Speaker
Description
Determination of the concentration and physical characteristics of a biomolecule with single molecule sensitivity is a fundamental measurement in biophysics, analytical chemistry, and central to diagnostic applications. In this work, we introduce a microfluidic device with an integrated nanopore sensor, which leverages sample compartmentalization in known pL volumes allowing direct molecular analysis and label free counting to depletion through resistive pulse sensing technique. For this purpose, the molecular concentration is determined by counting a finite number of molecules within a known volume, enabling direct quantification of pico- to nanomolar concentrations, 3 orders of magnitude dynamic range. Using this technique, we successfully determined the concentrations of double-stranded DNA (dsDNA) and green fluorescent protein (GFP) within minutes and without requiring any external calibration with a limit of detection of 1 pM that can potentially be improved to low fM. By mathematically modeling the kinetics of molecular depletion and stochasticity of molecular capture in finite volume, we show that the concentration can be inferred without the need to capture and count every molecule within the compartment. Apart from the concentration, molecular size, shape, volume, and charge can also be determined using this technique which is interesting for molecular fingerprinting applications. Moreover, we demonstrate the ability to preconcentrate the biomolecular sample up to 5-fold by real-time tuning of the microtrap volume, which can be used to reduce the measurement time and to lower the detection limit. We have shown that the response time for measurement can be further reduced through applying a salt concentration gradient across the nanopore. Overall, our approach enables reliable concentration and molecular physical characteristics measurements without the need for bulky or costly equipment and is compatible with potentially portable devices for point of need application.
| Keyword-1 | Molecular Biophysics |
|---|---|
| Keyword-2 | Microfluidic Nanopore sensor |
| Keyword-3 | Protein sensing |