Advances in Next Generation Sequencing (NGS) have greatly accelerated fundamental life science research but additional breakthroughs in technology are required to allow routine use of sequencing in the clinical setting for personalized medicine as a disease diagnostic, prognostic and therapeutic tool and other applications. Today's instrumentation and detection platforms allow sequencing of an entire genome for a cost of approximately $10,000 requiring large up-front investment into expensive equipment. The current bottlenecks include the use of expensive reagents and optical detection systems, large data storage and processing requirements, slow sample preparation and turnaround times. Therefore, the overall goal is to develop a low cost, real-time, fully-integrated sequencing system that encompasses the entire workflow and consists of several innovative sample preparation and sequencing modules that are being developed in parallel. One of the core components of the sequencing module will be two novel electric nano-biosensors that enable label free, accurate and sensitive detection of the pH change as an 'electrical signature'of the polymerization reaction during 'sequencing by synthesis'(SBS). Electronic sequencing offers several advantages over pyrosequencing and other existing techniques as it does not require fluorescent labels or reporter enzymes and optical detection systems. The innovative use of two independently operating electric nanosensors will generate significantly more accurate sequencing data at lower cost, while reducing the need for highly redundant sequence coverage. Dual sensing mechanism of detecting both steady state signal (e.g. nanoneedle) and transient signal (nanobridge) provides higher level of confidence and improves base calling accuracy, as two orthogonal sensing mechanisms with the two sensors are reporting the same event. The overall goal for this proposal is to demonstrate the feasibility of dual sensing for electronic DNA sequencing using the nanobridge and nanoneedle biosensors and developing a design for the dual sensor detection module. Basic functional testing and response to pH will be determined followed by a comparison of signal generation induced by pH changes during 'sequencing by synthesis'polymerization reaction. In the first phase, we will work on the characterization of nanobridge biosensor array, which is a depletion mode nano-resistor with low threshold and large linear detection range and the comparison of the properties of the two nanosensors with respect to response to pH or charge, buffer ionic strength and performance for DNA sequencing perspective. The results from the feasibility study will provide the basis for the design and modeling of a dual sensor array in the future.
The proposed project is to develop a highly sensitive, accurate dual electronic nano-biosensor embedded within a micro-channel structure to detect the pH change resulting from the DNA polymerization sequencing reaction in real time. The novel design of the two nanosensors will improve sensitivity and accuracy while relying on decades of experience in the semiconductor industry for their fabrication. The dual sensor will become an important building block for a novel 'sample to result'automated sequencing platform for routine medical sequencing and address the current bottleneck of cost/base through its label free electrical sensing mechanism, array format and the reduction of data storage needs normally associated with today's most commonly used optical readout systems.