Capture and separation of biomolecules using magnetic beads in a simple microfluidic channel without an external flow device.

The use of microfluidic devices and magnetic beads for applications in biotechnology has been extensively explored over the past decade. Many elaborate microfluidic chips have been used in efficient systems for biological assays. However most fail to achieve the ideal point of care (POC) status, as they require larger conventional external devices in conjunction with the microchip. This paper presents a simple technique to capture and separate biomolecules using magnetic bead movement on a microchip without the use of an external flow device. This microchip consisted of two well reservoirs (W1 and W2) connected via a tapered microchannel. Beads were dragged through the microchannel between the two wells at an equivalent speed to a permanent magnet that moved alongside the microchip. More than 95% of beads were transferred from W1 to W2 within 2 min at an average velocity of 0.7 mm s(-1). Enzymatic reactions were employed to test our microchip. Specifically, three assays were performed using the streptavidin coated magnetic beads as a solid support to capture and transfer biomolecules: (1) non-specific adsorption of the substrate, 6-8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), (2) capture of the enzyme, biotinylated alkaline phosphatase (AP), and (3) separation of AP from DiFMUP. Our non-specific adsorption assay indicated that the microchip was capable of transferring the beads with less than 0.002% carryover of DiFMUP. Our capture assay indicated efficient capture and transfer of AP with beads to W2 containing DiFMUP, where the transferred AP converted 100% of DiFMUP to DiFMU within 15 minutes. Our separation assay showed effective separation of AP from DiFMUP and elucidated the binding capacity of the beads for AP. The leftover unbound AP in W1 converted 100% of DiFMUP within 10 minutes and samples with less than the full bead capacity of AP (i.e. all AP was transferred) did not convert any of the DiFMUP. The immobilization of AP on the bead surface resulted in 32% reduced enzymatic speed compared to that of free AP in solution, as a result of altered protein conformation and/or steric hindrance of the catalytic site. Overall, this microfluidic platform was established as a simple, efficient and effective approach for separating biomolecules without any flow apparatus.

Engineering insights for multiplexed real-time nucleic acid sequence-based amplification (NASBA): implications for design of point-of-care diagnostics.

BACKGROUND: Nucleic acid sequence-based amplification (NASBA) offers huge potential for low-cost, point-of-care (POC) diagnostic devices, but has been limited by high false-positive rates and the challenges of primer design. OBJECTIVE: We offer a systematic analysis of NASBA design with a view toward expanding its applicability. METHODS: We examine the parameters that effect dimer formations, and we provide a framework for designing NASBA primers that will reduce false-positive results and make NASBA suitable for more POC diagnostic applications. Then we compare three different oligonucleotide sets to examine (1) the inhibitory effect of dimer formations, (2) false positives with poorly designed primers, and (3) the effect of beacon target location during real-time NASBA. The required T7 promoter sequence adversely affects the reaction kinetics, although the common abridged sequence can improve kinetics without sacrificing accuracy. RESULTS: We demonstrate that poorly designed primers undergo real-time exponential amplification in the absence of target RNA, resulting in false positives with a time to half of the peak value (t(1/2)) of 50 min compared to 45 min for true positives. Redesigning the oligonucleotides to avoid inhibitory dimers eliminated false positives and reduced the true positive t(1/2) by 10 min. Finally, we confirm the efficacy of two molecular beacon design schemes and discuss their multiplexing utility in two clinical scenarios. CONCLUSION: This study provides a pathway for using NASBA in developing POC diagnostic assays.

Detection of HIV-1 minority variants containing the K103N drug-resistance mutation using a simple method to amplify RNA targets (SMART).

The simple method for amplifying RNA targets (SMART) was used to detect K103N, a common HIV-1 reverse transcriptase drug-resistance mutation. Novel amplifiable SMART probes served as reporter molecules for RNA sequences that are captured and separated on a microfluidic platform under zero-flow conditions. Assays were performed both off chip and in a microchip reservoir using a modified version of real-time nucleic acid sequence-based amplification, without the noncyclic phase, and 65°C preheat. A total of 6000 copies/mL of the synthetic sequences were detected within 180 minutes of amplification. Although the sensitivity of research platforms is higher, SMART has the potential to offer comparable sensitivity and speed to commercially available viral load and HIV detection kits. Furthermore, SMART uses an inexpensive, practical, and more accurate isothermal exponential amplification technique. The use of molecular beacons resulted in relatively fast real-time detection (<180 minutes); however, they were also shown to hinder the amplification process when compared with end point detection. Finally, SMART probes were used for modeling of K103N concentrations within an unknown sample. Only 1% of the SMART probes was detected within the wild-type population (6 × 10(8) copies/mL). These results establish the groundwork for point-of-care drug resistance and viral load monitoring in clinical samples, which can revolutionize HIV patient care globally.

Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization.

The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein.