Digital and Absolute Assays for Low Abundance Molecular Biomarkers.
Digital and Absolute Assays for Low Abundance Molecular Biomarkers.
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低丰度分子生物标志物的数字和绝对测定。
DOI：
10.1021/acs.accounts.3c00030
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发表时间：
2023
影响因子：
18.3
通讯作者：
Smith,AndrewM
中科院分区：
文献类型：
--
作者：
Kuo,Chia-Wei;Smith,AndrewM
概述最近，基于离散分子靶标测量而不是分子整体平均信号的生物分子测定取得了巨大的进步。许多“数字”检测设计源自现已成熟的单分子成像和微流体技术，并提供了各种新的模式来量化生物样本（例如血液和组织匀浆）中的核酸和蛋白质。主要的新优势是能够对阿摩尔至飞摩尔浓度的痕量分析物进行稳健检测，而许多集成分析无法区分高于噪声水平的信号。此外，使用光学条形码可以区分混合物中的多个生物分子，与测序方法相比，读数更快、更简单。在理想的数字化验中，理论上，信号应该进一步代表绝对分子计数，而不是相对水平，从而消除对作为典型化验支柱的校准标准的需要。一些数字化验平台现已商业化，但挑战阻碍了这些新格式的采用和多样化，因为平衡检测的灵敏度和动态范围、增加分析物复用、提高样品通量和降低成本等方面存在广泛的需求。我们的实验室和其他实验室已经开发出技术来应对这些挑战，通过重新设计分子探针和标签，改善检测焦点体积内的分子传输，以及在流中应用基于解决方案的读出方法。本帐户描述了数字生物分子测定的原理、格式和设计限制，这些测定应用光学标签来实现简单和常规目标计数的目标，最终可能接近绝对读出标准。主要挑战可以从热力学和缔合反应动力学、传质和离散统计的基本概念来理解。主要进展包括 (1) 新型无机纳米晶体探针，与染料相比，计数更稳健；(2) 多种分子放大工具，可将众多标记附着到单个目标上；(3) 具有图案特征的专用表面，用于与标记电磁耦合以进行信号放大；(4) 表面捕获增强方法，通过破坏扩散耗尽区来浓缩目标；(5) 流式计数，其中分析物在溶液中快速计数，无需下拉到表面。这些生物分子计数工具的进一步进展和集成可以提高生命科学研究中实验室测量的精度，并有利于低丰度生物标志物的临床诊断测定，限制传统整体水平生物测定无法达到的生物样本体积。
ConspectusThere has been a recent surge of advances in biomolecular assays based on the measurement of discrete molecular targets as opposed to signals averaged across molecular ensembles. Many of these “digital” assay designs derive from now-mature technologies involving single-molecule imaging and microfluidics and provide an assortment of new modalities to quantify nucleic acids and proteins in biospecimens such as blood and tissue homogenates. A primary new benefit is the robust detection of trace analytes at attomolar to femtomolar concentrations for which many ensemble assays cannot distinguish signals above noise levels. In addition, multiple biomolecules can be differentiated within a mixture using optical barcodes, with much faster and simpler readouts compared with sequencing methods. In ideal digital assays, signals should, in theory, further represent absolute molecular counts, rather than relative levels, eliminating the need for calibration standards that are the mainstay of typical assays. Several digital assay platforms have now been commercialized but challenges hinder the adoption and diversification of these new formats, as there are broad needs to balance sensitivity and dynamic range of detection, increase analyte multiplexing, improve sample throughput, and reduce cost. Our lab and others have developed technologies to address these challenges by redesigning molecular probes and labels, improving molecular transport within detection focal volumes, and applying solution-based readout methods in flow.This Account describes the principles, formats, and design constraints of digital biomolecular assays that apply optical labels toward the goal of simple and routine target counting that may ultimately approach absolute readout standards. The primary challenges can be understood from fundamental concepts in thermodynamics and kinetics of association reactions, mass transport, and discrete statistics. Major advances include (1) new inorganic nanocrystal probes for more robust counting compared with dyes, (2) diverse molecular amplification tools that endow attachment of numerous labels to single targets, (3) specialized surfaces with patterned features for electromagnetic coupling to labels for signal amplification, (4) surface capture enhancement methods to concentrate targets through disruption of diffusion depletion zones, and (5) flow counting in which analytes are rapidly counted in solution without pull-down to a surface. Further progress and integration of these tools for biomolecular counting could improve the precision of laboratory measurements in life sciences research and benefit clinical diagnostic assays for low abundance biomarkers in limiting biospecimen volumes that are out of reach of traditional ensemble-level bioassays.