Xiaoqun Gong

A pivotal hurdle in traumatic brain injury (TBI) therapy is the self-perpetuating cycle between neuroinflammation and oxidative stress. Concurrent targeting of both pathways offers a promising strategy to overcome the key limitation in current therapy. Herein, a programmed streptavidin-condensed bifunctional nucleic acid nanoplatform (DZ-G4/H NPs) was developed via rolling circle amplification (RCA) to integrate numerous C1qa-cleaving DNAzymes with the peroxidase-like G-quadruplex/hemin (G4/H) complex. The platform ingeniously enhances the stability and drug-loading capacity of nucleic acid structures, enabling improved therapeutic efficacy through disruption of the self-perpetuating inflammation-oxidative stress cycle, which is superior to individual monotherapies. Consequently, it alleviated key pathophysiological hallmarks such as brain edema and cerebral blood flow (CBF) deficits, promoted holistic recovery of motor and cognitive functions, and ultimately improved survival in TBI mice. Beyond its promising application in TBI therapy that consolidates multiple therapeutic advantages, this nanostrategy offers a versatile method for streamlined programmed assembly of multifunctional modules, thereby providing an adaptable technical platform for the design and development of multifunctional nucleic acid drugs.

As an enzyme-free isothermal nucleic acid signal amplification technologies, Hybridization Chain Reaction (HCR) has emerged as versatile tools for molecular diagnostics in vitro and imaging in vivo. However, HCR itself has several limitations, including difficulties in quantification, slow reaction rates, insufficiently low detection limits, and the inability to use as homogeneous detection tools with simple molecular beacons for fluorescence characterization. Herein, we report an interesting finding that the introduction of DNA polymerase transformed the reaction from a linear mode to an exponential mode. Moreover, DNA polymerase-mediated hybridization chain reaction (pHCR) has resolved all the aforementioned problems. pHCR can be carried out in a homogeneous environment, featuring rapid and sensitive target detection capabilities, and is also easy to quantify. Through optimization of the reaction system, the detection limit for target nucleic acid I reached 290.4 amol, which is lower than traditional HCR, and it exhibits excellent specificity and good anti-interference capabilities. Additionally, the short output products facilitate the characterization of pHCR and its coupling with other reactions. In summary, pHCR holds great potential as a novel tool for the detection and analysis of nucleic acid biomarkers, with significant application prospects.

Fluorescence quenching lateral flow immunoassay (FQ-LFIA) has emerged as a powerful tool for the rapid detection of disease biomarkers due to its high signal-to-noise ratio and multimodality signal output. However, conventional fluorescent quenchers are constrained by specific distances and narrow absorption peaks, hindering their quenching efficiency and sensitivity for trace analyte detection. In this study, we synthesized bimetallic gold-iridium oxide nanoparticles (Au@IrO₂ NPs) via a seed-mediated growth method as the quenchers and constructed a distance-independent synergistic FQ-LFIA (SFQ-LFIA) platform. The Au@IrO₂ NPs significantly reduced the background fluorescence signal on the test line (T-line) through the inner filter effect (IFE), while their excellent catalytic activity oxidized the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to generate the colored product oxidized TMB (oxTMB), which further synergistically quenched background fluorescence via a secondary IFE pathway. Using creatine kinase-MB (CK-MB) as a model biomarker, the detection limit of the Au@IrO₂ NPs-based SFQ-LFIA in fluorescence mode (0.71 pg/mL) was 13-fold lower than in the precatalysis (8.91 pg/mL) and at least 215-fold lower than in colorimetric mode. The platform demonstrated high accuracy in quantifying CK-MB levels in clinical serum samples, providing a novel strategy for developing advanced in vitro rapid diagnostic systems.

CRISPR/Cas12a systems have emerged as versatile tools for molecular diagnostics, but directly detecting RNA and identifying specific DNA point mutations remain challenging. Herein, we report a simple engineering approach with a split site in the spacer sequence, enabling activation of CRISPR/Cas12a (LbCas12a) for trans-cleavage with similar efficiency to wild-type crRNA. The engineered crRNA facilitated RNA target recognition by replacing the 3'-end with RNA fragments, enhancing point mutation specificity for ssDNA targets. Based on this, we achieved amplification-free detection of microRNAs and DNA point mutations with high sensitivity and specificity. For clinical sample validation, we constructed reverse fluorescence-enhanced lateral flow test strips (rLFTS), which achieved femtomole-level detection. Moreover, the engineered crRNA-based CRISPR/Cas12a system also effectively recognized tumor cells via intracellular and in vivo imaging of miRNA-21. In conclusion, this engineered crRNA platform enhances CRISPR/Cas12a-based nucleic acid detection, promoting its wide application in molecular diagnostics and bioimaging.

A novel multi-site enzymatic repairing amplification method was developed for Tdt activity analysis with simplified design and outstanding sensitivity.

CRISPR/Cas12a systems have been repurposed as powerful tools for developing next-generation molecular diagnostics due to their trans-cleavage ability. However, it was long considered that the CRISPR/Cas12a system could only recognize DNA targets. Herein, we systematically investigated the intrinsic trans-cleavage activity of the CRISPR/Cas12a system (LbCas12a) and found that it could be activated through fragmented ssDNA activators. Remarkably, we discovered that the single-stranded DNA (ssDNA) activators in the complementary crRNA-distal domain could be replaced by target miRNA sequences without the need for pre-amplification or specialized recognition mechanisms. Based on these findings, we proposed the “Fragment Complementary Activation Strategy” (FCAS) and designed reverse fluorescence-enhanced lateral flow test strips (rFLTS) for the direct detection of miRNA-10b, achieving a limit of detection (LOD) of 5.53 fM and quantifying the miRNA-10b biomarker in clinical serum samples from glioma patients. Moreover, for the first time, we have developed the FCAS-based CRISPR/Cas12a system for miRNA in situ imaging, effectively recognizing tumor cells. The FCAS not only broadens the scope of CRISPR/Cas12a system target identification but also unlocks the potential for in-depth studies of CRISPR technology in many diagnostic settings.