Fluorescence Signal Enhancement-Based On-Site Single-Molecule Sensing: A Review

Compared to conventional sensing, single-molecule sensing is proven to have extremely high sensitivity and has thus been successfully applied in various fields. However, many single-molecule sensing techniques rely on precise but bulky instruments and complicated operations, thus limiting their scope in on-site applications. To solve the problem, we have reported a series of works on detecting chemicals, proteins, and nucleic acids on-site at a single-molecule level. Though these works deal with diverse targets (avian influenza virus (AIV), hepatitis B virus (HBV), and mercury ion (Hg ²⁺ )) and use different sensing devices, they have the same on-site single-molecule sensing tactic, which employs fluorescence-enhanced probes, thus supporting a portable fluorescence microscope equipped with an industrial camera to collect and analyze the single-molecule signal. We have optimized the sensing operations to guarantee that all the procedures can be conveniently implemented on-site using field-portable devices. Therefore, based on the fluorescence-enhanced probes, portable optical platform, and optimized sensing operations, our tactic is proven to have advantages such as high sensitivity, high accuracy, simple procedures, and fast speed. In this paper, we describe all the key components of our designed on-site single-molecule sensing tactic, including the sensing system, optical platform, and sensing operations. We compare this tactic with other reported single-molecule sensing approaches and discuss the pros and cons, as well as the prospect of our designed on-site single-molecule sensing. Our tactic will promote the high speed, convenience, and automation of single-molecule sensing and pave the way for the development of on-site single-molecule sensing approaches and instruments.