Protein Biosensor Arrays Based on Nanomaterials

描述(由申请人提供)：蛋白质测量在生物医学研究中至关重要，与疾病相关的应用包括早期检测、因果鉴定和治疗监测。灵敏、特异、快速、廉价地检测患者样本中的多种蛋白质对未来的公共卫生至关重要。该项目的广泛和长期目标是开发超灵敏的新阵列设备，用于精确测量蛋白质面板。最近的研究表明，由4到10个生物标记蛋白组成的小组，而不是现在使用的单一生物标记蛋白，将提供更可靠的癌症检测和监测。我们将开发和优化蛋白质微流控阵列，这种阵列准确、廉价，可以检测到比任何商业替代品低10-100倍的多种蛋白质。这个项目的目标是血清和组织中的生物标记物蛋白小面板阵列，而不是蛋白质组生物标记物的发现或分析。在过去的资助期间，我们开发了基于纳米颗粒的超灵敏蛋白质免疫阵列策略，使用数千种酶标记在磁性颗粒上进行电化学检测，或使用染料纳米颗粒标记进行光电检测。我们利用原型传感器和阵列高精度地检测前列腺癌患者血清中多达四种癌症生物标志物蛋白。我们最近将纳米结构传感器阵列与微流控技术和多标记磁性抗体颗粒的离线蛋白质捕获相结合。这一强大的方法极大地减少了干扰，可以在100fgmL-1(~3fm)的水平上检测到血清中的生物标记蛋白，比商业检测能力低10-100倍。如此高的灵敏度为检测固有的超低水平的生物标志物提供了新的机会。这一更新项目旨在将我们的超灵敏蛋白质检测方法转化为实现广泛的临床、外科和研究应用。可交付的产品是经过优化和验证的微流控阵列设备，可用于几乎任何一小块蛋白质面板的超灵敏检测。还将开发一种高速阵列，用于检测癌症手术期间癌细胞转移(扩散)到淋巴结的情况。在概念验证方面，将设计特定的设备，从收集的血清样本中预测患口腔癌的可能性，并明确区分病毒性(人乳头瘤病毒)和非病毒性口腔癌。我们的NIDCR/NIH合作者获得的患者和对照样本将用于建立免疫分析设备的临床灵敏度和选择性。具体目标总结如下：(1)优化用于8蛋白质口腔癌面板的纳米结构传感器芯片和多标记安培技术的微流控系统；(2)设计和优化使用电化学发光(ECL)检测到的纳米染料标记的简单电光微流控阵列；(3)建立用于口腔癌诊断的临床灵敏度和选择性最佳的阵列系统；(4)建立一种快速检测淋巴结转移生物标志物的微流控方法。1 公共卫生相关性：该项目将开发超灵敏设备来检测患者血清中的蛋白质生物标记物集合。它将为癌症的可靠早期诊断和监测提供新的工具，以促进个性化治疗。它还将开发一种快速检测癌症扩散(转移)的设备。

DESCRIPTION (provided by applicant): Measurements of proteins are of central importance in biomedical research, and disease-related applications include early detection, causal identification, and treatment monitoring. Sensitive, specific, fast, inexpensive detection of multiple proteins in patient samples is critically important for future public health. The broad, long term goals of this project are to develop ultrasensitive new array devices for accurate measurements of panels of proteins. Recent research has shown that panels of 4 to 10 biomarker proteins, as opposed to the single biomarkers now used, will provide much more reliable cancer detection and monitoring. We will develop and optimize microfluidic arrays for proteins that are accurate, cheap, and can detect multiple proteins at levels 10-100 fold lower than any commercial alternatives. This project targets arrays for small panels of biomarker proteins in serum and tissue, not proteomic biomarker discovery or analysis. In the past funding period, we developed nanoparticle-based strategies for ultrasensitive protein immunoarrays using many thousands of enzyme labels on magnetic particles for electrochemical detection, or dye-nanoparticle labels for electro-optical detection. We utilized prototype sensors and arrays to detect up to four cancer biomarker proteins in the serum of prostate cancer patients with high accuracy. We recently combined nanostructured sensor arrays with microfluidics and off-line protein capture by the multilabel magnetic-antibody particles. This powerful approach greatly decreases interferences and can detect biomarker proteins in serum at levels of 100 fg mL-1 (~3 fM), 10-100 fold below capabilities of commercial assays. Such high sensitivity provides new opportunities for detection of biomarkers with inherently ultralow levels. This renewal project seeks to translate our ultrasensitive protein detection approaches into the realization of widespread clinical, surgical, and research applications. The deliverables are optimized, validated microfluidic array devices for ultrasensitive detection of virtually any small panel of proteins. A high-speed array for detection of cancer cell metastasis (spreading) to lymph nodes during cancer surgery will also be developed. For proof-of-concept, specific devices will be designed to predict the probability of oral cancer from collections of serum samples, and to clearly distinguish between viral (humanpapilloma virus) and non-viral oral cancers. Patient and control samples obtained by our NIDCR/NIH collaborators will be used to establish clinical sensitivity and selectivity of the immunoassay devices. Summarized specific aims are: (1) Optimize microfluidic systems featuring nanostructured sensor chips and multilabel amperometry for an 8-protein oral cancer panel; (2) Design and optimize a simple electro-optical microfluidic array using dye-nanoparticle labels detected by electrochemiluminescence (ECL); (3) Establish clinical sensitivity and selectivity for oral cancer diagnosis for the best array systems; (4) Develop a rapid microfluidic assay for detection of a metastasis biomarker in lymph nodes. 1 PUBLIC HEALTH RELEVANCE: This project will develop ultrasensitive devices to detect collections of protein biomarkers in patient serum. It will provide new tools for reliable early diagnosis and monitoring of cancer to facilitate personalized therapy. It will also develop a rapid device to detect the spread (metastasis) of cancer.