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，因果鉴定和治疗监测。敏感、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，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组。最近的研究表明，与目前使用的单一生物标志物相比，4至10种生物标志物蛋白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，并且可以检测比任何商业替代品低10-100倍的多种蛋白质。该项目的目标是血清和组织中的小组生物标志物蛋白的阵列，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灵敏蛋白质免疫阵列策略，使用磁性颗粒上的数千种酶标记进行电化学检测，或使用染料纳米颗粒标记进行电光检测。我们利用原型传感器和阵列以高精度检测前列腺癌患者血清中多达四种癌症生物标志物蛋白。我们最近结合了纳米结构的传感器阵列与微流体和离线蛋白质捕获的多标记磁性抗体颗粒。这种强大的方法大大降低了干扰，可以检测血清中的生物标志物蛋白，水平为100 fg mL-1（~3 fM），比商业测定的能力低10-100倍。这种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。 该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，手术和研究应用。交付的产品是经过优化和验证的微流控阵列设备，可用于超灵敏检测几乎任何一小组蛋白质。还将开发用于检测癌症手术期间癌细胞转移（扩散）到淋巴结的高速阵列。为了验证概念，将设计特定的设备来预测收集的血清样本中口腔癌的概率，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测定设备的临床灵敏度和选择性。 具体目标概括如下：（1）优化微流控系统，其具有纳米结构传感器芯片和用于8蛋白口腔癌面板的多标记电流分析法;（2）设计和优化使用通过电化学发光（ECL）检测的染料纳米颗粒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，以获得最佳阵列系统;（4）建立淋巴结转移生物标志物的快速微流控检测方法。1 公共卫生相关性：该项目将开发超灵敏设备，以检测患者血清中蛋白质生物标志物的集合。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）的装置。

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.