Protein Biosensor Arrays Based on Nanomaterials

蛋白质的测量在生物医学研究中具有核心重要性， 应用包括早期检测、病因鉴定和治疗监测。灵敏，特异，快速， 患者样品中多种蛋白质的廉价检测对于未来的公共卫生是至关重要的。的 该项目的广泛，长期目标是开发超灵敏的新阵列设备，用于精确 蛋白质组的测量。最近的研究表明，4至10个生物标志物蛋白质的小组，如 与现在使用的单一生物标志物相反，它将提供更可靠的癌症检测和监测。 我们将开发和优化用于蛋白质的微流控阵列，这些微流控阵列准确，廉价，并且可以检测多种蛋白质。 蛋白质水平比任何商业替代品低10-100倍。该项目的目标阵列为小型 血清和组织中的生物标志物蛋白质组，而不是蛋白质组学生物标志物发现或分析。 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， - 免疫阵列，其使用磁性颗粒上的数千种酶标记用于电化学检测，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的四种癌症生物标志物蛋白质具有高准确性。我们最近 具有微流体和通过多标记的离线蛋白质捕获的组合纳米结构传感器阵列 磁性抗体粒子。这种强大的方法大大减少了干扰，可以检测生物标志物 血清中的蛋白质水平为100 fg mL-1（~3 fM），比商业测定的能力低10-100倍。等 高灵敏度为检测具有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实现 广泛的临床、外科和研究应用。交付物经过优化、验证 微流控阵列装置，用于超灵敏检测几乎任何一小组蛋白质。高速阵列 用于在癌症手术期间检测癌细胞向淋巴结的转移（扩散）， 开发为了验证概念，将设计特定的设备来预测口腔癌的概率， 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建立 免疫测定装置的临床灵敏度和选择性。 概括的具体目标是：（1）优化具有纳米结构传感器芯片的微流体系统 （2）设计并优化了一个简单的电光-荧光检测系统， 使用通过电化学发光（ECL）检测的染料-纳米颗粒标记的微流控阵列;（3）建立 最佳阵列系统的口腔癌诊断的临床灵敏度和选择性;（4）开发快速 用于检测淋巴结中转移生物标志物的微流体测定。 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