The drive underlying my research has been the development of analytical methods for the detection and identification of infection that are rapid and convenient enough to deploy directly at the point-of-care. To this end, my studies have focused on the use of electrochemical DNA biosensors for the detection of both amplified pathogen DNA and host-produced, pathogen-specific antibodies. Here I describe the characterization and application of these E-DNA sensors for the detection of various amplified products indicative of infection.

A significant hurdle in the point-of-care detection of PCR products is that they are double-stranded, rendering them difficult to detect directly using traditional hybridization methods. In response, we have developed a novel biosensor platform capable of detecting double stranded DNA. This sensor comprises a linear E-DNA probe that employs triplex-forming nucleotides as its recognition element. Specifically, I developed sensors containing a single stranded DNA probe that selectively hybridizes within the major groove of a specific double stranded DNA target via reverse Hoogsteen base pairing to form a triple helix. Here we demonstrate the versatility of this detection platform by creating sensors that can bind to either polypurine or polypyrimidine tracts present in double stranded DNA. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ∼10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. To illustrate this, I have demonstrated the sensor's ability detect unpurified, double-stranded PCR amplicons containing the relevant conserved HIV1 sequence.

Our second biosensor system avoids the problem of detecting double-stranded amplification products by employing an amplification method that produces single- stranded targets. Specifically, I have integrated such amplification with sequence- specific E-DNA detection in a single, monolithic microfluidic chip. This Integrated Microfluidic Electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method that produces single-stranded products readily amenable to hybridization-based detection. To demonstrate the clinical diagnostic utility of the IMED chips, which conferred rapid (under 2 hr) detection and discrimination of specific Salmonella serovars at clinically relevant < 1000 CFU/ml levels, I have demonstrated their ability to monitor pathogens in whole, unprocessed blood collected from an animal model of sepsis.

Moving beyond the detection of pathogen-specific nucleic acids, I closed my thesis work with the development of a sensor architecture for the detection of anti- pathogen antibodies. Termed scaffold sensors, these detect antibodies by displaying an antigen (or epitope) on the distal end of the DNA probe. This approach supports the rapid (< 10 min), single-step measurement of antibodies directly in seroconverted human serum at concentrations more than a thousand-fold below those found in authentic patient samples. This sensor also offers quantitative measurements of antibody concentrations in seroconverted patient sera in the days following infection.