CRISPR Microfluidic Array for Simultaneous Multiplexed Sensing of Proteins and miRNAs

测量血液中的生物分子，如蛋白质和核酸，称为生物标记物，是检测癌症和其他疾病的有价值的方法。由几种蛋白质和核酸组成的样本显示出实现这一目的的希望，但目前必须使用单独的、耗时的方法进行测量。该项目使用一种名为CRISPR(集群规则间隔短回文重复)的生物结构来实现测量。CRISPR可以被设计成与核酸或标记了核酸的蛋白质结合，只有当特定的核酸或标记与CRISPR结合时才会发出信号。这种方法将被开发成一种快速、低成本的3D打印半自动设备，在同一测试中测量几种核酸和几种蛋白质生物标志物。该项目还将开发一种名为电化学发光(ECL)的灵敏电驱动发光，作为一种更简单的检测方法。使用3D打印设备的血液测试将被设计为在诊所或医生办公室由训练有素的技术人员在短时间内运行。这将允许在所谓的护理点模式下快速周转。结果将在患者仍在他或她的办公室时向医生提供，并可在那时用作诊断的一部分。该项目将利用联合国大学的方案。康涅狄格州大学支持研究生、本科生、科学教师和高中生的新CRISPR技术教育。活动包括通过探索化学研究经验和培训学院举办CRISPR检测研讨会，为中学生和科学教师开发大学早期体验计划，促进对CRISPR阵列设计和制造的博士生培训，并通过ACS种子计划为康涅狄格州大学本科生和康涅狄格州NSF REU项目的本科生以及高中生提供研究机会。分子生物标记物在许多疾病的医学诊断中变得越来越有价值。与单一生物标记物相比，生物标记物小组显示出优越的诊断实用价值，而蛋白质-miRNA组合小组比单独的蛋白质或miRNA小组显示出显著改善的诊断预测。该项目将开发第一个使用CRISPR技术同时测量miRNAs和蛋白质组合的分析阵列。新的3D打印阵列将能够在护理点(POC)模式下分析用于广泛疾病诊断的miRNA-蛋白质生物标志物面板，以及在其他生物医学研究应用中。除了荧光检测外，还将使用CDC和iPhone相机检测电化学发光(ECL)。重点将放在miRNA和蛋白质生物标记物上，它们是侵袭性前列腺癌的高效生物标记物。检测将通过测量来自高级别前列腺癌患者和对照组的130个现有人类血清样本中的目标miRNAs和蛋白质来验证。该项目的智力优势包括设计和开发第一个工程战略，使用新的CRISPR分析技术在POC同时检测miRNAs和蛋白质面板。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Measurement of biomolecules like proteins and nucleic acids, called biomarkers, in blood, is a valuable approach to detect cancers and other diseases. Samples consisting of several proteins and nucleic acids show promise for this purpose, but at present must be measured using separate, time-consuming methods. This project uses a biological construct called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to enable measurements. CRISPR can be designed to bind a nucleic acid or a protein labeled with a nucleic acid and give a signal only when that specific nucleic acid or label binds to CRISPR. This approach will be developed into a fast, low-cost 3D-printed, semi-automated device to measure several nucleic acids and several protein biomarkers in the same test. The project will also develop sensitive electrically-driven light emission called electrochemiluminescence (ECL) as a simpler detection method. The blood test using the 3D-printed device will be designed to run in a short time in a clinic or physician’s office by a lightly trained technician. This will allow fast turnaround in what is called point of care mode. Results will be available to the physician while the patient is still in his or her office and can be used at that time as part of the diagnosis. This project will utilize programs at Univ. of Connecticut to support education in new CRISPR technology for graduate students, undergraduates, science teachers and high school students. Activities include holding workshops on CRISPR detection through SECRET (School of Exploratory Chemistry Research Experience and Training), developing Early College Experience programs for secondary school students and science teachers, facilitating training of Ph.D. students in CRISPR array design and fabrication, and providing research opportunities for undergraduates at UCONN and in UCONN’s NSF REU program, as well as high school students through the ACS SEED program.Molecular biomarkers have become increasingly valuable in medical diagnostics for many diseases. Panels of biomarkers, as opposed to single biomarkers, show superior diagnostic utility and combined protein-miRNA panels have shown significantly improved diagnostic predictions over separate protein or miRNA panels. This project will develop the first analytical array using CRISPR technology to simultaneously measure combinations of miRNAs and proteins. The new 3D-printed array will be able to analyze miRNA-protein biomarker panels for a wide range of disease diagnostics in point-of-care (POC) mode, as well as in other biomedical research applications. In addition to fluorescent detection, electrochemiluminescence (ECL) will be explored with CDC and iPhone camera detection. The focus will be on miRNA and protein biomarkers that are high performing biomarkers for aggressive prostate cancer. Assays will be validated by measuring target miRNAs and proteins in 130 existing human serum samples from patients with high grade prostate cancers and controls. The intellectual merit of this project involves design and development of the first engineering strategy using a new CRISPR assay technology to detect panels of miRNAs and protein simultaneously at POC.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.