(253f) Designing an Integrated Biosensing Platform for Sample-to-Answer Solution

Real time PCR (rt-PCR), the current gold standard for pathogen detection, is expensive and requires trained personnel/bulky instrumentation making it impractical for field applications. We envision to develop a push-button sample-to-answer low-cost, self-powered and portable instrument with a slot where an integrated microfluidic chip is inserted for detection of any target pathogen of interest. The objective is to use our patented nanomembrane-based biosensing technology to develop a low-cost, rapid (45 min), specific (single base mismatch) and sensitive (1 fM) pathogen detection platform without any PCR amplification. The integrated microfluidic biochip consists of a sample pretreatment unit to isolate nucleic acid from the target sample and nanomembrane-based molecular sensing/pre-concentration unit.

The sample pretreatment unit is fabricated based on gel electrophoresis where an agar gel is used to separate sample reservoir from microfluidic channel. The DC field is applied to draw the negatively charged nucleic acid molecules to a microfluidic channel from chemically lysed target sample. This allows to filter out the negatively charged biomolecules from the cell debris. This sample preparation is then integrated with an ion-selective nanomembrane based preconcentration unit to concentrate the isolated nucleic acid at a specific position in a microfluidic channel and biosensor is placed in that location for detection of target pathogen of interest. Several interesting phenomena are known to take place at the interface of an ion selective environment (nanochannel or ion exchange membrane or particle) and the surrounding electrolyte in DC or AC electric fields. These phenomena include formation of depletion and concentration regions around the ion selective nanoporous materials, development of a space charge region, development of electrically driven vortices, water splitting reaction etc. The concentration of biomolecules in our integrated platform is due to the depletion phenomenon developed at the cation-exchanged membrane interface and application of pressure driven flow in the opposite direction of depletion front. The preconcentration makes the biosensing rapid and sensitive by reducing the diffusion time and also concentrating the target molecules by 200x. The anion-exchanged nanomembrane based biosensing is based on a charge inversion phenomenon that occurs when negatively charged DNA/RNA molecules hybridize with specific probes attached to nanoporous materials bearing a positive fixed charge. This is detected by measuring a change in current-voltage characteristics of the system before and after hybridization. Instead of the classical electrochemical sensing technique, we utilize a much more sensitive conductance signature based on sensitivity of ion current across the nanoporous membrane to surface charge density change effected by hybridized molecules. The sensitivity involves a nonlinear correction to the conductance and is hence much more sensitive than EIS or other linear electrochemical sensing techniques.