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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI076899-04
Application #
8423403
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (03))
Program Officer
Gondre-Lewis, Timothy A
Project Start
2010-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2013
Total Cost
$335,848
Indirect Cost
$90,445
Name
University of California Santa Barbara
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
094878394
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Idili, Andrea; Plaxco, Kevin W; Vallee-Belisle, Alexis et al. (2013) Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches. ACS Nano 7:10863-9
Porchetta, Alessandro; Vallee-Belisle, 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
Perez Rafael, Sandra; Vallee-Belisle, Alexis; Fabregas, Esteve et al. (2012) Employing the metabolic "branch point effect" to generate an all-or-none, digital-like response in enzymatic outputs and enzyme-based sensors. Anal Chem 84:1076-82
Vallee-Belisle, Alexis; Ricci, Francesco; Plaxco, Kevin W (2012) Engineering biosensors with extended, narrowed, or arbitrarily edited dynamic range. J Am Chem Soc 134:2876-9
Watkins, Herschel M; Vallee-Belisle, 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
Plaxco, Kevin W; Soh, H Tom (2011) Switch-based biosensors: a new approach towards real-time, in vivo molecular detection. Trends Biotechnol 29:1-5
Lubin, Arica A; Plaxco, Kevin W (2010) Folding-based electrochemical biosensors: the case for responsive nucleic acid architectures. Acc Chem Res 43:496-505
Cho, Minseon; Xiao, Yi; Nie, Jeff et al. (2010) Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing. Proc Natl Acad Sci U S A 107:15373-8