SBIR-STTR Award

Pancreatic cancer (pac) has a poor prognosis in most cases due to the lack of early detection owing to its nonspecific, asymptomatic nature. An effective, inexpensive screening tool is needed for early diagnosis (within 15 years of tumor initiation). It is well accepted that genomic instability is a hallmark of cancer. Recent research indicates that less than hundred microrna (mirna) sequences specifically generated by tumors are sufficient for early detection of pac and other cancers for effective intervention with excellent prognosis. Mirna can be extracted from urine and blood using standard kits. The key challenge is to detect specific biomarkers in the thousand-fold large background of mirna sequences that body normally produces. While quantitative reverse transcribe-polymerase chain reaction (qrt-pcr) is an effective tool for mirna profiling, it is prohibitively expensive fo screening. A microarray is an inexpensive alternative. However, due to the small size of mirna, a conventional microarray is not reliable due to large background from nonspecific binding. A disruptive technology is needed to read microarrays of mirna at high specificity with minimal background from the normal mirna sequences and high sensitivity to avoid pcr amplification. Owing to the small size of mirna, pcr is an added expensive complexity. It is well known that electrochemical detection has excellent specificity with virtually no background from nonspecific binding of targets to microarray of probes. The key limitations of this active detection method are: (a) only one target sequence per electrode can be detected, and (b) the redox current decreases as the sensor electrode size diminishes , making multiplexing difficult. A method developed in saraf's lab, at the university of nebraska-lincoln, can electrochemically "read" microarray spots on a monolith electrode by simply scanning a laser with a beam size of ~10 µm to quantitatively measure the local redox current. Published studies indicate that scanning electrometer for electrical double-layer (seed) has (conservative) responsivity of

Public Health Relevance Statement:


Public Health Relevance:
profiling circulating mirna without pcr for early detection of pancreatic cancer narrative microrna circulating in blood have the potential to effectively diagnose pancreatic cancer and other cancers before clinical signs appear. The key is to detect less than 30 mirna sequences in the midst of thousand fold large background of other mirna sequences that the body normally produces. The current technologies are either difficult to multiplex or too expensive to be used for screening. The proposed proof-of-concept study is, to directly measure the tumor specific mirna extracted from about 1 ml of serum or plasma using a disruptive technology called seed. Measurement by seed is virtually blind to non-specific binding with 100% consistency. Owing to its electrochemical nature the binding time of targeted mirna to the probe will reduce from ~18 hours for conventional microarray methods to below one hour.

NIH Spending Category:
bioengineering; biotechnology; cancer; digestive diseases; pancreatic cancer; prevention; rare diseases

Project Terms:
automation; base; binding (molecular function); biological markers; blind; blood; blood specimen; cancer patient; cancer type; circulating microrna; clinical; complex; cost; data; design; detection; diagnosis; early diagnosis; effective intervention; electric field; electrodes; electronics; fluorescence; foot; genomic instability; goals; gold; growth; heart; hour; housekeeping; human; improved; innovation; instrument; lasers; legal patent; length; licensing; malignant neoplasm of pancreas; malignant neoplasms; marketing; measurement; measures; medical center; metastatic to; methods; micrornas; microscope; mole the mammal; nature; nebraska; needles; neoplastic cell; next generation sequencing; nucleotides; optics; outcome forecast; oxidation-reduction; pancreas; pancreatitis; patients; performance; persons; phase; phase 1 study; phase 2 study; plasma; polymerase chain reaction; prototype; public health relevance; publishing; reading; relative (related person); reporting; research; sampling; scanning; screening; sensitivity and specificity; sensor; serum; signal transduction; specificity; spottings; staging; statistics; success; survival rate; targeted sequencing; technology; time; tool; translations; tumor; tumor initiation; tumor markers; universities; untranslated rna; urine

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 7%. The success in developing therapies for other cancers over the last two decades has not improved the dismal overall survival (OS) rate for PDAC. It is now becoming increasingly apparent that personalized medicine based on the genetics of a tumor will be essential for developing a treatment strategy. Thus, there is an urgent need to develop a noninvasive, genetic biomarker-based surrogate monitoring test to personalize therapy by rapidly assessing treatment efficacy to make early decisions on therapy strategy and to minimize the cost of failure. Meta-analysis combining several studies shows that profiling dysregulation of a large panel of circulating microRNA (miRNA) sequences in blood will potentially have over 85% specificity and sensitivity to detect PDAC-related alteration due to therapy. For the Phase II study, 22 circulating miRNAs impacted by PDAC have been chosen to validate a blood test technology. The technology is a quantitative microarray where binding as low as 0.4 zeptomoles of target molecules to a microspot of immobilized probes will be measured at 100% specificity, zero background, 10 attomolar sensitivity, and a dynamic range of five orders of magnitude. The novel detection method, called Scanning Electrometer for Electrical Double-layer (SEED), will measure local redox on individual microarray spots (patterned on a monolith electrode) to quantify miRNA expression levels without PCR and cDNA conversion. The signal is zero for no binding (i.e., absolute). The binding is by an electrochemical redox enhanced binding (EREB) process to focus as few as 1,800 copies of target molecules to probe molecules immobilized on a Au electrode in <30 min. A linear semi-logarithmic response, similar to a standard curve of qPCR, is obtained for a target concentration ranging from 10-2 to 103 fM. Analysis on plasma from a healthy donor shows a remarkable quantitative correlation between synthetic miRNA in buffer and spiked in plasma ranging from 10 aM to 1 pM. Analysis of 5 miRNA sequences on a chip in patient plasma shows multiplexing and a quantitative correlation with qPCR. The two-year program to validate the EREB/SEED technology will be organized into three specific aims: (1) perform direct miRNA analysis using plasma from a healthy subject that will be spiked with a mixture of synthetic miRNA to evaluate the accuracy of the technology in a biospecimen; validate the technology on plasma from patient undergoing, (2) chemotherapy and (3) radiation therapy, to study the efficacy of the therapy by profiling miRNA at different time points. The validation will be based on a quantitative figure of merit.

Thesaurus Terms:
Aftercare; Attention; Base; Binding; Bioinformatics; Biometry; Blood; Blood Tests; Body Fluids; Buffers; Cancer Center; Cancer Therapy; Characteristics; Chemoradiation; Chemotherapy; Circulating Microrna; Clinic; Clinical; Clinical Research; Clinical Trials; Cohort; Complementary Dna; Complex; Computer Software; Cost; Data; Design; Detection; Disease; Disease Diagnosis; Disease Progression; Early Detection Research Network; Early Diagnosis; Efficacy Study; Electrodes; Evolution; Failure; Future; Genetic; Genetic Markers; Goals; Gold; Hematology; Immobilization; Immunotherapy; Improved; Individual; Instrument; Invention; Label; Legal Patent; Letters; Logarithm; Malignant Neoplasm Of Pancreas; Malignant Neoplasms; Measurement; Measures; Meta-Analysis; Methods; Microarray Analysis; Micrornas; Molecular; Molecular Biology; Monitor; Nature; Novel; Novel Therapeutics; Oncology; Operative Surgical Procedures; Outcome; Outcome Forecast; Oxidation-Reduction; Pancreatic Ductal Adenocarcinoma; Pathology; Pathway Analysis; Patients; Pattern; Personalized Medicine; Phase; Phase 2 Study; Physicians; Plasma; Point Of Care; Privatization; Process; Prognostic; Programs; Progression-Free Survivals; Prototype; Publishing; Radiation Therapy; Reading; Reagent; Repository; Research; Response; Sampling; Scanning; Sensitivity And Specificity; Severities; Signal Transduction; Specificity; Spottings; Statistics; Success; Survival Rate; Technology; Technology Validation; Testing; Therapy Development; Time; Translating; Treatment Efficacy; Treatment Strategy; Tumor; Tumorigenesis; Validation;