This paper reviews the latest research progress of surface-enhanced Raman spectroscopy (SERS) microfluidic chips in the field of biosensing. Due to its single-molecule sensitivity, selectivity, minimal or no preprocessing, and immediacy, SERS is considered a promising biosensing technology. However, the nondirectional interactions between biological samples and the substrate, as well as fluctuations in the sample environment temperature during signal acquisition, can affect the stability and reproducibility of SERS signals. Integrating SERS spectroscopy with microfluidic chips not only leverages the continuous sample flow, high reaction efficiency, high throughput, and multifunctionality of microfluidic chips to address challenges in biosensing applications but also expands the scope of microfluidic technology by providing a novel on-chip optical detection method. The combination of SERS and microfluidic chips not only enables the complementary advantages of both technologies but also offers a highly promising "combined technology" for the field of biosensing. This paper starts by introducing the enhancement mechanisms of SERS and presents both labeled and label-free SERS strategies. Based on the differences in substrate properties, we broadly categorize SERS microfluidic chips into colloidal nanoparticle-based SERS microfluidic chips and fixed substrate-based SERS microfluidic chips. Finally, we review the latest research progress on SERS microfluidic chips for biosensing biological targets such as nucleic acids, proteins, small biomolecules, and live cells. In the conclusion and outlook section, we summarize the challenges faced by SERS microfluidic chips in biosensing and propose feasible solutions. To better leverage the role of SERS microfluidic chips in biosensing, we also present an outlook on the future development of this combined technology.

Electrochemical techniques are commonly used to analyze and screen various environmental pathogens. When used in conjunction with other optical recognition methods, it can extend the sensing range, lower the detection limit, and offer mutual validation. Nowadays, electrochemical-optical dual-mode biosensors have ensured the accuracy of test results by integrating two signals into one, indicating their potential use in primary food safety quantitative assays and screening tests. Particularly, visible optical signals from electrochemical/colorimetric dual-mode biosensors could meet the demand for real-time screening of microbial pathogens. While electrochemical-optical dual-mode probes have been receiving increasing attention, there is limited emphasis on the design approaches for sensors intended for microbial pathogens. Here, we review the recent progress in the merging of optical and electrochemical techniques, including fluorescence, colorimetry, surface plasmon resonance (SPR), and surface enhanced Raman spectroscopy (SERS). This study particularly emphasizes the reporting of various sensing performances, including sensing principles, types, cutting-edge design approaches, and applications. Finally, some concerns and upcoming advancements in dual-mode probes are briefly outlined.

Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing system was based on two DNA hybrid probes, each of which was constructed by annealing a reconfigurable DNA hairpin with single-stranded DNA. Upon target recognition by the first DNA hybrid probe, a reconfigurable DNA switch was liberated, triggering a toehold-mediated strand displacement reaction (TSDR) with the second DNA hybrid probe, which was accompanied by the release of another reconfigurable DNA switch. This released allosteric DNA switch could further interact with the first hybrid DNA probe via the TSDR strategy to form a reciprocal strand displacement network between the two DNA hybrid probes. Theoretically, this reciprocal strand displacement reaction would continue till the complete consumption of the reaction substrates. Thus, it provides a new signal amplification method leading toward target recognition. More interestingly, it creates a ratiometric signal response mode for target recognition, which involves the fluorescence increment of one fluorophore (Cy5) and concurrent decrement of another fluorophore (Cy3) accompanied by the target-triggered reciprocal strand displacement reaction. This process could achieve a low detection limit of about 0.1 pM toward the target nucleic acid and selective discrimination toward different mismatched targets. It could also be applied for detection in a serum sample. Thus, the developed catalytic DNA circuit reaction strategy together with ratiometric signal readout provides a new avenue for programmable, reliable and sensitive detection of nucleic acids and might also pave the way for developing more advanced DNA circuits or biosensors.

The real-time imaging of low-abundance tumor-related microRNAs (miRNAs) in living cells holds great potential for early clinical diagnosis of cancers. However, the relatively low detection sensitivity and possible false-positive signals of a probe in complex cellular matrices remain critical challenges for accurate RNA detection. Herein, we developed a novel aptamer-functionalized cruciate DNA probe that enabled amplified multiple miRNA imaging in living cells via catalytic hairpin assembly (CHA). The cross-shaped design of the cruciate DNA probe improved the stability against nucleases and acted as a modular scaffold for CHA circuits for efficient delivery into tumor cells. The cruciate DNA probe allowed self-assembly through thermal annealing and displayed excellent performance for sensitive miRNA detection in vitro. The cruciate DNA probe could be internalized into nucleolin-overexpressed cells specifically via cell-targeting of the AS1411 aptamer, achieving amplified fluorescence imaging and quantitative evaluation of the expression of miRNAs in living cells. Through the simultaneous detection of intracellular multiple miRNAs, the developed cruciate DNA probe could provide more accurate information and reduce the chances of false positive signals for cancer diagnosis. This approach offers a new opportunity for promoting the development of miRNA-related biomedical research and tumor diagnostic applications.

The sensitive detection of cancer-associated exosomal microRNAs shows enormous potential in cancer diagnosis. Herein, a ratiometric fluorescent biosensor based on self-assembled fluorescent gold nanoparticles (Au NPs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for sensitive detection of colorectal cancer (CRC)-associated exosomal miR-92a-3p. In this biosensing system, the hairpin DNA modified with sulfhydryl and fluorescent dye Atto-425 at both ends is conjugated to fluorescent Au NPs through Au-S bonds, resulting in the quenching of Atto-425. The miR-92a-3p can open the hairpin of DNA and forms an miR-92a-3p/DNA heteroduplex, triggering the specific cleavage of DSN for the DNA in the heteroduplex. As a result, Atto-425 leaves the fluorescent Au NPs and recovers the fluorescence emission. The released miR-92a-3p can hybridize with another hairpin DNA and lead to a stronger fluorescence recovery of Atto-425 to form a signal amplification cycle. The stable fluorescence of Au NPs and the changing fluorescence of Atto-425 constitute a ratiometric fluorescent system reflecting the concentration of miR-92a-3p. This biosensor exhibits excellent specificity and can distinguish CRC patients from healthy individuals by detecting miR-92a-3p extracted from clinical exosome samples, showing the potential in CRC diagnosis.