The crisis in the management of infectious disease for the developed world and in the developing world (global health arena) requires rapid, easy to use, integrated, and inexpensive diagnostic devices for the detection of agents of infectious diseases, i.e. bacteria, viruses. In this application, the development of a ?Lab-on-a-Transistor? is proposed for cell capture and thermal lysing, and ultra rapid techniques for performing nucleic acid amplification on silicon transistors with a direct, rapid electrical detection of the amplified products. The grand challenge of making nucleic acid amplification truly a point-of-care test will be addressed, where the results can be obtained with high accuracy and reliability in less than 5 minutes on a silicon sensor array. To address these challenges, the bacteria Listeria monocytogenes will be used as the model system within the three years of this proposed grant but the technology platform can be applied to other pathogenic microorganisms as well.
Intellectual Merit: The proposed concept of developing a ?Lab-on-a-Transistor? has the following intellectual merit; (a) concentrating single bacteria on individual field effect transistors with in a linear array using dielectrophoresis within a microfluidic channel. Then ultra-localized heating on the surface of the field effect sensor using an ac voltage will be explored. Using this method, thermal lysing of bacteria that are attached on the surface of the transistors will be performed by achieving temperatures of 95oC or higher, (b) the same sensor would be used as a heater to perform nucleic acid amplification reactions using either a polymerase chain reaction or a rolling circuit amplification method, and (c) exploring the use of the transistors themselves for label free electrical detection of the PCR products.
Broad Impact: The proposed work will have a broad impact in the area of silicon based biosensors and the proposed technology could provide significant advances in developing point of care sensors. Graduate students will be involved in summer lecture and hands-on workshops to be held at the Micro and Nanotechnology Laboratory at the University of Illinois. REU summer support will be requested for hiring additional undergraduates to work with the graduate students on the research project during the summer, which will also help to develop a pipeline of graduate student researchers for the future.
The crisis in management of infectious disease for the developed and developing world requries rapid, easy to use, integrated, and inexpensive diagnostic devices for the detection of bacterial and viral agents. The urgency of this critical need cannot be over-emphasized since millions will benefit from the use of rapid diagnostic technologies. Specifically, nucleic acid based methods are still considered the gold standard for detection and identification of microorganisms and viruses due to their high specificity and selectivity as compared to antibody-based assays. The recent technological advances in micro-fluidics and micro/nanotechnology present new opportunities for development of small, sensitive, single-use, point-of-care Lab-on-Chip diagnostics devices that are capable of providing a rapid analysis of nucleic acid amplification for the global health applications. The major goals of this project were to establish the feasbility of using silicon transistors as biosensors to be used in point-of-care devices for detection of bacteria. We envisioned the concept of a lab-on-transistor where the silicon transistor functions as the main element for performing various steps in the biochemical assays. While much more work needs to be done to fully realize the potential of this technology, we have demonstrated and published on approaches for cell capture and thermal lysing, heating of small droplets to perform DNA detection, ultra-rapid techniques for performing nucleic acid amplification, and rapid electrical detection of amplification of DNA products on silicon transistors. Specifically, (i) we developed a new method that positions droplets on an array of individual silicon microwave heaters on chip to precisely control temperature of droplets in air, allowing us to perform biochemical reactions (Figure 1), (ii) we reported a method to position and lyse individual cells on silicon nanowires and nanoribbon biological field effect transistors. This method allows rapid and simple single cell lysis and analysis with potential applications to medical diagnostics, proteome analysis, and developmental biology studies (Figure 2), and (iii) We demonstrated that we can perform multiplexed detection of pathogens through loop mediated isothermal amplification on a silicon chip. We show that dehydrated primers dried on the chip can resuspend when other reagents are microinjected, and specifically amplify the target genes, allowing for multiplexed screening of pathogens (Figure 3). Our long term goal is to address this grand challange of making nucleic acid amplification a point-of-care test where the results can be obtained with high accuracy and reliability in less then 5 minutes at a doctor's office, at bed side, or at home. During the research, we also trained PhD students at this cutting edge of bioengineering and biology. This project provided excellent interdiscplinary opportunities for the trainees involved as they were exposed to micro and nano-fabrication and also to micro biology and cellular biology through these projects. They also collanborated with colleagues at Purdue University Food Science Depatment to get a better understanding of bacterial pathogenesis and amplification techniques. They also collaborated with companies including Abbott and TSMC (Taiwan Semiconductor Manufacturing Corp.). The outreach activities included presenting the results to broader audience at the annnual summer bionanotechnology summer school at UIUC. Collaborations also resulted in leveraged funding from Abbott, USDA and TSMC and further industrial contacts and experiences for students. We published 5 papers credited to this grant, many conference papers and invited talks.
