Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection

Abstract

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the singlestranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Dual-Mode Logic Gate for Intelligent and Portable Detection of MicroRNA Based on Gas Pressure and Lateral Flow Assay

“…The lateral flow strip (LFS), as an attractive mini-device, has been used for developing portable lateral flow assays (LFA) due to its portability, affordability, quick response, and ease of use. − Due to the merits of the LFS, DNA logic gates combined with the LFS were fabricated to develop portable and intelligent biosensing for detection of DNA, proteins, and small molecules. − However, visual detection is qualitative, which makes it difficult to obtain the actual concentration of targets. Moreover, visual readouts may be inaccurate due to personal judgment, and the sensitivity of colorimetric biosensing is limited.…”

Light-Driven and Metal–Organic Framework Synergetic Loaded DNA Tetrahedral Amplifier for Exonuclease III-Powered All-in-One Biosensing and Chemotherapy in Live Biosystems

“…As a result of their excellent signal amplification, designability, and biocompatibility, DNA amplifiers have become powerful biosensing tools for the analysis of low-abundance targets, especially when combined with fluorescence imaging to analyze live biosystems. − Further, in recent years, a large variety of nucleases have been discovered, and the introduction of exonuclease or endonuclease can offer higher sensitivity. − In particular, E. coli-derived exonuclease III (Exo III) has not only high catalytic activity for stepwise removal (from the 3′ to 5′ terminal) of the blunt end of a double-stranded nucleic acid but also does not degrade the protruding single-stranded part.…”

Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance

“…Invasive reactions − are of signal amplification using Archaeoglobus fulgidus (Afu) FEN1 , to recognize the invasive structure formed between the target, upstream probe (UP), and downstream probe (DP), and the DP could be cleaved into the flap (5′ terminus of the DP) at the overlap structure. The flap further initiates a secondary invasive reaction with a fluorescence quencher hairpin probe (HP) to produce an amplified fluorescence signal.…”