Label-Free Oligonucleotide-Based SPR Biosensor for the Detection of the Gene Mutation Causing Prothrombin-Related Thrombophilia.

Rodrigo Sierpe,Marcelo J Kogan,Soledad Bollo

doi:10.3390/s20216240

Abstract

Prothrombin-related thrombophilia is a genetic disorder produced by a substitution of a single DNA base pair, replacing guanine with adenine, and is detected mainly by polymerase chain reaction (PCR). A suitable alternative that could detect the single point mutation without requiring sample amplification is the surface plasmon resonance (SPR) technique. SPR biosensors are of great interest: they offer a platform to monitor biomolecular interactions, are highly selective, and enable rapid analysis in real time. Oligonucleotide-based SPR biosensors can be used to differentiate complementary sequences from partially complementary or noncomplementary strands. In this work, a glass chip covered with an ultrathin (50 nm) gold film was modified with oligonucleotide strands complementary to the mutated or normal (nonmutated) DNA responsible for prothrombin-related thrombophilia, forming two detection platforms called mutated thrombophilia (MT) biosensor and normal thrombophilia (NT) biosensor. The results show that the hybridization response is obtained in 30 min, label free and with high reproducibility. The sensitivity obtained in both systems was approximately 4 ΔμRIU/nM. The dissociation constant and limits of detection calculated were 12.2 nM and 20 pM (3 fmol), respectively, for the MT biosensor, and 8.5 nM and 30 pM (4.5 fmol) for the NT biosensor. The two biosensors selectively recognize their complementary strand (mutated or normal) in buffer solution. In addition, each platform can be reused up to 24 times when the surface is regenerated with HCl. This work contributes to the design of the first SPR biosensor for the detection of prothrombin-related thrombophilia based on oligonucleotides with single point mutations, label-free and without the need to apply an amplification method.

Highlights

Thrombophilia corresponds to an abnormality in coagulation or in the fibrinolytic system that increases the risk of coagulation leading to a thrombotic event
Thrombophilia can be due to genetic or acquired factors, and the second most frequent factor corresponds to the mutation of the prothrombin gene G202210A, called prothrombin-related thrombophilia (PRT). This mutation is found in 5% to 10% of patients presenting with venous thrombosis and in approximately 15% of patients being investigated for thrombophilia [2]
The construction of each biosensor began by forming a self-assembled monolayer of 4MBA on the gold surface of the chip outside the equipment via drop coating, as has been previously reported [40,41]

Summary

Introduction

Thrombophilia corresponds to an abnormality in coagulation or in the fibrinolytic system that increases the risk of coagulation leading to a thrombotic event. Thrombophilia can be due to genetic or acquired factors, and the second most frequent factor corresponds to the mutation of the prothrombin gene G202210A, called prothrombin-related thrombophilia (PRT). This mutation is found in 5% to 10% of patients presenting with venous thrombosis and in approximately 15% of patients being investigated for thrombophilia [2]. PRT is produced by a substitution of a single base pair, replacing guanine with adenine, at the position of nucleotide 20,210 on chromosome 11p-q12, increasing the level of prothrombin (precursor of thrombin) and the risk of venous thrombosis or pulmonary embolism [5,6]. A suitable alternative that does not require sample amplification and that has the potential to detect the mutation in a single base pair is the surface plasmon resonance (SPR) technique

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