Multiplexed Surface Plasmonic Biomarker Detection with Enzymatic Amplification

描述（由申请人提供）：拟议研究的目标是开发可靠且广泛适用的方法，用于在微升样品体积中极低（飞摩尔）浓度的蛋白质生物标志物的多重体外检测。这些新型的超灵敏微阵列生物传感测量将通过结合使用表面酶化学、生物功能化纳米颗粒和独特的基于表面等离子体的生物亲和吸附检测技术来实现。这些新方法的关键组成部分是(i)利用酶促反应在纳米颗粒表面和微阵列元件上附着、修饰和扩增生物分子，以增加生物亲和感测过程的选择性和灵敏度；（ii）将化学和酶化的生物功能纳米颗粒结合到微阵列感测测量中，以增强对目标生物分子的捕获和产生的信号。（iii）新型光学检测方法的实施，如表面等离子体共振相位成像和纳米粒子增强光学衍射，以多路复用格式提供额外的生物传感灵敏度。适配体、抗体和酶促附着反应的组合将用于捕获和检测存在于生物流体样品中zeptomole水平的蛋白质（例如，VEGF、PSA、肌钙蛋白）和microRNA生物标志物靶标。这些新的检测方法可以在飞摩尔及以下浓度下识别和定量多种蛋白质，是发现新的蛋白质生物标志物的基本技术，在如此低的水平下跟踪这些新的生物标志物（例如，心血管和癌症生物标志物）的能力将有助于创造更好的早期疾病检测方法和治疗后患者监测策略。

DESCRIPTION (provided by applicant): The goal of the proposed research is the development of reliable and widely applicable methods for the multiplexed in vitro detection of protein biomarkers at extremely low (femtomolar) concentrations in microliter sample volumes. These novel ultrasensitive microarray biosensing measurements will be achieved through the combined use of surface enzyme chemistries, biofunctionalized nanoparticles, and unique surface plasmon-based bioaffinity adsorption detection techniques. Key components in these new methodologies are (i) the use of enzymatic reactions for the attachment, modification and amplification of biomolecules on both nanoparticle surfaces and microarray elements to increase the selectivity and sensitivity of the bioaffinity sensing process, (ii) the incorporation of chemically and enzymatically biofunctionalized nanoparticles into the microarray sensing measurements in order to enhance the capture of and signal generated by target biomolecules, and (iii) the implementation of novel optical detection methods such as surface plasmon resonance phase imaging and nanoparticle-enhanced optical diffraction that provide additional biosensing sensitivity in a multiplexed format. A combination of aptamers, antibodies and enzymatic attachment reactions will be used to capture and detect protein (e.g., VEGF, PSA, troponin) and microRNA biomarker targets that exist in biofluid samples at zeptomole levels. These new detection methodologies that can identify and quantitate multiple proteins at femtomolar concentrations and below are essential technologies for the discovery of new sets of protein biomarkers, and the ability to track these new biomarkers (e.g., cardiovascular and cancer biomarkers) at such low levels will facilitate the creation of better methods for early disease detection and strategies for post-treatment patient monitoring.