A paper-embedded thermoplastic microdevice integrating additive-enhanced allele-specific amplification and silver nanoparticle-based colorimetric detection for point-of-care testing

Abstract

This study introduces a thermoplastic microdevice integrated with additive-enhanced allele-specific amplification and hydrazine-induced silver nanoparticle-based detection of single nucleotide polymorphism (SNP) and opportunistic pathogens. For point-of-care testing of SNP, an allele-specific loop-mediated isothermal amplification reaction using nucleotide-mismatched primers and molecular additives was evaluated to discriminate single-nucleotide differences in the samples. The microdevice consists of purification and reaction units that enable DNA purification, amplification, and detection in a sequential manner. The purification unit enables the silica-based preparation of samples using an embedded glass fiber membrane. Hydrazine-induced silver nanoparticle formation was employed for endpoint colorimetric detection of amplicons within three min at room temperature. The versatile applicability of the microdevice was demonstrated by the successful identification of SNPs related to sickle cell anemia, genetically-induced hair loss, and Enterococcus faecium. The microdevice exhibited a detection limit of 10³ copies per μL of SNP targets in serum and 10² CFU mL⁻¹ of Enterococcus faecium in tap water within 70 min. The proposed microdevice is a promising and versatile platform for point-of-care nucleic acid testing of different samples in low-resource settings.