(334g) A Nanoporous Membrane Molecular Sensing Platform

We report a new molecular (nucleic acid and protein) sensor platform that is economical to mass produce, label-free,  multi-target (>5), rapid (<20 min), selective (SNP) and sensitive (pM)----and is hence most suitable for portable diagnostics.   In addition to the difficulty in labeling targets, optical detection also suffers from the small number of simultaneous detectable targets, as most fluorescent reporters occupy a bandwidth of more than 50 nm in the narrow 300 nm visible spectrum.  Classical electrochemical sensors, on the other hand, suffer from many robustness issues due to spurious electron transfer reactions and capacitance and conductance signals due to conformation change in the target, probe, or Self-Assembled Monolayers in samples of different ionic strengths and counterion valencies.  For both optical and electrochemical sensing, the assay time is often in excess of several hours for < nM concentrations due to the diffusion limitation.  Our new membrane sensor alleviates all these difficulties.  We exploit the fact that when a DC field is applied across a nanoporous ion-selective membrane, a molecularly small charge polarized layer develops on the depletion side after concentration polarization.  The presence of molecules in this polarized layer can hence sensitively change the limiting and overlimiting currents of the membrane.  The overlimiting current shift is particularly large (~1 V) because large molecules can change the hydrodynamic slip length and hence suppress the surface vortices responsible for the overlimiting current.   Furthermore, oppositely charged small molecule targets can produce a bipolar membrane, like a P-N semiconductor junction, that can produce a large water-splitting signal when a high field develops at the carrier-depleted junction with a reverse bias.  By forgoing the method of electron transfer to detect a target, we eliminate the need for SAM and the robustness problems that arise from spurious electrochemical reactions.  This new molecular sensing technology also accelerates the assay time because the high field in the polarized layer can attract large nucleic acids by dielectrophoresis and the depletion region can concentrate the charged molecules near the sensor surface in a flowing stream.   Our platform utilizes DNA probes or aptamers to achieve high specificity and enable sensitive detection of the target molecule down to pM concentrations. We also used flow to remove non-specifically bound molecules and have developed calibration curves for the proper shear rate for a particular target size and probe length.   The entire platform is fabricated onto PDMS or polymer chips, and we have tested it for detection of various targets, including nucleic acid sequences (both DNA and RNA) from 20 bp to >1 kb and several proteins.