxyl-MoS₂-based SPR biosensors can be used as a new diagnostic technology to respond to the increasing need for fetal Down's syndrome screening in maternal serum samples. Our results demonstrated that the carboxyl-MoS₂-based SPR biosensor was capable of determining PAPP-A2 levels with acceptable accuracy and recovery. We hope that this technology will be investigated in diverse clinical trials and in real case applications for screening and early diagnosis in the future.

Keywords: DS; Down’s syndrome; FDS; PAPP-A2; SPR; carboxyl-MoS2; carboxyl-functionalized molybdenum disulfide; fetal Down’s syndrome; pregnancy-associated plasma protein A2; surface plasmon resonance.

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.

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).

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.

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.

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.

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.

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.

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.

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.