ABSTRACT
Timely biomarker quantitation has potential to improve human health but current methods have disadvantages either in terms of cost and complexity for benchtop instruments, or reduced performance in quantitation and/or multiplexing for point-of-care systems. We previously developed microfluidic devices wherein visually observed flow distances correlated with a model analyte's concentration.1 Here, we significantly expand over this prior result to demonstrate the measurement of unamplified DNA analogues of microRNAs (miRNAs), biomarkers whose levels can be altered in disease states. We have developed a method for covalently attaching nucleic acid receptors on poly(dimethylsiloxane) microchannel surfaces by silane and cross-linker treatments. We found a flow distance dependence on target concentrations from 10 µg/mL to 10 pg/mL for DNA in both buffer and synthetic urine. Moreover, flow time in addition to flow distance is correlated with target concentration. We also observed longer flow distances for single-base mismatches compared to the target sequence at the same concentration, indicating that our approach can be used to detect point mutations. Finally, experiments with DNA analogues of miRNA biomarkers for kidney disease (mir-200c-3p) and prostate cancer (mir-107) in synthetic urine showed the ability to detect these analytes near clinically relevant levels. Our results demonstrate that these novel microfluidic assays offer a simple route to sensitive, amplification-free nucleic acid quantitation, with strong potential for point-of-care application.
ABSTRACT
Simplified analysis systems that offer the performance of benchtop instruments but the convenience of portability are highly desirable. We have developed novel, miniature devices that feature visual inspection readout of a target's concentration from a ~1 µL volume of solution introduced into a microfluidic channel. Microchannels are constructed within an elastomeric material, and channel surfaces are coated with receptors to the target. When a solution is flowed into the channel, the target cross-links multiple receptors on the surface, resulting in constriction of the first few millimeters of the channel and stopping of flow. Quantitation is performed by measuring the distance traveled by the target solution in the channel before flow stops. A key advantage of our approach is that quantitation is accomplished by simple visual inspection of the channel, without the need for complex detection instrumentation. We have tested these devices using the model system of biotin as a receptor and streptavidin as the target. We have also characterized three factors that influence flow distance: solution viscosity, device thickness, and channel height. We found that solution capillary flow distance scales with the negative logarithm of target concentration and have detected streptavidin concentrations as low as 1 ng/mL. Finally, we have identified and evaluated a plausible mechanism wherein time-dependent channel constriction in the first few millimeters leads to concentration-dependent flow distances. Their simplicity coupled with performance makes these "flow valve" systems especially attractive for a host of analysis applications.
