Ultrasensitive Detection of Single-Walled Carbon Nanotubes Using Surface Plasmon Resonance.

Because single-walled carbon nanotubes (SWNTs) are known to be a potentially dangerous material, inducing cancers and other diseases, any possible leakage of SWNTs through an aquatic medium such as drinking water will result in a major public threat. To solve this problem, for the present study, a highly sensitive, quantitative detection method of SWNTs in an aqueous solution was developed using surface plasmon resonance (SPR) spectroscopy. For a highly sensitive and specific detection, a strong affinity conjugation with biotin-streptavidin was adopted on an SPR sensing mechanism. During the pretreatment process, the SWNT surface was functionalized and hydrophilized using a thymine-chain based biotinylated single-strand DNA linker (B-ssDNA) and bovine serum albumin (BSA). The pretreated SWNTs were captured on a sensing film, the surface of which was immobilized with streptavidin on biotinylated gold film. The captured SWNTs were measured in real-time using SPR spectroscopy. Specific binding with SWNTs was verified through several validation experiments. The present method using an SPR sensor is capable of detecting SWNTs of as low as 100 fg/mL, which is the lowest level reported thus far for carbon-nanotube detection. In addition, the SPR sensor showed a linear characteristic within the range of 100 pg/mL to 200 ng/mL. These findings imply that the present SPR sensing method can detect an extremely low level of SWNTs in an aquatic environment with high sensitivity and high specificity, and thus any potential leakage of SWNTs into an aquatic environment can be precisely monitored within a couple of hours.

Multiplex Assay for Rapid Detection and Analysis of Nucleic Acid Using Barcode Receptor Encoded Particle (BREP).

Several multiplex nucleic acid assay platforms have been developed in response to the increasing importance of nucleic acid analysis, but these assays should be optimized as per the requirements of point-of-care for clinical diagnosis. To achieve rapid and accurate detection, involving a simple procedure, we propose a new concept in the field of nucleic acid multiplex assay platforms using hydrogel microparticles, called barcode receptor-encoded particles (BREPs). The BREP assay detects multiple targets in a single reaction with a single fluorophore by analyzing graphically encoded hydrogel particles. By introducing sets of artificially synthesized barcode receptor and barcode probes, the BREP assay is easily applicable in multiplexing any genetic target; sets of barcode receptors and barcode probes should be designed delicately for universal application. The performance of the BREP assay was successfully verified in a multiplex assay for the identification of different malaria species with high sensitivity, wide dynamic range, fast detection time, and multiplexibility.

Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics.

Cell-free DNA (cfDNA) has been implicated as an important biomarker in cancer management. Thus, efficient techniques for cfDNA extraction are necessary for precision medicine. We developed a centrifugation-free cfDNA extraction microfluidic chip capable of extracting cfDNA from plasma samples through microfluidic circuits within 15 min under vacuum pressure using an immiscible solvent. The microfluidic chip had excellent performance that was comparable to the most widely used commercial product (QIAamp kit) in terms of extraction efficiency, purity, and quality of DNA samples. The microfluidic chip was validated for the continuous monitoring of HER-2 type breast cancer and was able to successfully detect a point mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA) during severe liver metastasis. The chip effectively eliminates the repetitive centrifugation processes and dramatically shortened the sample preparation time. The proposed platform could facilitate the development of a sample-to-answer system for use in liquid biopsy of cancers.

Rapid molecular diagnosis of infectious viruses in microfluidics using DNA hydrogel formation.

There has been an urgent need to quickly screen and isolate patients with viral infections from patients with similar symptoms at point-of-care. In this study, we introduce a new microfluidic method for detection of various viruses using rolling circle amplification (RCA) of pathogens on the surface of thousands of microbeads packed in microchannels. When a targeted pathogen meets the corresponding particular template, the DNAs are rapidly amplified into a specific dumbbell shape through the RCA process, forming a DNA hydrogel and blocking the flow path formed between the beads. Due to the significant increase in reaction surface area, the detection time was shortened to less than 15 min and the detection limit of various pathogens has been reached to 0.1 pM. By injecting the stained liquid, the existence of the target pathogens in a sample fluid can be determined with the naked eye. Furthermore, by integrating multi-channel design, simultaneous phenotyping of various infective pathogens (i.e., Ebola, Middle East respiratory syndrome (MERS), and others) in biological specimens can be performed at a point-of-care.

Centrifugation-free extraction of circulating nucleic acids using immiscible liquid under vacuum pressure.

Extraction of cell-free DNA (cfDNA), which exists at an extremely low concentration in plasma, is a critical process for either targeted-sensing or massive sequencing of DNAs. However, such small amount of DNA cannot be fully obtained without high-speed centrifugation (<20,000 g). Here, we developed a centrifugation-free cfDNA extraction method and system that utilizes an immiscible solvent under single low vacuum pressure throughout the entire process. It has been named Pressure and Immiscibility-Based EXtraction (PIBEX). The amounts of extracted cfDNA by PIBEX were compared with those extracted by the conventional gold standards such as QIAGEN using quantitative PCR (qPCR). The PIBEX system showed equal performance regarding extraction amount and efficiency compared to the existing method. Because the PIBEX eliminates the troublous and repetitive centrifugation processes in DNA extraction, it can be further utilized in microfluidic-sample preparation systems for circulating nucleic acids, which would lead to an integrated sample-to-answer system in liquid biopsies.