Pushing the detection limits: strategies towards highly sensitive optical-based protein detection.

Proteins are one of the main constituents of living cells. Studying the quantities of proteins under physiological and pathological conditions can give valuable insights into health status, since proteins are the functional molecules of life. To be able to detect and quantify low-abundance proteins in biofluids for applications such as early disease diagnostics, sensitive analytical techniques are desired. An example of this application is using proteins as biomarkers for detecting cancer or neurological diseases, which can provide early, lifesaving diagnoses. However, conventional methods for protein detection such as ELISA, mass spectrometry, and western blotting cannot offer enough sensitivity for certain applications. Recent advances in optical-based micro- and nano-biosensors have demonstrated promising results to detect proteins at low quantities down to the single-molecule level, shining lights on their capacities for ultrasensitive disease diagnosis and rare protein detection. However, to date, there is a lack of review articles synthesizing and comparing various optical micro- and nano-sensing methods of enhancing the limits of detections of the antibody-based protein assays. The purpose of this article is to critically review different strategies of improving assay sensitivity using miniaturized biosensors, such as assay miniaturization, improving antibody binding capacity, sample purification, and signal amplification. The pros and cons of different methods are compared, and the future perspectives of this research field are discussed.

Recent advances on protein-based quantification of extracellular vesicles.

Extracellular vesicles (EVs) are secreted by almost all cells into the circulatory system and have the important function of intercellular communication. Ranging in size from 50 to 1000 nm, they are further classified based on origin, size, physical properties and function. EVs have shown the potential for studying various physiological and pathological processes, such as characterizing their parent cells with molecular markers that could further signify diseases. Proteins within EVs are the building blocks for the vesicles to function within a biological system. Isolation and proteomic profiling of EVs can advance the understanding of their biogenesis and functions, which can give further insight of how they can be used in clinical settings. However, the nanoscale size of EVs, which is much smaller than that of cells, comprises a major challenge for EV isolation and the characterization of their protein cargos. With the recent advances of bioanalytical techniques such as lab-on-a-chip devices and innovated flow cytometry, the quantification of EV proteins from a small number of vesicles down to the single vesicle level has been achieved, shining light on the promising applications of these small vesicles for early disease diagnosis and treatment monitoring. In this article, we first briefly review conventional EV protein determination technologies and their limitations, followed by detailed description and analysis of emerging technologies used for EV protein quantification, including optical, non-optical, microfluidic, and single vesicle detection methods. The pros and cons of these technologies are compared and the current challenges are outlined. Future perspectives and potential research directions of the EV protein analysis methods are discussed.