An integrated and multi-functional droplet-based microfluidic platform for digital DNA amplification.

Digital DNA amplification is a powerful method for detecting and quantifying rare nucleic acids. In this study, we developed a multi-functional droplet-based platform that integrates the traditional digital DNA amplification workflow into a one-step device. This platform enables efficient droplet generation, transition, and signal detection within a 5-min timeframe, distributing the sample into a uniform array of 4 × 104 droplets (variation <2%) within a chamber. Subsequent in-situ DNA amplification, fluorescence detection, and signal analysis were carried out. To assess the platform's performance, we quantitatively detected the human epidermal growth factor receptor (EGFR) mutation and human papillomavirus (HPV) mutation using digital polymerase chain reaction (dPCR) and digital loop-mediated isothermal amplification (dLAMP), respectively. The fluorescence results exhibited a positive, linear, and statistically significant correlation with target DNA concentrations ranging from 101 to 105 copies/µL, demonstrating the capability and feasibility of the integrated device for dPCR and dLAMP. This platform offers high-throughput droplet generation, eliminates droplet fusion and transition, is user-friendly, reduces costs compared to current methods, and holds potential for thermocycling and isothermal nucleic acid quantification with high sensitivity and accuracy.

PEG-PtS2 nanosheet-based fluorescence biosensor for label-free human papillomavirus genotyping.

A simple and efficient ultrasonication-assisted liquid exfoliation method is proposed to produce PtS2 nanosheets on a large scale and improve their dispersion in aqueous solution by surface polyethylene glycol modification. The interaction of polyethylene glycol-modified PtS2 (PEG-PtS2) nanosheets with fluorescent labeled DNA and the fluorescence quenching mechanism using FAM-labeled hpv16e6 gene fragment as a probe was investigated. The excitation and emission wavelengths were 468 and 517 nm, respectively. The fluorescence quenching mechanism of PEG-PtS2 nanosheets for double-stranded DNA (dsDNA) might stem from the static quenching effect. Based on the difference in fluorescence quenching capability of PEG-PtS2 nanosheets in fluorescent probe tagged single-stranded DNA (ssDNA) and dsDNA, a mix-and-detect method was proposed for determination of DNA. Without the need for probe immobilization and tedious washing steps, the genotyping of human papillomavirus (HPV) was easily achieved. The limit of detection was calculated to 0.44 nM, showing a good linear range within 0.05-10 nM. We believe this biosensor provides opportunities to develop a simple and low-cost strategy for molecular diagnostics. Graphical abstract.

Single layer linear array of microbeads for multiplexed analysis of DNA and proteins.

In this study, a microfluidic platform was developed to generate single layer, linear array of microbeads for multiplexed high-throughput analysis of biomolecules. The microfluidic device is comprised of eight microbead-trapping units, where microbeads were immobilized in a linear array format by the exertion of a negative pressure in the control channel connected to each sieving microstructure. Multiplexed assays were achieved by using a mixture of different spectrally-encoded microbeads functionalized with specific probes, followed by on-chip reaction and detection. The microfluidic-based microbeads array platform was employed for multiplexed analysis of DNA and proteins, as demonstrated by the simultaneous discrimination of four HPV genotypes and the parallel detection of six different proteins. Compared with the off-chip protocols, the on-chip analysis exhibited better reaction efficiency, higher sensitivity and wider linear detection range. Visual inspection and identification of functionalized microbeads were facilitated by the single layer arrangement of microbeads so that accurate data acquisition can be performed during the detection process.

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels.

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/µL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.

A new method of screening human papillomavirus genotypes and clinical validation.

Human papillomavirus (HPV) infection is a necessary factor in the development of cervical cancer. A new HPV screening method, "Human Papillomavirus Genotyping (HPG)", was developed to detect 29 HPV genotypes distribution in China. The utility of HPG was compared to Hybrid Capture 2 High-Risk HPV DNA test (HC2), and it was determined that the HPG test had been proven to be a more credible and sensitive screening HPV method than the HC2 test. HPV16, HPV 52, HPV 56, and HPV 58 were the four most common HPV genotypes in women who have suffered chronic cervicitis or abnormal vaginal bleeding in China. HPV 16 (28.57%) and 18 (17.86%) were more likely to infect multiple HPV genotypes than other HPV genotypes. Age group more than 50 years had a higher risk than other age groups.

Patterned growth of vertically aligned silicon nanowire arrays for label-free DNA detection using surface-enhanced Raman spectroscopy.

Patterning is of paramount importance in many areas of modern science and technology. As a good candidate for novel nanoscale optoelectronics and miniaturized molecule sensors, vertically aligned silicon nanowire (SiNW) with controllable location and orientation is highly desirable. In this study, we developed an effective procedure for the fabrication of vertically aligned SiNW arrays with micro-sized features by using single-step photolithography and silver nanoparticle-induced chemical etching at room temperature. We demonstrated that the vertically aligned SiNW arrays can be used as a platform for label-free DNA detection using surface-enhanced Raman spectroscopy (SERS), where the inherent "fingerprint" SERS spectra allows for the differentiation of closely related biospecies. Since the SiNW array patterns could be modified by simply varying the mask used in the photolithographic processing, it is expected that the methodology can be used to fabricate label-free DNA microarrays and may be applicable to tissue engineering, which aims to create living tissue substitutes from cells seeded onto 3D scaffolds.