Microfluidic Biosensor for Rapid Nucleic Acid Quantitation Based on Hyperspectral Interferometric Amplicon-Complex Analysis

Abstract

Nucleic acid detection plays a vital role in both biomedical research and clinical medicine. The temperature circulation changes of the widely used polymerase chain reaction technique are time-consuming and technically challenging for system development. Recombinase polymerase amplification (RPA) is an isothermal method for rapid nucleic acid detection. However, current RPA amplicon detection methods are complicated and expensive and easily generate false positives, restricting the promotion of RPA techniques. In this work, a hyperspectral interferometric amplicon-complex quantitation method is presented, combined with asymmetric dipole complex strategy optical scattering analysis. GelRed dye was utilized to form amplicon-complex particles, and the Fourier domain spectrum computation contributed to complex scattering quantitation. With this method, a supporting microfluidic chip and automatic system were developed to achieve integrated, rapid, quantitative, and miniscule nucleic acid detection. The Plasmodium falciparum dhfr gene was utilized as an example for targeted nucleic acid quantitation and single nucleotide polymorphism detection. The total reaction time was decreased to merely 20 min, and the limit of detection was only 3.17 ng/μL. The minimum measurable concentration of target was 1.68 copies/ μL, 31.67 times more sensitive than turbidity detection, and the single reaction chamber was only 9.33 μL. No scattering increase occurred for template-free control, and thus, false positives caused by primer dimers and nonspecific products could be avoided. The experimental results prove that the provided method and system can detect single-base mutations in the dhf r gene and is a reasonable technique for rapid, automatic, and low-cost nucleic acid detection.

Nanogels designed for cell-free nucleic acid sequestration

“…The intercalation of this probe between the base pairs of DNA molecules leads to a significant increase in photoluminescence intensity. 41,42 Competitive binding of DNA to other species (such as cNGs) suppresses GelRed intercalation and results in the reduction in photoluminescence signal. Thus, cfDNA binding to cNGs was monitored by measuring concomitant decrease in GelRed photoluminescence.…”

Label-free recombinase polymerase amplification with hyperspectral digital optofluidics

“…Interference, a prevalent physical phenomenon, offers the prospect of revealing nanoscale fluctuations on sample surfaces through quantitative analysis of interference signals [7,8] . Noteworthy is that this technique neither demands precision instruments for the generation of distinguishable coherent signals nor imposes a requirement for expensive optical apparatus or constituents.…”

A sample-to-answer DNA detection microfluidic system integrating sample pretreatment and smartphone-readable gradient plasmonic photothermal continuous-flow PCR

“…Specifically, compared with the widely recommended PCR, isothermal amplifications including RPA and LAMP (loop-mediated isothermal amplification) have the limitations of insufficient detection accuracy and requiring intricate reagents and an irreducible reaction time. [23][24][25] The recently emerging nanomaterial-mediated photothermal PCRs prove the feasibility of completing 35 cycles within minutes, but most time-domain PCRs require complex thermal control and even thermally synchronized fluorescence detection. In addition, the wavelength (450 nm) of gold nanofilm-based photothermal PCR can cause fluorescence dye bleaching, so DNA was only roughly characterized by adding the SYBR green dye to the product after PCR.…”