A nucleic acid-based biosensor is a type of biosensor that uses nucleic acids, such as DNA or RNA, as the recognition element for detecting specific analytes or molecules. Nucleic acid-based biosensors can detect various types of targets, such as pathogens, genetic mutations, environmental pollutants, and drugs. Thus, they can also be used for a variety of applications, including medical diagnosis, environmental monitoring, and food safety testing. The property of being easy to design and produce makes them an attractive option for a wide range of applications. In this study, visual quantification of analytes (copper ion and flap endonuclease 1) based on nucleic acid biosensor has been realized on microfluidic particle dam for point-of-care testing. Additionally, new approaches combining nucleic acid probes and CRISPR/Cas12a biosensing technology for detecting biomarkers such as flap endonuclease 1 and hOGG1 were also explored.

First, contamination of copper ions in drinking water has become a severe hazard due to the use of copper water pipes and discharge of industrial wastewater globally. Therefore, there is a need for easy-to-use platforms that can quantitatively analyze trace amounts of copper ions (Cu²⁺) in drinking water to ensure water safety on daily basis. In this study, we present a microfluidic particle accumulation technique integrated with Cu(II)-catalyzed Fenton reaction for the visual and quantitative detection of copper ions. Microparticles (MMPs) and polystyrene microparticles (PMPs) are connected via a single strand DNA, MB155. However, with the presence of Cu²⁺, hydroxyl free radicals (•OH) produced from Cu²⁺/hydrogen peroxide (H₂O₂) Fenton reactions cleave MB155, causing an increased amount of free PMPs. To count these particles, the solution is loaded onto a microfluidic chip where free MMPs and MMPs-MB155-PMPs can be collected by a magnetic separator, while free PMPs continue flowing until they accumulate at the particle dam. The results showed a good linear relationship between the trapping length of PMP accumulation and the Cu²⁺ concentration from 0 to 300 nM. A limit of detection (LOD) of 70.1 nM was achieved, which is approximately 449 times lower than the 2 × 10³ µg/L (~31.5 µM) required by the World Health Organization (WHO). The technique also showed high selectivity and good tolerance to pH and hardness, indicating its compatibility for detecting copper contamination in tap water. This suggests that the platform could be potentially used for routine monitoring of copper contamination in drinking water.

Second, flap endonuclease 1 (FEN1) is an endonuclease that plays a crucial role in various DNA metabolism pathways by removing 5′ single-stranded overhangs of branched duplex DNA (5′ flap) to prevent malignant cell transformation. However, abnormal expression of FEN1 is often linked to tumor progression, making it a potential cancer biomarker for diagnosis and treatment. In this study, we present a multimodal approach to detect FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-stranded DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5′ flap was designed to be cleaved by FEN1, and the resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA upon binding to CRISPR/Cas12a-crRNA. The trans-cleavage occurs only once and is independent of the length or sequence of the ssDNA, allowing for efficient signal amplification and multimodal signals, such as fluorescence or a cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) which alters solution turbidity after magnetic separation. Furthermore, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are collected at the particle dam, allowing for a visible PMP accumulation length that is proportional to FEN1 activity. This multimodal detection is selective to FEN1 activity and achieves a low limit of detection (LOD) with only 40 minutes of reaction time. When applied to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting its potential as a diagnostic tool for cancer.

Third, human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a critical enzyme in the base excision repair pathway that removes 8-oxoguanine (8-oxoG), a common DNA damage resulting from oxidative stress. Abnormal expression of hOGG1 has been implicated in various diseases, including cancer and neurodegenerative disorders, making it a potential diagnostic and therapeutic target. Here, we designed a dumbbell DNA probe with an 8-oxo modification and a 3’ overhang, intended to be recognized by hOGG1 and cleaved. The resulting duplex DNA is closed after ligated by T4 DNA ligase and features a specific protospacer adjacent motif (PAM), which triggers trans-cleavage of ssDNA modified with fluorophore and quencher when CRISPR/Cas12a-crRNA binds to it, producing fluorescence signal and realizing hOGG1 fluorescent detection. The technique can attain a limit of detection of 0.00037 U/mL while being selective to hOGG1. Additionally, it exhibits excellent compatibility for identifying hOGG1 in bio-samples, indicating promising potential for use in disease diagnosis and scientific research.

The above research expands the application of nucleic acid-based biosensor, demonstrating its great potential in biosensing.