Sequenece-specific detection of proteases using electronic p-Chips in multiplex format

使用多重格式的电子 p-Chips 进行蛋白酶的序列特异性检测

• 批准号: 8853166
• 负责人: Rafal Fudala
• 金额: $ 22.5万
• 依托单位: PHARMASEQ, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8853166
• 关键词: Affect; American Cancer Society; Biological Assay; Biological Markers; Bladder; Blood; Blood Circulation; Cancer Detection; Cancer Patient; Cancer cell line; Carcinoma in Situ; Cells; Cleaved cell; Clinical; Consensus; Databases; Deposition; Detection; Diagnosis; Diagnostic; Disease; Disease Progression; Dyes; Electronics; Elements; Energy Transfer; Enzymes; Evaluation; Family; Film; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gelatinase A; Gelatinase B; Generations; Goals; Health; Health Sciences; Human; Hyaluronic Acid; Hyaluronidase; Inflammatory; Invaded; Label; Lasers; Libraries; Light; Link; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Matrix Metalloproteinases; Measurement; Measures; Medical; Metalloproteases; Metals; Methods; Monitor; Neoplasm Metastasis; Outcome; Patients; Peptide Hydrolases; Peptides; Preparation; Procedures; Process; Reader; Relative (related person); Research; Sampling; Signal Transduction; Silicon; Silver; Source; Staging; Symptoms; Technology; Testing; Texas; Tissues; Tumor Markers; Universities; Urine; Variant; Work; base; cancer cell; cyanine dye 3; cyanine dye 5; density; design; enzyme activity; fluorophore; glycylproline; instrumentation; isoleucylvaline; nanoparticle; novel; novel strategies; plasmonics; prolyl-tyrosine; prolylarginine; protein aminoacid sequence; public health relevance; ratiometric; response; screening; tumor; urinary

 DESCRIPTION (provided by applicant): The main goal of this project is to demonstrate the feasibility of combining novel cancer testing using Förster resonance energy transfer (FRET) with an emerging platform for clinical diagnostics. The intent is to develop an assay capable of detecting and monitoring bladder cancer (BlCa) using a urine sample. The assay targets a family of matrix metalloproteinases (MMPs) and hyaluronidase (HA-ase). The presence and relative distribution of these enzyme markers is strongly correlated with progression of BlCa. The principle of the assay involves the preparation of immobilized peptide substrates that are dual-labeled with fluorescent dyes and the determination of the rate at which they are cleaved by MMPs or HA-ase present in a patient sample. This will be done by measuring the extent of FRET between the two fluorescent moieties linked through the target substrates for each respective MMPs or HA-ase. In order to differentiate between sequences, each substrate will be immobilized on different p-Chips, which are ultra-small integrated circuits with unique serial numbers. Invented by PharmaSeq, Inc., each p-Chip's distinctive identification number (ID) is transmitted to an analyzer when the p-Chip is illuminated with laser light. The association of a specific substrate with an ID allows construction of a p-Chip-mounted database in which the identity of a target can be determined by the ID of the p-Chip on which it resides. Multiple p-Chips carrying different substrates for different enzymes can be mixed together in a single assay ("multiplexing"). Results are derived by sequentially measuring the fluorescence and obtaining the ID of each p-Chip in the assay. This is accomplished by passing the p-Chips through a flow-based reader ("Simuplex", also manufactured by PharmaSeq). Sensitivity of the assay can be increased using modified p-Chips onto which a silver nanoparticle film has been deposited prior to peptide-dye conjugation. The benefit of the proposed work is the creation of a highly sensitive multiplex, enzymatic assay capable of simultaneously detecting low concentrations of several enzyme biomarkers that are indicative of cancer. The research plan involves 1) an evaluation of the suitability of the silver nanoparticle film prepared on the p-Chip for maximum fluorescence enhancement, and 2) the application of the selected silver nanoparticle configuration toward enhanced detection of metastasis markers for bladder cancer. We will use the capability of the PharmaSeq Simuplex flow reader to simultaneously excite and detect fluorescence at two wavelengths suited for two dyes, Cy3 and Cy5, and evaluate the FRET ratio changes resulting from the release of a target fragment in the substrate cleaving process. Our final goal is to take advantage of the metal-fluorophore interactions and develop a highly sensitive assay on the p-Chip platform to simultaneously detect the activity of MMP-2, MMP-9, as well as HA-ase in the urine of bladder cancer patients being monitored for disease progression.

 描述（由申请人提供）：该项目的主要目标是证明使用Förster共振能量转移（FRET）将新型癌症检测与新兴的临床诊断平台相结合的可行性。目的是开发一种能够使用尿样检测和监测膀胱癌（BlCa）的测定法。该检测试剂盒靶向基质金属蛋白酶（MMP）和透明质酸酶（HA-酶）家族。这些酶标志物的存在和相对分布与BlCa的进展密切相关。该测定法的原理涉及制备用荧光染料双标记的固定化肽底物，并测定它们被患者样品中存在的MMP或HA酶切割的速率。这将通过测量通过每个相应MMP或HA酶的靶底物连接的两个荧光部分之间的FRET程度来完成。为了区分序列，每个底物将被固定在不同的p-Chip上，p-Chip是具有唯一序列号的超小型集成电路。由PharmaSeq，Inc.发明，当用激光照射p-芯片时，每个p-芯片的独特识别号（ID）被传送到分析器。特定衬底与ID的关联允许构建p芯片安装的数据库，其中靶的身份可以通过其所驻留的p芯片的ID来确定。携带不同酶的不同底物的多个p-芯片可以在单个测定中混合在一起（“多路复用”）。通过依次测量荧光并获得测定中每个p-Chip的ID得出结果。这通过使p-芯片通过基于流动的读取器（“Simuplex”，也由PharmaSeq制造）来实现。使用修饰的p-芯片可以增加测定的灵敏度，在所述修饰的p-芯片上在肽-染料缀合之前已经沉积了银纳米颗粒膜。所提出的工作的好处是创建一个高度敏感的多重，酶测定能够同时检测低浓度的几种酶生物标志物，是癌症的指示。该研究计划包括：1）评估在p-Chip上制备的银纳米颗粒膜对于最大荧光增强的适用性，以及2）将所选银纳米颗粒配置应用于膀胱癌转移标志物的增强检测。我们将使用PharmaSeq Simuplex流式读取器的能力，在适合两种染料Cy 3和Cy 5的两个波长处同时激发和检测荧光，并评估在底物裂解过程中释放靶片段导致的FRET比率变化。我们的最终目标是利用金属-荧光团相互作用，并在p-Chip平台上开发一种高灵敏度的检测方法，以同时检测膀胱癌患者尿液中MMP-2、MMP-9以及HA酶的活性，以监测疾病进展。