Jun Chen

Real-time monitoring of natural killer (NK) cell-mediated cancer immunotherapy efficacy at the cellular and organismal levels remains a critical challenge, primarily due to the ultra-low abundance of Granzyme A (GzmA) delivered into target cells, its rapid spatiotemporal dynamics, and the scarcity of suitable in situ imaging probes. Here, we present a DNA nanowire-based biosensor that leverages a spatially confined catalytic hairpin assembly (SCCHA) system for the real-time in situ imaging of immunotherapy efficacy, achieved by sensing GzmA-induced apurinic/apyrimidinic endonuclease 1 (APE1) inactivation. This biosensor integrates an APE1-responsive molecular beacon (MB) with a spatially organized CHA amplification circuit on a rigid DNA nanowire scaffold. In tumor cells with high endogenous APE1 activity, cleavage of the MB initiates a confined CHA reaction, yielding a strong fluorescence readout. Upon NK cell attack, delivered GzmA specifically inactivates intracellular APE1, thereby suppressing CHA signal amplification and producing a distinct fluorescence attenuation that dynamically visualizes NK cell cytotoxicity. Owing to the spatial confinement effect of the DNA nanowire, the biosensor achieved a 6.44-fold improvement in sensitivity over conventional free-diffusion CHA system. This platform enabled real-time visualization of NK cell−tumor cell interactions, and accurately reported NK cell cytotoxicity through GzmA-mediated APE1 inactivation. In vivo experiments further demonstrated its capability in evaluating immunotherapy efficacy. Collectively, this DNA nanowire-based biosensor provides a highly sensitive and reliable in situ imaging toolkit for evaluating cancer immunotherapy in real time, offering a promising translational approach for preclinical drug screening and personalized immunotherapeutic monitoring.

Extracellular vesicles (EVs) secreted by glioblastoma (GBM) cells carry tumor-specific biomarkers such as microRNA-1246 (miR-1246), offering significant potential for noninvasive GBM detection. However, the efficient isolation of EVs from complex biofluids and ultrasensitive detection of their low-abundance miRNAs encapsulated in EVs remain formidable challenges. To address this, we designed a multifunctional nanoplatform of UiO-66-NH2@Au@MSDC (UAM), which integrates EV capture and in situ miRNA analysis. The initial construction of UiO-66-NH2@Au (UA) provided a high density of thiol-binding sites for the attachment of mercapto (SH)-spacer-DNA-cholesterol (MSDC) probes to form UiO-66-NH2@Au@MSDC (UAM). The abundant cholesterol motifs on these probes subsequently enabled highly efficient EV capture, achieving a recovery rate of 84.8 ± 2.4% under a low centrifugal force of 8000g while preserving EV structural integrity and bioactivity. For the downstream analysis of EVs, we developed an innovative EXPAR-based strategy, termed Y-EXPAR, to address the specificity challenge in detecting miR-1246. The entire detection system employs a temperature-synergistic strategy at 55 °C, which simultaneously triggers the membrane fusion of EVs with synthetic cationic liposomes (preloaded with Y-EXPAR reagents) and activates the Y-EXPAR. The resulting fusion creates confined nanoreactors that concentrate trace amounts of miR-1246, thereby enabling ultrasensitive detection with detection limits of 1.4 fM for miR-1246 and 22 particles/μL for EVs. This integrated platform overcomes key bottlenecks in EV-based liquid biopsy, providing a practical, low-equipment-demand approach for the early diagnosis of GBM and laying a foundation for advancing precision oncology diagnostics.

Developing highly sensitive and specific imaging strategies for long noncoding RNAs (lncRNAs) is crucial for disease diagnosis and therapeutic research. However, lncRNAs are characterized by long sequence lengths (>200 nt), low abundance, and complex secondary structures, which make their sensitive and specific imaging challenging. To address these challenges, we developed a hybridization chain reaction (HCR) strategy based on dual-site cooperative recognition for lncRNA imaging, termed dual-site cooperative recognition HCR (DCHCR). Through multisite cooperative recognition, the adverse effects of lncRNA secondary structures on probe hybridization are alleviated, thereby significantly improving probe-target binding efficiency and enhancing detection sensitivity and accuracy. Compared with conventional single-site recognition HCR strategies, DCHCR exhibits improved reaction kinetics and higher sensitivity, achieving a detection limit of 47.1 pM. DCHCR also demonstrates strong practical applicability, enabling effective discrimination of lncRNA expression levels between cancer and normal cells at the level of living cells. Furthermore, this strategy enables effective imaging of lncRNAs in tumors of living mice with high contrast. Overall, this strategy is expected to serve as a powerful tool for lncRNA analysis and may contribute to the clinical diagnosis and therapeutic development of lncRNA-related diseases.

Highly sensitive analysis of exosomal microRNAs (miRNAs) holds significant implications for early cancer diagnosis and prognostic evaluation, yet the low abundance, high homology, and short sequence length of exosomal miRNAs present critical challenges for achieving both sensitivity and specificity in detection. Herein, a dual signal amplification (DSA) strategy integrating an APE1 enzyme-assisted DNA walker with rolling circle amplification (RCA) was developed for highly sensitive, specific, and label-free analysis of exosomal miRNAs. Using miR-10b as a breast cancer proof-of-concept target, the system integrated an APE1 enzyme-assisted DNA walker with rolling circle amplification (RCA). Target miR-10b triggered the APE1-assisted DNA walker immobilized on magnetic beads, generating abundant RCA primer probes. Following magnetic separation, the obtained RCA primers were incubated with the RCA reaction mixture to initiate the RCA reaction, yielding long single-stranded DNA products with copious G-quadruplex (G4). Thioflavin T (ThT) then selectively bound to G4 structures, forming fluorescent G4-ThT complexes that enabled a label-free fluorescence readout. This approach achieved a detection limit of 0.27 fM with exceptional specificity, demonstrating the capacity to discriminate single-nucleotide variants. To validate its applicability, the DSA strategy successfully distinguished miR-10b expression between breast cancer-cell-derived and normal cell-derived exosomes. Furthermore, clinical validation revealed marked differences in serum exosomal miR-10b between breast cancer patients and healthy controls, exhibiting remarkable diagnostic accuracy (AUC = 0.990). This innovative DSA strategy provides a robust platform for ultrasensitive miRNA detection and holds promise for advancing early cancer diagnosis and therapeutic development.

Bst polymerase is a large fragment of DNA polymerase I with strong strand displacement activity and broad substrate adaptability. It is one of the most commonly used enzymes in developing isothermal DNA amplification reactions for trace nucleic acid detection. Although many of these methods are ultrasensitive and specific, they often suffer from complex primer design, multienzyme dependency, and complicated amplification systems relating to unexpected side reactions and false reported signals. In this work, we designed a simple, rapid, and accurate Bst polymerase-catalyzed isothermal exponential amplification reaction (named BIEAR) for multiplex detection of microRNAs and tried it in diabetic retinopathy (DR) screening and progression risk assessment. The BIEAR system relies solely on one DNA hairpin triggering probe, one double-stranded DNA signal probe, and one type of Bst polymerase to drive bidirectional strand extension and displacement reaction cycles, which produce exponentially amplified fluorescence signals quantitatively dependent on the target microRNAs. The system has achieved pM-graded detection limits in individual and multiplex detection of miR-21 and miR-93, and the reaction time for detecting miR-93 is only 10 min. It exhibited high accuracy with 99.20-105.6% recovery rates in measuring miR-21 in human plasma samples. We have also applied BIEAR to detect concentrations of blood circulating miR-21 and miR-93 and combined them as an indicator for screening DR, achieving a diagnostic sensitivity of 93.80%, a specificity of 84.80%, and an area under the curve of 0.952. This novel method provides a simple, convenient, and accurate approach for potential molecular diagnosis such as early screening of DR.

Sensitive and specific imaging of long noncoding RNAs (lncRNAs) in situ in living cells is severely challenged by their lengthy sequences and trace amount. Enzyme-free isothermal nucleic acid amplification methods, particularly hybridization chain reactions (HCRs), have been widely applied for intracellular nucleic acid fluorescence imaging. However, HCR methods suffer from a low linear amplification efficiency and prolonged procedure time, limiting their application in rapid diagnostics. In this work, we propose a synergistic dual hybridization chain reaction (SDHCR) strategy, integrating two rounds of HCRs with dual chain displacement actions (CDAs), tailored for ultrasensitive and enzyme-free detection of lncRNA detection. This SDHCR approach markedly enhances both the amplification dynamics and sensitivity compared to conventional HCR techniques. By delivering SDHCR probes into diverse cancer cell lines via manganese dioxide nanoparticles (MnO₂ nanosponges, MNS), we achieved specific and sensitive imaging of a key lncRNA biomarker, ABHD11-AS1. We posit that the advanced amplification strategy holds significant promise as a potent yet user-friendly tool for both fundamental research and clinical diagnostics.