Protein Biosensor Arrays Based on Nanomaterials

蛋白质的测量在生物医学研究中至关重要，而且与疾病相关 应用包括早期检测、病因识别和治疗监测。敏感，具体，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。这个 该项目广泛的、长期的目标是开发超灵敏的新阵列器件，以精确地 蛋白质面板的测量。最近的研究表明，由4到10个生物标记蛋白组成的小组，如 与现在使用的单一生物标记物相反，它将提供更可靠的癌症检测和监测。 我们将开发和优化准确、廉价、可以检测到多种蛋白质的微流控阵列 蛋白质水平比任何商业替代品低10-100倍。该项目以小型阵列为目标 血清和组织中的生物标志物蛋白质组，而不是蛋白质组生物标志物的发现或分析。 在过去的资助期间，我们开发了基于纳米颗粒的超敏感蛋白质策略。 使用数千种酶标记在磁性颗粒上进行电化学检测的免疫阵列，或 用于光电检测的染料纳米颗粒标记。我们利用原型传感器和阵列来检测多达 前列腺癌患者血清中四种肿瘤生物标志物蛋白的高准确度我们最近 将纳米结构传感器阵列与微流控技术相结合，并通过多标签离线捕获蛋白质 磁性抗体颗粒。这种强大的方法大大减少了干扰，可以检测到生物标志物 血清中蛋白质的水平为100fgmL-1(~3fm)，比商业检测能力低10-100倍。是这样的 高灵敏度为检测固有的超低水平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。交付成果经过优化、验证 微流控阵列设备，用于几乎任何一小块蛋白质的超灵敏检测。一种高速阵列 用于检测癌细胞在癌症手术过程中转移(扩散)到淋巴结的情况 发展起来的。对于概念验证，将设计特定的设备来预测口腔癌的可能性 收集血清样本，并明确区分病毒(人乳头瘤病毒)和非病毒 口腔癌。我们的NIDCR/NIH合作者获得的患者和对照样本将用于确定 免疫分析装置的临床灵敏度和选择性。 总结的具体目标是：(1)优化具有纳米结构传感器芯片的微流控系统 和用于8蛋白口腔癌面板的多标记安培法；(2)设计并优化了一种简单的光电探测器 利用电化学发光(ECL)检测染料-纳米颗粒标记的微流控阵列； 临床灵敏度和选择性用于口腔癌诊断的最佳阵列系统；(4)快速开发 微流控技术检测淋巴结转移生物标志物。 1

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