Protein Biosensor Arrays Based on Nanomaterials

描述（由申请人提供）：蛋白质的测量在生物医学研究中至关重要，疾病相关的应用包括早期检测、因果识别和治疗监测。对患者样本中多种蛋白质进行敏感、特异、快速、廉价的检测，对未来的公共卫生至关重要。该项目的长远目标是开发超灵敏的新型阵列设备，用于精确测量蛋白质面板。最近的研究表明，与目前使用的单一生物标志物相比，由4到10个生物标志物蛋白质组成的小组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的微流体阵列，这些阵列准确，便宜，并且可以检测比任何商业替代品低10-100倍的多种蛋白质。该项目针对血清和组织中生物标记蛋白的小面板阵列，而不是蛋白质组学生物标记的发现或分析。在过去的资助期内，我们开发了基于纳米颗粒的超灵敏蛋白质免疫阵列策略，使用成千上万的酶标记在磁颗粒上进行电化学检测，或染料纳米颗粒标记用于光电检测。我们利用原型传感器和阵列检测前列腺癌患者血清中多达四种癌症生物标志物蛋白，准确度高。我们最近将纳米结构传感器阵列与微流体和多标签磁性抗体颗粒的离线蛋白质捕获相结合。这种强大的方法大大减少了干扰，可以在100 fg mL-1 （~3 fM）的水平上检测血清中的生物标志物蛋白，比商业分析的能力低10-100倍。这种高灵敏度为检测固有超低水平的生物标志物提供了新的机会。这个更新项目旨在将我们的超灵敏蛋白质检测方法转化为广泛的临床，外科和研究应用的实现。交付物是优化的，经过验证的微流控阵列设备，用于几乎任何小块蛋白质的超灵敏检测。在癌症手术过程中，用于检测癌细胞转移（扩散）到淋巴结的高速阵列也将被开发出来。对于概念验证，将设计特定的设备来从收集的血清样本中预测口腔癌的可能性，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测定装置的临床敏感性和选择性。总结的具体目标是：(1)优化具有纳米结构传感器芯片和多标签安培法的微流控系统，用于8蛋白口腔癌小组；(2)利用电化学发光技术（ECL）检测染料纳米颗粒标记，设计并优化了一种简单的电光微流控阵列；(3)建立最佳阵列系统诊断口腔癌的临床敏感性和选择性；(4)建立一种快速的微流体检测方法，用于检测淋巴结转移的生物标志物。1

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.