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Early detection, diagnosis and treatment of human immune deficiency virus (HIV) infection is the key to reduce acquired immunodeficiency syndrome (AIDS) mortality. In our research, an innovative surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions (ESDRs) and double-layer DNA tetrahedrons (DDTs). ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA, leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA (dsDNA) products. Subsequently, the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures. Due to the high efficiency of ESDRs and large molecular weight of DDTs, the SPR response signal was enhanced dramatically. The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1pM to 150nM with a detection limit of 48fM. In addition, the whole detecting process can be accomplished in 60min with high accuracy and duplicability. In particular, the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample, indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection.

A novel colorimetric biosensing strategy, employing coupling spontaneous cascade catalytic hairpin assembly (CHA) circuits and mismatched hairpin, has been developed successfully for ultrasensitive and specific detection of target microRNA (miRNA). This system is composed of two layers of CHA. While target miRNA exists, the toehold-based assembly of the first-layer hairpins comes into being, followed by the generation of the first-layer CHA products and the cyclic release of the target miRNA. Then the first-layer CHA products can act as initiator for the assembly of the second-layer hairpins to produce the second-layer CHA products, with the first-layer CHA products recovered simultaneously. Finally, the second-layer CHA products can combine with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, for catalyzing a colorimetric reaction. Under the optimal experimental conditions, the established biosensor can detect target miRNA down to 36.2 fM (S/N = 3) with a linear range from 100 fM to 10 nM, and discriminate target miRNA from mismatched miRNA with high selectivity. It was also applied to test the concentration of miRNA spiked into salmon sperm samples. Therefore, this biosensing strategy may become an alternative tool for the detection of miRNA in biomedical research.

Sensitive and specific detection of HIV-related DNA is of great importance for early accurate diagnosis and therapy of HIV-infected patients. Here, we developed a one-step and rapid fluorescence strategy for HIV-related DNA detection based on strand displacement amplification and a Mg2+-dependent DNAzyme reaction. In the presence of target HIV DNA, it can hybridize with template DNA and activate strand displacement amplification to generate numerous DNAzyme sequences. With the introduction of Mg2+, DNAzyme can be activated to circularly cleave the substrate DNA, which leads to the separation of fluorophore reporters from the quenchers, resulting in the recovery of the fluorescence. Under the optimal experimental conditions, the established biosensing method can detect target DNA down to 61 fM with a linear range from 100 fM to 1 nM, and discriminate target DNA from mismatched DNA perfectly. In addition, the developed biosensing strategy was successfully applied to assay target DNA spiked into human serum samples. With the advantages of fast, easy operation and high-performance, this biosensing strategy might be an alternative tool for clinical diagnosis of HIV infection.

The authors describe an electrochemical strategy for ultrasensitive and specific detection of microRNA (miRNA). It is based on both multicomponent nucleic acid enzyme (MNAzyme) amplification and rolling circle amplification (RCA). In the presence of target miRNAs, partial enzyme A (partzyme A) and partial enzyme B (partzyme B) are assembled to form active MNAzymes. Once formed, the MNAzymes catalyze the cleavage of the hairpin substrates to liberate biotinylated fragments which hybridized with the capture probes immobilized on a gold electrode. The RCA is then initiated to form a product that binds many detection probes. Finally, the amperometric signal (best acquired at a working voltage of 0.22 V vs. Ag/AgCl) is obtained by employing the streptavidinylated alkaline phosphatase as the enzyme. This biosensor has a 1.66 fM detection limit, and a dynamic range that extends from 10 fM to 1 nM. It displays specificity down to single mismatch discrimination of target miRNAs and good reproducibility. It was successfully applied to the determination of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells.

The authors describe an electrochemical sensing strategy for highly sensitive and specific detection of target (analyte) DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme (MNAzyme). MNAzymes were formed by multicomponent complexes which produce amplified “output” signals in response to specific “input” signal. In the presence of target nucleic acid, multiple partial enzymes (partzymes) oligonucleotides are assembled to form active MNAzymes. These can cleave H0 substrate into two pieces, thereby releasing the activated MNAzyme to undergo an additional cycle of amplification. Here, the two pieces contain a biotin-tagged sequence and a byproduct. The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode. By employing streptavidinylated alkaline phosphatase as an enzyme label, an electrochemical signal is obtained. The electrode, if operated at a working potential of 0.25 V (vs. Ag/AgCl) in solution of pH 7.5, covers the 100 pM to 0.25 μM DNA concentration range, with a 79 pM detection limit. In our perception, the strategy introduced here has a wider potential in that it may be applied to molecular diagnostics and pathogen detection.

DNA methyltransferase (MTase) activity assay and its inhibitor screening are important for the diagnosis and treatment of methylation-related diseases. Herein, a label-free and highly sensitive biosensing method based on entropy-driven reaction and toehold-initiated rolling circle amplification (TIRCA) was developed for DNA MTase activity detection and inhibitor screening. Briefly, in the presence of MTase, the triple-stranded complex (TSC) could be methylated to avoid cleaving by MboI endonuclease. The complete TSC was able to initiate downstream entropy-driven reaction and TIRCA to produce G-quadruplex that can bind with Thioflavin T (ThT) to output fluorescent signal. Under the optimal experimental conditions, the established biosensing strategy could detect Dam MTase down to 0.06 U/mL with a linear range from 0.1 U/mL to 40 U/mL. Importantly, the signal-to-noise (S/N) of this biosensing strategy was extremely high. This strategy could discriminate target Dam MTase from another two MTases efficiently. Moreover, this developed biosensing method was applied to screen DNA MTase inhibitors. Therefore, we anticipate this unique strategy will serve as an alternative tool to detect DNA MTase activity and screen its inhibitors in clinical diagnosis and therapeutics.