Development of colorimetric biosensor for detection of pathogen and single nucleotide polymorphism using gold nanoparticles

Abstract

Colorimetric biosensors for the detection of biologically important molecules such as DNA, protein, ligands, and so on have been intensively investigated since it enables visual detection without the need for any detection equipment. Recently, noble metal nanoparticles especially gold nanoparticles (AuNPs) have been developed as a new family of biomarkers for the colorimetric detection of DNA. In this study, the development of colorimetric detection method for polymerase chain reaction (PCR)-amplified nucleic acids and its application for the diagnosis of pathogen infection and single nucleotide polymorphisms (SNPs) were described. Firstly, we have developed a greatly simplified colorimetric detection method to identify PCR-amplified nucleic acids. Our method relies on the PCR product having thiol group at one end, which is generated by employing thiolated PCR primer. After PCR amplification reaction, unmodified AuNPs are added into the reaction tube followed by the addition of NaCl to induce the aggregation of AuNPs. The PCR products strongly bind to the surface of AuNPs through the interaction of the thiol group at the end and the abundant negative charges of the bound DNAs enhance the electrostatic repulsion among AuNPs, which consequently leads to the prevention of the salt-induced aggregation. As a result, the color of AuNPs remains red in the presence of the PCR-amplified nucleic acids, while the AuNPs change its color from red to blue due to the salt-induced aggregation in the absence of the PCR products. This simple but very efficient colorimetric strategy was successfully demonstrated by diagnosing Chlamydia infection using a real human urine sample. Since the results can be clearly seen with the naked eye without any complicated step such as surface modification of AuNPs or PCR product purification, this method can be easily applied to point-of-care diagnosis. Furthermore, simple colorimetric method for SNPs detection using AuNPs and thiolated PCR...