The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 µl in each operation, which is remarkably less than that of a macro system.

DNA diagnosis in a microseparator based on particle aggregation.

A novel aggregation-based biosensing method to achieve detection of oligonucleotides in a pinched-flow fractionation (PFF) microseparator was developed. Employing functionalized polystyrene microspheres, this method is capable of the direct detection of the concentration of a specific DNA sequence. The label-free target DNA hybridizes with probe DNA of two kinds on the surface of the microspheres and causes the formation of an aggregate, thus increasing the average size of the aggregate particles. On introducing the sample into a PFF microseparator, the aggregate particles locate at a specific position depending on the size of the aggregate. Through a multi-outlet asymmetric PFF microseparator, the aggregate particles become separated according to outlets. Because the size of the aggregate particles is proportional to the concentration of the target DNA, a rapid quantitative analysis is achievable with an optical microscope. A biological dose-response curve with concentration in a dynamic range 0.33-10nM has been achieved; the limit of detection is between 33 and 330 pM. The specificity of the method and the potential to detect single-nucleotide polymorphism (SNP) of known concentration were examined. The method features simple, direct and cheap detection, with a prospect of detecting other biochemical samples with distinct aggregation behavior, such as heavy-metal ions, bacteria and proteins.

Screening of aptamers on microfluidic systems for clinical applications.

The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications.

An integrated microfluidic system for rapid screening of alpha-fetoprotein-specific aptamers.

The systematic evolution of ligands by exponential enrichment (SELEX) is a screening technique that involves the progressive selection of highly specific ligands via repeated rounds of partition and amplification from a large random pool of nucleic acid sequences. The products of this selection process are called aptamers and are either short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules with a high binding affinity to a large variety of target analytes. However, SELEX is a lengthy, labor-intensive, iterative process requiring multiple rounds of extraction and polymerase chain reaction (PCR) amplification. In order to address these problems, this study presents a new integrated microfluidic system consisting of a magnetic bead-based microfluidic SELEX chip and a competitive assay chip to automate the aptamer screening process. More importantly, the selected ssDNA sequences were confirmed to have a high affinity and specificity to the target molecules, using the developed competitive assay chip. With this approach, an aptamer specific to alpha-fetoprotein (AFP), which is a biomarker for liver cancers, has been successfully selected. The screened aptamer was used as a recognition molecule for AFP and has a linear detection range from 12.5 to 800 ng/mL, which was suitable for rapid clinical applications.