BIOSENSOR ARRAYS BASED ON SINGLE WALL CARBON NANOTUBES

描述(申请人提供)：蛋白质和细菌的灵敏检测在生物医学研究中具有核心重要性，应用于阐明疾病途径、免疫学、医学诊断、系统生物学、功能蛋白质组学和新药开发。鉴于生物医学科学的新兴领域和日益加剧的世界生物恐怖主义，高选择性和高灵敏度的结合分析阵列变得越来越重要。灵敏、快速、特异、多重检测生物和环境样品中的蛋白质、细菌和毒素对公众健康至关重要。这一提议的广泛、长期目标是利用图案化的单壁碳纳米管(SWNTs)和生物分子识别元件开发用于蛋白质和细菌的高灵敏微纳生物传感器阵列。主要的具体应用包括同时测量血清中的一整套蛋白质生物标记物，用于早期癌症检测。直径约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，这些碳纳米管站在直径20-200纳米的束中，称为单壁碳纳米管森林，与导电表面有电连接。这些高比表面积、可图案化、导电的纳米结构为高灵敏生物传感器阵列提供了新的机会。在这个项目中，我们将纳米管的电传导与针对蛋白质和细菌的抗体的特异性分子识别相结合。作为分子导线的单壁碳纳米管将在其末端的酶标记和外部电子连接之间矢量地传输电子。在这里，我们将重点放在过氧化物酶连接的安培免疫分析中，其中酶催化的过氧化氢还原提供通过单壁碳纳米管传递的电信号。在前期工作中，我们将抗体附着在SWNT森林上，并实现了对几种范围在ng/mL及以下的抗原的检测。使用新开发的具有高标记与AB2比率的纳米管、过氧化物酶和二次抗体(AB2)与SWNT免疫传感器的结合物，我们最近检测到人血清中的前列腺特异性抗原(PSA)为0.01 ng/mL(0.25fmoL/mL)，检测限好于最好的商业化PSA检测方法。超低NSB与纳米管连接的导电聚合物布线以及多酶标记策略相结合，有望实现最高的灵敏度。因此，我们将优化新的AB2纳米结合物，探索导电聚合物布线策略，并通过竞争性吸附的蛋白质/表面活性剂添加剂和在纳米管末端合成新型低NSB有机表面来消除干扰的非特异性结合(NSB)。最后，我们将利用图案化的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。除了实现我们的具体目标外，该项目还应为制造用于各种生物医学应用的敏感微阵列器件提供基本指导。

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.