Clinical Application for Screening Down's Syndrome by Using Carboxylated Graphene Oxide-Based Surface Plasmon Resonance Aptasensors

Abstract

Background: Advanced medical detection technology requires high sensitivity and accuracy to increase the disease detection rate. We showed that carboxyl-functionalized graphene oxide (carboxyl-GO) biosensing materials are capable of accurate detection.

Methods: We developed a carboxylated GO-based surface plasmon resonance (SPR) aptasensor suitable for screening Down's syndrome in clinical serum. This biosensing material could rapidly and accurately detect hCG protein with a low concentration to identify fetal Down's syndrome. The developed carboxyl-GO-based SPR aptasensor showed excellent sensitivity and limit of detection without the use of antibodies and without any specific preference.

Results: hCG protein detection limits of 1 pM in buffer samples and 1.9 pM in clinical serum samples were achieved. The results showed that the carboxyl-GO-based chip could detect hCG well below the normal physiological level of serum protein (5.0 mIU/mL). High affinity, sensitivity, and better detection limit were obtained in the range of 1.9 pM to 135 pM. The results showed a 5k-fold dilution factor, and that an SPR angle shift of more than 20 millidegrees (m^o) was associated with a significant risk of fetal Down's syndrome compared to normal pregnant women. The results clearly showed that the detection of hCG protein in serum samples from pregnant women at 12-19 weeks could be used to screen Down's syndrome with high selectivity and sensitivity.

Conclusion: Our findings suggest the potential application of carboxyl-GO film in proof-of-concept studies for serum assays as a new type of SPR material. In addition, peptide and carboxyl-GO films may be conducive to the development of future point of care testing and rapid diagnostic devices for other diseases such as cancer.

Keywords: DS; Down’s syndrome; GO-COOH; SPR; aptasensor; carboxyl-functionalized graphene oxide; hCG; human chorionic gonadotropin; peptides; surface plasmon resonance.

Figure 1

A schematic diagram of chemical coupling to carboxyl-GO sheets and peptide probe immobilization on the surfaces of SPR aptasensors to produce a thiol-reactive coupling surface. (A) The self-assembled monolayer linker of a bare Au surface via Cys thiol-Au interactions. (B) The carboxyl-GO sheets were immobilized on the Au surface via NH₂-COOH interactions. (C) EDC/NHS was used to activate the -COOH functional groups. (D) The EDC/NHS that activated the carboxyl-GO films was covalently attached to the amine group of Cys. Ethylenediamine was used to block unreacted linkers, and the amino group of peptide was covalently attached to -COOH of GO. (E) Monoclonal peptides were immobilized on carboxyl-GO surfaces via NH₂-COOH interactions. (F) The interaction between the hCG analyte and biorecognition peptide created a signaling event SPR angle shift by the interfaced carboxyl-GO film.

Figure 2

Photograph and profile of synthesized carboxyl-GO films for (A) SEM (B) TEM, and (C) AFM images.

Figure 3

Electrical properties of fabricated carboxyl-GO sheets in solution and films. (A) Zeta potential distribution curve of GO and carboxyl-GO sheets in aqueous suspensions. (B) Zeta potential analysis at different pH values of carboxyl-GO sheets synthesized using chloroacetic acid. (C) FTIR curves of GO and carboxyl group-modified GO. (D) Survey XPS spectrum of GO and carboxyl-GO sheets. High-resolution XPS scan spectra of the (E) C1s and (F) O1s regions of the carboxyl-GO sheets.

Figure 4

(A) Absorption spectra and band-gap of GO, carboxyl-GO dispersions in DI water. (B) SPR reflectance curve for the GO and carboxyl-GO sensing chips in comparison to the conventional Au chip. (C) Characterization of absorption properties at multilayer interface absorption spectra corresponding to Cr/Au, Au/Cys and BK7/Au/Cr/Cys/carboxyl-GO in water. (D) Dispersion relationship of SP propagating along a flat modulation at the BK7/Au/Cr/Cys/carboxyl-GO structure in contact with water at the interface.

Figure 5

Sensorgrams for the SPR responses of specificity binding of peptide-hCG interactions to the (A) carboxyl-GO and (B) GO sheets immobilized SPR sensing surface. (C) Linear calibration curve based on the relationship between SPR angle and hCG protein concentration on carboxyl-GO and GO chip responses. The average SPR response to various hCG concentrations ranging from 1 pM to 1nM. (D) Linear curve at low concentration (linear range between 1 pM to 100 pM). The specificity of the aptasensor tested the SPR responses to interfering proteins. The SPR responses to BSA, HSA, PAPP-A, PAPP-A2, CK-19 and hCG were investigated under (E) PBS buffer and (F) spiked 10% serum buffer experimental conditions.

Figure 6

SPR sensorgram data showing a comparison of binding interactions of a peptide probe with hCG protein in clinical serum samples. The hCG concentrations in the women with normal pregnancy (NPW) were (A) 163.64 nM at 15 weeks, (B) 170.47 nM at 12 weeks, (C) 126.50 nM at 14 weeks, and (D) 185.34 nM at 17 weeks.

Figure 7

SPR sensorgram data showing a comparison of binding interactions of a peptide probe with hCG protein in clinical serum samples. The hCG concentrations in the women with fetal Down’s syndrome (FDSW) were (A) 381.74 nM at 19 weeks, (B) 674.83 nM at 14 weeks, (C) 488.92 nM at 12 weeks, and (D) 418.47 nM at 12 weeks, (E) 430.82 nM at 16 weeks, and (F) 525.65 nM at 17 weeks.

Figure 8

Plot of the results of the calibration curve of clinical serum samples and analysis of SPR angle shift with assay specificity using the peptide-based SPR biosensor for carboxyl-GO film. (A) Calibration curves of average SPR detection with various hCG protein concentrations ranging from 1.9 pM to 135 pM in the serum samples. The error bars represent the SD of three replicates. (B) Correlation between two variables of the NPW and FDSW groups in serum samples. The proportion of healthy NPW group were significantly reduced in the 5k and 10k-fold dilution factor compared to the FDSW group. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. * and ** significantly different from NPW group, P= 0.004 and P=0.013, respectively. Correlating SPR and ELISA concentrations at (C) for patients of FDSW group (R² = 0.92) and (D) for healthy pregnant (NPW group) (R² =0.93) samples.