Aptamer-Based Nanotechnology for Detection of Plasma Melanoma Markers

基于适体的纳米技术检测血浆黑色素瘤标记物

• 批准号: 7740000
• 负责人: GARY A CLAWSON
• 金额: $ 19.46万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740000
• 关键词: Affinity; American Joint Committee on Cancer; Antisense Oligonucleotides; Area; Benchmarking; Binding; Biological Assay; Blood; C-terminal; Cancer Diagnostics; Chemistry; Clinical Trials; Coupled; Coupling; DNA; DNA Binding; Data; Deposition; Detection; Development; Discrimination; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Epitopes; Equilibrium; Event; Fluorescence; Frequencies; Funding; In Vitro; Individual; Investigation; Label; Libraries; Ligands; Link; Malignant Neoplasms; Measurable; Measures; Methods; Modification; Molecular; Nanotechnology; Nucleotides; Patients; Plasma; Process; Property; Proteins; Protocols documentation; RNA; Reagent; Research Personnel; Resistance; Sampling; Scheme; Screening procedure; Sensitivity and Specificity; Specificity; Spottings; Staging; Stretching; Sulfhydryl Compounds; Technology; Testing; Translations; Tumor Markers; aptamer; base; cancer type; clinically relevant; design; interest; melanoma; neoplastic cell; novel marker; nuclease; public health relevance; sensor; single molecule; tool

DESCRIPTION (provided by investigator): Although major advances have occurred toward understanding the molecular events in various cancers, their translation into therapies has with few exceptions been limited, and early detection remains the cornerstone of successful treatment. Consequently, there is a great deal of interest in identifying sensitive and specific markers for individual cancers, particularly in blood. Two areas of intense investigation are identification and quantification of circulating tumor cells (CTCs), as well as identification of sensitive and specific tumor markers in plasma. We are developing a chip-based RNA sensor platform using derivatized nanowires (NWs) for early detection of CTCs. This sensor platform, which relies on formation of a hybridization "sandwich", possesses excellent specificity (single nucleotide mismatch discrimination for 2 recognition events) as well as sensitivity (data indicate each single "sandwich" binding induces a resonance frequency shift of ~ 1 kHz, which is easily measurable). The platform is being constructed in "bottom-up" fashion, and we have shown that NWs derivatized with antisense oligonucleotides fully retain their functionality after integration on chip. Here we propose to derivatize NWs with modified DNA aptamers, specific for the clinically relevant plasma melanoma tumor markers S100B and MIA, extending the functionality of our chip- based sensor and considerably expanding its versatility while retaining multiplex capabilities and exquisite sensitivity/specificity. The Specific Aims of the application encompass: 1) Make constructs and express S100B and MIA and purify them in vitro. 2) Perform Aptamer selection using our Primer-Free protocol until ~ 5-10 high-affinity aptamers are identified for each protein, and characterize binding properties. Introduce various modifications to aptamers to enhance stability/nuclease resistance and re-test binding parameters. 3) Perform pair-wise testing of fluorescent "sandwich" binding of spotted microarrays with purified S100B/MIA to determine the optimal pair of aptamers (Ap1 and Ap2) recognizing distinct epitopes. 4) Use the optimal pairs of Ap1/Ap2 on microarrays to detect various concentrations of S100B and MIA spiked into fractionated control plasma samples, to determine non-specific binding and ~ limits of detection. 5) Derivatize NWs with Ap1, and perform sandwich binding assays with: a) fluorescently-labeled Ap2; and b) Ap2 covalently linked to 50-nm AuNPs, with spiked plasma samples as in 4). 6) Electrofluidically deposit derivatized NWs on chips, perform sandwich binding assays as in 4), and measure shifts in resonance frequency of individual NWs: Benchmark vs. conventional ELISA. 7) Quantify S100B and MIA levels in plasma from melanoma patients using conventional ELISA-based reagents for benchmarking, compare with CTC levels from the same patient samples, and relate to AJCC Stage. Bank the balance of the samples for subsequent testing with the chip-based sensor. PUBLIC HEALTH RELEVANCE: This application, which is focused on detection of tumor markers in blood from melanoma patients, seeks to develop a nanotechnology-based platform with wide-ranging applicability in cancer diagnostics. The methods to be utilized can be applied to any cancer for which suitable markers are available, will be easily adaptable to new markers as they are discovered. The platform is designed to allow simultaneous detection of a number of different tumor markers, and we envision its development as a screening" tool for early detection of all major cancer types.

描述(由研究人员提供)：尽管在理解各种癌症的分子事件方面取得了重大进展，但它们在治疗中的转化受到限制，几乎没有例外，早期发现仍然是成功治疗的基石。因此，人们对确定个别癌症的敏感和特异性标记物非常感兴趣，特别是在血液中。两个密集的研究领域是循环肿瘤细胞(CTCs)的鉴定和定量，以及血浆中敏感和特异的肿瘤标志物的鉴定。我们正在开发一种基于芯片的RNA传感器平台，使用衍生纳米线(NWS)来早期检测CTCs。该传感器平台依赖于杂交“三明治”的形成，具有良好的特异性(两个识别事件的单核苷酸错配识别)和灵敏度(数据表明，每个单一的“三明治”结合都会导致~1 kHz的共振频率漂移，这一点很容易测量)。该平台是以“自下而上”的方式构建的，我们已经证明了反义寡核苷酸衍生的NWS在集成到芯片后完全保留了它们的功能。在这里，我们建议使用针对临床相关血浆黑色素瘤肿瘤标志物S100B和MIA的修饰DNA适配子来衍生NWS，从而扩展我们基于芯片的传感器的功能并显著扩展其多功能性，同时保留多路复用功能和精致的灵敏度/特异性。应用的具体目的包括：1)构建S100B和MIA的表达载体，并在体外进行纯化。2)使用我们的无引物法进行适配子选择，直到为每个蛋白质鉴定出5-10个高亲和力适配子，并表征结合性质。引入各种适体修饰以增强稳定性/核酸酶抗性并重新测试结合参数。3)对斑点微阵列与纯化的S100B/MIA的荧光“夹心”结合进行配对测试，以确定识别不同表位的最佳适配子对(AP1和AP2)。4)在芯片上使用最优的AP1/AP2对来检测不同浓度的S100B和MIA，以确定非特异性结合和检出限。5)用AP1衍生NWS，并进行夹心结合分析：a)荧光标记的AP2；和b)共价连接到50 nm AuNPs的AP2，与添加的血浆样品，如4)。6)电流体在芯片上沉积衍生的NW，执行如4)中的三明治结合分析，并测量单个NW的共振频率的偏移：基准与传统的ELISA法。7)用常规的EL ISA试剂定量检测黑色素瘤患者血浆中S100B和MIA的水平，并与同一患者样本中的CTC水平进行比较，并与AJCC分期相关。将剩余的样品储存起来，以便随后使用基于芯片的传感器进行测试。 公共卫生相关性：这一应用程序专注于检测黑色素瘤患者血液中的肿瘤标志物，旨在开发一个在癌症诊断中具有广泛适用性的基于纳米技术的平台。所使用的方法可以应用于任何有合适标记物可用的癌症，随着新标记物的发现，这些方法将很容易适应。该平台旨在允许同时检测许多不同的肿瘤标志物，我们设想它的发展将成为一种筛查工具，用于所有主要癌症类型的早期检测。