Chiu Hoi Kei, Kartanas Tadas, Saar Kadi L, Luxhøj Carina Mouritsen, Devenish Sean, Knowles Tuomas P J
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Fluidic Analytics Ltd., Unit A The Paddocks Business Centre, Cherry Hinton Road, Cambridge CB1 8DH, United Kingdom.
Biomicrofluidics. 2021 Apr 29;15(2):024113. doi: 10.1063/5.0039872. eCollection 2021 Mar.
Protein detection and quantification is a routinely performed procedure in research laboratories, predominantly executed either by spectroscopy-based measurements, such as NanoDrop, or by colorimetric assays. The detection limits of such assays, however, are limited to M concentrations. To establish an approach that achieves general protein detection at an enhanced sensitivity and without necessitating the requirement for signal amplification steps or a multicomponent detection system, here, we established a chemiluminescence-based protein detection assay. Our assay specifically targeted primary amines in proteins, which permitted characterization of any protein sample and, moreover, its latent nature eliminated the requirement for washing steps providing a simple route to implementation. Additionally, the use of a chemiluminescence-based readout ensured that the assay could be operated in an excitation source-free manner, which did not only permit an enhanced sensitivity due to a reduced background signal but also allowed for the use of a very simple optical setup comprising only an objective and a detection element. Using this assay, we demonstrated quantitative protein detection over a concentration range of five orders of magnitude and down to a high sensitivity of , corresponding to pM concentrations. The capability of the platform presented here to achieve a high detection sensitivity without the requirement for a multistep operation or a multicomponent optical system sets the basis for a simple yet universal and sensitive protein detection strategy.
蛋白质检测和定量是研究实验室中经常进行的一项操作，主要通过基于光谱的测量方法（如NanoDrop）或比色测定法来执行。然而，这些测定方法的检测限仅限于微摩尔浓度。为了建立一种能够在提高灵敏度的情况下实现通用蛋白质检测，且无需信号放大步骤或多组分检测系统的方法，在此，我们建立了一种基于化学发光的蛋白质检测方法。我们的方法专门针对蛋白质中的伯胺，这使得任何蛋白质样品都能被表征，此外，其潜在特性消除了洗涤步骤的需求，提供了一条简单的实施途径。此外，基于化学发光的读数确保了该测定可以在无激发源的方式下进行操作，这不仅由于降低了背景信号而提高了灵敏度，还允许使用仅由一个物镜和一个检测元件组成的非常简单的光学装置。使用这种测定方法，我们展示了在五个数量级的浓度范围内进行定量蛋白质检测，最低灵敏度达到纳摩尔浓度，即皮摩尔浓度。本文介绍的平台能够在无需多步操作或多组分光学系统的情况下实现高检测灵敏度，为一种简单、通用且灵敏的蛋白质检测策略奠定了基础。
