Advanced fabrication of biosensor on detection of Glypican-1 using S-Acetylmercaptosuccinic anhydride (SAMSA) modification of antibody

Abstract

Glypican-1 (GPC-1) has been recognized as biomarker of pancreatic cancer. Quantification of GPC-1 level is also pivotal to breast cancer and prostate cancer's patients. We hereby report the first biosensor for GPC-1 detection. Instead of using crosslinking technique and surface immobilization of antibody, we applied a novel method for biosensor fabrication, using S-Acetylmercaptosuccinic anhydride (SAMSA) to modify the Anti-GPC-1 producing a thiol-linked Anti-GPC-1. The thiol-linked Anti-GPC-1 was then directly formed a single-layer antibody layer on the gold biosensor, minimizing the biosensor preparation steps significantly. Time of Flight Secondary Ions Mass Spectroscopy (TOF-SIMS) characterization verified the thiol-linked antibody layer and demonstrated a unique perspective for surface protein characterization. Differential pulse voltammetry (DPV) was applied to quantify GPC-1 antigen in undiluted human serum with a concentration range of 5,000 pg/µL to 100 pg/µL. The performance of this newly designed biosensor was also compared with modified self-assembled monolayer system fabricated biosensor, demonstrating the high-sensitivity and high-reproducibility of the SAMSA modified antibody based biosensor. This simple fabrication method can also expand to detection of other biomolecules. The simplified operation process shows great potential in clinical application development.

Figure 1

Reaction process of SAMSA and Anti-GPC-1 producing thiol-linked Anti-GPC-1.

Figure 2

(a) Nyquist plot for impedance characterization of bare electrode, Anti-GPC-1 incubated electrode, and thiol-linked Anti-GPC-1 incubated electrode.

Figure 3

TOF-SIMS analysis of C-N bond based on two biosensors, (a) unmodified Anti-GPC-1 incubated electrode surface; (b) thiol-linked Anti-GPC-1 incubated electrode surface.

Figure 4

(a)CV characterization of GPC-1 biosensor based on scan rates of 30 mV/s–100 mV/s. (b) Linear calibration curves of the oxidation peaks and the reduction peaks against the square root of scan rate based on CV characterization of GPC-1 biosensor.

Figure 5

(a) Investigation of different antigen incubation time on GPC-1 biosensor. (b) Modified self-assembled monolayer system (3-MPA & DTT) based GPC-1 biosensor (Biosensor 1) on DPV measurements of multiple concentrations of GPC-1 antigen in undiluted human serum. (c) SAMSA modified antibody based GPC-1 biosensor (Biosensor 2) on DPV measurements of multiple concentrations of GPC-1 antigen in undiluted human serum. (d) The calibration curve based on Biosensor 1 and Biosensor 2. (e) Exhibition of change of current response at different concentrations of GPC-1 antigens based on two different biosensors. (f) Interference study based on HE4 antigen.