BIOSENSOR ARRAYS BASED ON SINGLE WALL CARBON NANOTUBES

描述（由申请人提供）：蛋白质和细菌的敏感检测在生物医学研究中至关重要，可用于阐明疾病途径，免疫学，医学诊断，系统生物学，功能蛋白质组学和新药开发。随着生物医学领域的发展和世界生物恐怖主义的加剧，高选择性和高灵敏度的结合分析阵列变得越来越重要。对生物和环境样品中的蛋白质、细菌和毒素进行灵敏、快速、特异、多重检测对公共卫生至关重要。本研究的长远目标是利用单壁碳纳米管（SWNTs）和生物分子识别元件，为蛋白质和细菌开发高灵敏度的微纳米生物传感器阵列。主要的具体应用包括同时测量血清中用于早期癌症检测的蛋白质生物标志物。直径约为1.4 nm的单壁碳纳米管（SWNTs）具有世界上最高的单位质量电导率，在生物分子的电传感方面具有良好的潜力。我们已经组装了大约30纳米长的碳纳米管，这些碳纳米管被称为SWNT森林，直径为20-200纳米，与导电表面有电连接。这些高表面积、可模式化、导电的纳米结构为高灵敏度的生物传感器阵列提供了新的机会。在这个项目中，我们将纳米管电转导与蛋白质和细菌抗体的特异性分子识别结合起来。作为分子导线的单壁碳纳米管将在其尖端的酶标记和外部电连接之间进行电子矢量传输。我们将重点放在过氧化物酶连接的安培免疫分析上，其中酶催化H2O2的还原提供了通过swnt传输的电信号。在前期工作中，我们将抗体附着在SWNT森林上，并检测到ng/mL及以下的几种抗原。利用新开发的纳米管、过氧化物酶和二抗（Ab2）的偶联物与SWNT免疫传感器，我们最近以0.01 ng/mL （0.25 Fmol/mL）检测了人血清中的前列腺特异性抗原（PSA），其检测限优于最佳的商业PSA检测方法。超低NSB结合纳米管连接处的导电聚合物布线，以及多酶标签策略，保证了最高的灵敏度。因此，我们将优化新的Ab2纳米偶联物，探索导电聚合物布线策略，并通过竞争性吸附蛋白质/表面活性剂添加剂和在纳米管末端合成新的低NSB有机表面来消除非特异性结合（NSB）干扰。最后，我们将利用图案swnt抗体组件将优化的制造/检测方法集成到多分析物传感器阵列中。除了实现我们的特定目标，这个项目应该为制造用于各种生物医学应用的敏感微阵列设备提供基本指导。

DESCRIPTION (provided by applicant): Sensitive detection of proteins and bacteria is of central importance in biomedical research, with applications to elucidating disease pathways, immunology, medical diagnostics, systems biology, functional proteomics, and new drug development. Highly selective and sensitive binding-assay arrays are increasingly important in light of emerging areas of biomedical science and intensified world bioterrorism. Sensitive, rapid, specific, multiple detection of proteins, bacteria and toxins in biological and environmental samples is critically important for public health. The broad, long term goals of this proposal are to develop highly sensitive micro- and nano-biosensor arrays for proteins and bacteria using patterned single-walled carbon nanotubes (SWNTs) and biomolecule recognition elements. Major specific applications include simultaneous measurement of suites of protein biomarkers in serum for early cancer detection. Single walled carbon nanotubes (SWNTs) with approximately 1.4 nm diameters have the world's highest conductivity per unit mass and have excellent potential for electrical sensing of biomolecules. We have assembled approximately 30 nm long carbon nanotubes standing in 20-200 nm diameter bundles called SWNT forests with electrical links to conductive surfaces. These high surface area, patternable, conductive nanostructures provide new opportunities for highly sensitive biosensor arrays. In this project, we combine nanotube electrical transduction with specific molecular recognition of antibodies for proteins and bacteria. SWNTs as molecular wires will shuttle electrons vectorially between enzyme labels at their tips and external electrical connections. We focus here on peroxidase-linked amperometric immunoassays, in which enzyme catalyzed reduction of H2O2 provides electrical signals transmitted via SWNTs. In preliminary work, we attached antibodies to SWNT forests and achieved detection of several antigens in the ng/mL range and below. Using newly developed conjugates of nanotubes, peroxidases and secondary antibodies (Ab2) with high label-to-Ab2 ratios with the SWNT immunosensors, we recently detected prostate specific antigen (PSA) in human serum at 0.01 ng/mL (0.25 Fmol/mL), a detection limit better than the best commercial assays for PSA. Ultra-low NSB combined with conductive polymer wiring of nanotube junctions, along with multi-enzyme label strategies, promise the highest possible sensitivity. Thus, we will optimize the new Ab2 nanoconjugates, explore conductive polymer wiring strategies, and eliminate non-specific binding (NSB) of interferences via competitively adsorbed protein/surfactant additives and synthesis of novel low NSB organic surfaces on nanotube ends. Finally, we will integrate optimized fabrication/detection approaches into multi-analyte sensor arrays utilizing patterned SWNT-antibody assemblies. In addition to achieving our specific aims, this project should provide fundamental guidance for fabrication of sensitive microarray devices for a wide variety of biomedical applications.