Here we propose the development of molecular diagnostics that can accurately and quantitatively measure multiple protein biomarkers at the point-of-care, within the 15-minute timeframe of a typical patient/provider interaction. Specifically, we propose major technological advancements in our E-AB sensing platform to achieve this goal. The E-AB sensor is based on the binding-induced folding of electrode- bound DNA aptamers. Significant preliminary data indicates that the E-AB platform is sensitive (nanomolar to tens of picomolar), rapid (seconds to minutes), and selective enough to employ directly in urine, serum and other complex, unprocessed clinical materials. E-AB sensors are likewise reagentless, reusable, low volume (sub-microliter) and fully electronic (electrochemical), and are thus likely adaptable to point-of-care applications. The key hypothesis underlying this proposal is that, with sufficiently improved sensitivity and detection confidence, we can adapt the E-AB sensor platform to provide therapeutically actionable diagnostic information from clinical samples. And while our long-term objective is the fabrication of E-AB sensors suitable for the detection of a wide range of diagnostic proteins, our initial efforts will focus on a specific, representative application for which comparison with standard clinical approaches is readily achieved: the measurement of urinary and serum chemokines diagnostic of episodes of acute renal allograft rejection. Toward this end, we propose here improvements in the sensitivity and sample-to-sample stability of the E-AB sensors such that they reach the detection limits and reproducibility required for routine clinical use. We will achieve these goals by 1) selection of higher affinity aptamers (via a novel, ultra-efficient microfluidic- SELEX technology), 2) significant improvements in E-AB gain (via optimization of probe density and geometry, redox and attachment chemistry, and other important fabrication variables), and 3) development of control sensing elements that will enable precise background subtraction and correction. We have assembled an innovative and collaborative team with significant expertise in high-throughput aptamer selection, electrochemical detection and clinical practice. We believe that the success of this project will have significant ramifications -it will represent a large step toward a simple, reagentless, low cost diagnostic platform that is broadly applicable to molecular diagnostics at the point of care.
Current methods for the detection of diagnostic proteins require cumbersome, resource-intensive laboratory procedures that typically require hours or days to provide clinicians with therapeutically actionable diagnostic information. Here we propose the development of a new class of electronic (electrochemical) biosensors aimed at the rapid (<15 minutes), point-of-care quantification of multiple protein biomarkers directly in unprocessed clinical samples (i.e., urine and finger-lance samples of blood). The successful implementation of such a technology would significantly shorten the time between examination and treatment, which, in turn, will improve healthcare efficacy and transform the healthcare cost structure. While our long-term objective is the development of devices suitable for the detection of any of a wide range of diagnostic proteins, our initial efforts will focus on a representative and particularly pressing application for which comparison with standard clinical approaches is readily achieved: the measurement of urinary and blood-borne chemokines diagnostic of kidney transplant rejection.
|Ferguson, Brian Scott; Hoggarth, David A; Maliniak, Dan et al. (2013) Real-time, aptamer-based tracking of circulating therapeutic agents in living animals. Sci Transl Med 5:213ra165|
|Porchetta, Alessandro; Vallée-Bélisle, Alexis; Plaxco, Kevin W et al. (2013) Allosterically tunable, DNA-based switches triggered by heavy metals. J Am Chem Soc 135:13238-41|
|Bonham, Andrew J; Paden, Nicole G; Ricci, Francesco et al. (2013) Detection of IP-10 protein marker in undiluted blood serum via an electrochemical E-DNA scaffold sensor. Analyst 138:5580-3|
|Idili, Andrea; Plaxco, Kevin W; Vallée-Bélisle, Alexis et al. (2013) Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches. ACS Nano 7:10863-9|
|Ricci, Francesco; Vallee-Belisle, Alexis; Porchetta, Alessandro et al. (2012) Rational design of allosteric inhibitors and activators using the population-shift model: in vitro validation and application to an artificial biosensor. J Am Chem Soc 134:15177-80|
|Vallée-Bélisle, Alexis; Ricci, Francesco; Uzawa, Takanori et al. (2012) Bioelectrochemical switches for the quantitative detection of antibodies directly in whole blood. J Am Chem Soc 134:15197-200|
|White, Ryan J; Kallewaard, Hannah M; Hsieh, Wen et al. (2012) Wash-free, electrochemical platform for the quantitative, multiplexed detection of specific antibodies. Anal Chem 84:1098-103|
|Watkins, Herschel M; Vallée-Bélisle, Alexis; Ricci, Francesco et al. (2012) Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study. J Am Chem Soc 134:2120-6|
|Kang, Di; Vallee-Belisle, Alexis; Porchetta, Alessandro et al. (2012) Re-engineering electrochemical biosensors to narrow or extend their useful dynamic range. Angew Chem Int Ed Engl 51:6717-21|
|Bonham, Andrew J; Hsieh, Kuangwen; Ferguson, B Scott et al. (2012) Quantification of transcription factor binding in cell extracts using an electrochemical, structure-switching biosensor. J Am Chem Soc 134:3346-8|
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