Recent advancements in nucleic acid-based nanoprobes have revolutionized targeted subcellular imaging, offering unprecedented precision and versatility in visualizing cellular components. This review highlights the design and application of these probes, which leverage their high specificity to bind target molecules, enabling accurate imaging and dynamic tracking of organelles such as mitochondria, lysosomes, and the endoplasmic reticulum. By integrating signal amplification and fluorescence modules, these probes significantly enhance imaging sensitivity, making them invaluable tools in disease diagnosis, drug development, and biomedical research. Furthermore, their cross-disciplinary potential extends to gene expression regulation, disease mechanism elucidation, and personalized therapy. This comprehensive overview underscores the transformative impact of nucleic acid-based probes in advancing analytical chemistry and life sciences, paving the way for future innovations in subcellular imaging and therapeutic applications.

Traditional responsive fluorescent probes are predominantly restricted to qualitative biomarker detection, incapable of delivering real-time quantitative analysis or spatial mapping of protease activity in vivo, which is essential for elucidating disease progression. To overcome this, a ratiometric second near-infrared region (NIR-II) fluorescent (FL) probe (DCNP@IR-806) was developed by conjugating caspase-3-specific peptide substrates and sensitizer molecules (IR-806) to lanthanide-doped down-conversion nanoparticles (DCNP). DCNP@IR-806 achieves single-channel emission at 1550 nm under dual excitation, facilitating self-calibrated quantification and real-time monitoring of activated caspase-3 in vivo. Radiotherapy induces tumor cell apoptosis, thereby activating caspase-3, which subsequently triggers a ratiometric NIR-II FL signal change of DCNP@IR-806. The ratiometric signal demonstrates a linear correlation with caspase-3 concentration, achieving a detection limit of 9.96 U mL −1 . Then, an early efficacy assessment system capable of predicting radiotherapy outcomes within 12 h post-treatment was constructed, markedly expediting evaluation compared to traditional methods that require weeks. This rapid, precise, and user-friendly assessment facilitates timely optimization of therapeutic regimens to enhance efficacy while minimizing side effects. This platform represents a significant advancement in precision oncology by transitioning from qualitative imaging to in situ quantitative biomarker tracking.

Accurate differentiation of benign and malignant breast tumors is paramount for establishing schemes of breast cancer treatment and prognosis. Here we report a near-infrared (NIR) fluorescence probe (YF-1) with the overexpressed cathepsin C (CTSC) in metastatic breast tumors as the detecting substrate. This probe allows accurate identification of malignant tumor tissue specimens among tumor tissue specimens with unknown properties in a blind study. Importantly, a series of visible to NIR CTSC-activated fluorescence probes based on the same strategy realize effective identification of malignant tumor tissues, suggesting that CTSC could be the specific identification substrate of malignant breast tumors. Furthermore, a hydrophilic PEG moiety is coupled into YF-1, producing another CTSC-activated NIR probe (YF-2). YF-2 has excellent tumor-targeting capability, enabling the visualization of lung-metastatic breast tumors. The excellent detection accuracy and construction versatility of CTSC probes pave the way for preoperative diagnosis of malignant breast tumors.

BACKGROUND:Escherichia coli (E. coli) is a model microorganism extensively used in microbial research due to its well-characterized physiology and genetic tractability. Monitoring its physiological indicators, such as metabolic activity and stress responses, is essential for understanding microbial life processes. However, existing methods often lack sensitivity, rapidity, and biocompatibility. This study addresses these limitations by combining electrochemiluminescence (ECL) biosensing with a biocompatible interface, to enable precise and efficient monitoring of E. coli's physiological state. RESULTS:This study presents a novel ECL biosensor that leverages the biocompatibility of bovine serum albumin (BSA) with E. coli. The biosensor employs horseradish peroxidase (HRP) and 4-chloro-1-naphthol (4-CN) to induce a precipitation reaction that quenches the ECL signal of CdS-K2S2O4, enabling the sensitive detection of endogenous hydrogen peroxide (H2O2) produced by E. coli. The sensor exhibits excellent linearity, stability, and reproducibility, with a detection limit of 54 nM for H2O2. Additionally, the biosensor effectively distinguishes between antibiotic-resistant and sensitive strains of E. coli, demonstrating its potential for comprehensive physiological assessment. SIGNIFICANCE:This innovative ECL biosensor represents a significant advancement in microbial monitoring, combining high sensitivity, rapidity, and biocompatibility, offering a powerful tool for comprehensive physiological monitoring in bioanalytical sciences. Its potential applications in biotechnology, food industry, and drug research highlight its practical relevance.

Evaluating tumor radiosensitivity is beneficial for the prediction of treatment efficacy, customization of treatment plans, and minimization of side effects. Tracking the mitochondrial DNA (mtDNA) repair process helps to assess tumor radiosensitivity as mtDNA repair determines the fate of the cell under radiation-induced mtDNA damage. However, current probes developed to monitor levels of DNA repair enzymes suffered from complex synthesis, uncontrollable preparation, limited tumor selectivity, and poor organelle-targeting ability. Especially, the correlation between mtDNA repair activity and inherent radiosensitivity of tumors has not yet been explored. Here, we present a mitochondria-targeted DNA-based nanoprobe (TPP-Apt-tFNA) for in situ monitoring of the activity of the mtDNA repair enzyme and evaluating tumor radiosensitivity. TPP-Apt-tFNA consists of a DNA tetrahedral framework precisely modified with three functional modules on each of the three vertexes, that is, the tumor cell-targeting aptamer, the mitochondrion-targeting moiety, and the apurinic/apyrimidinic endonuclease 1 (APE1)-responsive molecule beacon. Once selectively internalized by tumor cells, the nanoprobe targeted the mitochondrion and specifically recognized APE1 to activate fluorescence, allowing the observation of mtDNA repair activity. The nanoprobe showed elevated APE1 levels in the mitochondria of tumor cells under oxidative stress. Moreover, the nanoprobe enabled the illumination of different levels of APE1-mediated mtDNA repair activity in different cell cycle phases. Furthermore, using the nanoprobe in vitro and in vivo, we found that tumor cells with high activity of mtDNA repair, which allowed them to recover from radiation-induced mtDNA lesions, had low sensitivity to radiation and an unsatisfactory radiotherapy outcome. Our work provides a new imaging tool for exploring the roles of mtDNA repair activity in diverse biological processes and for guiding tumor radiation treatment.

Benefiting from the unique properties of ionizing radiation, such as high tissue penetration, spatiotemporal resolution, and clinical relevance compared with other external stimuli, radiotherapy-induced drug release strategies are showing great promise in developing effective and personalized cancer treatments. However, the requirement of high doses of X-ray irradiation to break chemical bonds for drug release limits the application of radiotherapy-induced prodrug activation in clinics. Recent advances in nanomaterials offer a promising approach for radiotherapy sensitization as well as integrating multiple modalities for improved therapy outcomes. In particular, the catalytic radiosensitization that utilizes electrons and energy generated by nanomaterials upon X-ray irradiation has demonstrated excellent potential for enhanced radiotherapy. In this Review, we summarize the design principles of X-ray-responsive chemical bonds for controlled drug release, strategies for catalytic radiosensitization, and recent progress of X-ray-responsive nanoradiosensitizers for enhanced radiotherapy by integration with chemotherapy, chemodynamic therapy, photodynamic therapy, photothermal therapy, gas therapy, and immunotherapy. Finally, we discuss the challenges of X-ray-responsive nanoradiosensitizers heading toward possible clinical translation. We expect that emerging strategies based on radiotherapy-triggered drug release will facilitate a frontier in accurate and effective cancer therapy in the near future.

MicroRNA-21 (MiR-21) has been confirmed to be upregulated in tumors, and its abnormal expression is closely associated with tumor occurrence. However, the traditional imaging methods are limited to qualitative imaging of miR-21, and no effective strategy has been developed for monitoring its concentration in vivo during cancer initiation and progression. Herein, a biosensor is created utilizing a NIR-II ratiometric fluorescent nanoprobe to quantitatively monitor dynamic alterations in miR-21 levels in vivo. The nanoprobe (termed DCNP@DNA2@IR806) is constructed by introducing IR806 as a donor and down-conversion nanoparticles (DCNP) as the acceptor, using DNA as linkers. Upon miR-21-responsive initiation of the nanoprobe, the 1550 nm fluorescent signal of DCNP stimulated by a 808 nm laser (F1550, 808Ex) increased because of the close proximity of IR806 to the DCNP and the subsequent non-radiative energy transfer (NRET). Meanwhile, the 1550 nm fluorescent signal of DCNP stimulated by a 980 nm laser (F1550, 980Ex) remained stable because of the absence of NRET. This ratiometric NIR-II fluorescent signal has been confirmed to be a reliable indicator of miR-21 concentration in vivo. The strategy holds promise for further enhancing the understanding of microRNAs-based molecular mechanisms underlying cancer progression, laying a foundation for the early diagnosis of microRNAs-related diseases. A miR-21-activated ratiometric NIR-II fluorescence nanoprobe (DCNP@DNA2@IR806) for early diagnosis and real-time monitoring of miR-21 levels during cancer initiation and progression via enhanced ratiometric fluorescence signals in vivo. The content of activated miR-21 is positively correlated with tumor volume in the development of tumors. image

Chemical warfare agents represent a severe threat to mankind and their efficient decontamination is a global necessity. However, traditional disposal strategies have limitations, including high energy consumption, use of aggressive reagents and generation of toxic byproducts. Here, inspired by the compartmentalized architecture and detoxification mechanism of bacterial micro-compartments, we constructed oil-in-water Pickering emulsion droplets stabilized by hydrogen-bonded organic framework immobilized cascade enzymes for decontaminating mustard gas simulant (2-chloroethyl ethyl sulfide, CEES) under sweet conditions. Two exemplified droplet systems were developed with two-enzyme (glucose oxidase/chloroperoxidase) and three-enzyme (invertase/glucose oxidase/chloroperoxidase) cascades, both achieving over 6-fold enhancement in decontamination efficiency compared with free enzymes and >99% selectivity towards non-toxic sulfoxide. We found that the favored mass transfer of sugars and CEES from their respective phases to approach the cascade enzymes located at the droplet surface and the facilitated substrate channeling between proximally immobilized enzymes were key factors in augmenting the decontamination efficacy. More importantly, the robustness of immobilized enzymes enabled easy reproduction of both the droplet formation and detoxification performance over 10 cycles, following long-term storage and in far-field locations.

Oxidative stress, orchestrated by myeloperoxidase (MPO), plays crucial roles in the progression of many diseases. Nonetheless, the role of MPO-mediated oxidative stress in distinct factor-induced acute liver injuries (ALI) is still up for dispute, mainly due to the lack of probes for in vivo monitoring of MPO releases. Here, a highly selective MPO probe (CSQ) based on the epoxidation biochemical reaction within the MPO-H2O2-Cl- system is screened to construct dual near infrared-IIb (NIR-IIb) ratiometric (F-1550Em,F- 808Ex/F-1550Em,F- 980Ex) luminescence nanoprobes by integrating CSQ onto down conversion nanoparticles (DCNPs) with and without liver-targeting moiety (twin NIR-IIb nanoprobes). Liver-targeting probes are employed to monitor MPO release, whereas non-liver-targeting probes are utilized to assess MPO activity across all cell types. Using twin NIR-IIb nanoprobes, the MPO-mediated oxidative stress progressively increased are observed in carbon tetrachloride (CCl4)-induced ALI over 12 h. In contrast, the MPO-mediated oxidative stress in acetaminophen (APAP)-induced ALI initially increased, peaked within 3 h, and then rapidly weakened to normal levels within 12 h. Importantly, the differential release of MPO from neutrophils/Kupfer cells to extracellular fluids in the two types of ALI is revealed. This work reveals significant differences in MPO distribution and the role of MPO-mediated oxidative stress in CCl4 and APAP-induced ALI.

Zero-dimensional (0D) halide perovskites have garnered significant interest due to their novel properties in optoelectronic and energy applications. However, the mechanisms underlying their phase transformations and fluorescence properties remain poorly understood. In this study, we have synthesized a micron-scale 0D perovskite observable under confocal laser scanning microscopy (CLSM). This approach enables us to trace the phase transformation process from 0D to three-dimensional (3D) structures, offering a deeper understanding of the underlying mechanisms. Remarkably, we discovered that this in situ transformation is highly sensitive to water, allowing for label-free fluorescent analysis of trace amounts of water in organic solvents through the phase transformation process. Additionally, we have designed a reusable paper strip for humidity analysis leveraging this sensitivity as an application of the micron scale material. Our findings not only elucidate the physicochemical properties of perovskites but also expand the potential of halide perovskite materials in analytical chemistry.

Helicobacter Pylori infection is drawing increasing attentions in public health, especially the drug resistance problems induced by Single-Nucleotide Variants (SNV). Diagnosis of H. Pylori remains challenging for its requirement in selectivity and sensitivity. Herein an initial check-reexamination strategy is designed for analysis of H. Pylori DNA and SNV. At the first stage, target DNA with all genotypes is captured to form a Y-shaped structure, resulting in an electrochemiluminescence (ECL) signal recovered from quenched states. Then Cas9 assisted cleavage processes are followed to cut off the Y-shaped structure, resulting in corresponding signal decrease. By means of these two stages with different selectivity, both the total amount of H. Pylori DNA and the ratio of SNV can be clarified. To expand its capacity, a large-scale screening assay is carried out on chip. Array detection improves the reliability and the following PCA analysis confirms the otherness. This approach improved the work efficiency and reduced the cost, which may offer an appealing option for the prevention and cure of H. pylori infections in the future.

The abnormal fluctuation of temperature in vivo usually reflects the progression of inflammatory diseases. Noninvasive, real-time, and accurate monitoring and imaging of temperature variation in vivo is advantageous for guiding the early diagnosis and treatment of disease, but it remains difficult to achieve. Herein, we developed a temperature-activated near-infrared-II fluorescence (NIR-II FL) and surface-enhanced Raman scattering (SERS) nanoprobe for long-term monitoring of temperature changes in rat arthritis and timely assessment of the status of osteoarthritis. The thermosensitive polymer bearing NIR-II FL dye was grafted onto the surface of nanoporous core-satellite gold nanostructures to form the nanoprobe, wherein the nanoprobe contains NIR-II FL and Raman reference signals that are independent of temperature change. The ratiometric FL1150/FL1550 and S-1528/S-2226 values of the nanoprobe exhibited a reversible conversion with temperature changes. The nanoprobe accurately distinguishes the temperature variations in the inflamed joint versus the normal joint in vivo by ratiometric FL and SERS imaging, allowing for an accurate diagnosis of inflammation. Meanwhile, it can continuously monitor fluctuations in temperature over an extended period during the onset and treatment of inflammation. The tested temperature change trend could be used as an indicator for early diagnosis of inflammation and real-time evaluation of therapeutic effects.

Background Cell surface enzymes are important proteins that play essential roles in controlling a wide variety of biological processes, such as cell-cell adhesion, recognition and communication. Dysregulation of enzyme-catalyzed processes is known to contribute to numerous diseases, including cancer, cardiovascular diseases and neurodegenerative disease. From the perspective of drug discovery and development, there is a growing interest in detecting the cell surface enzyme activity, propelled by the arising need for innovative diagnostic and therapeutic approaches to address various health conditions. Results In this review, we focus on advances in chemical strategies for the detection of cell surface enzyme activity. Firstly, this comprehensive review delves into the diverse landscape of cell surface enzymes, detailing their structural features and diverse biological functions. Various enzyme families on the cell surface are examined in depth, elucidating their roles in cellular homeostasis and signaling cascades. Subsequently, various biosensors, including electrochemical biosensors, optical biosensors and dual-mode biosensors, used for detecting the cell surface enzyme activity are described. Exemplars are provided to illustrate the mechanisms, limit of detection and prospective applications of these different biosensors. Furthermore, this review unravels the intricate interplay between cell surface enzymes and cellular physiology, contributing to the development of novel diagnostic and therapeutic strategies for various diseases. In the end, the review provides insights into the ongoing challenges and future prospects associated with the detection of cell surface enzyme activity. Significance Detecting cell surface enzyme activity holds pivotal significance in biomedical research, offering valuable insights into cellular physiology and disease pathology. Understanding enzyme activity aids in elucidating signaling pathways, drug interactions and disease mechanisms. This knowledge informs the development of diagnostic tools and therapeutic interventions targeting various ailments, from cancer to neurodegenerative disease. Additionally, it contributes to the advancement of drug screening and personalized medicine approaches.

It remains a challenge to use a single probe to simultaneously detect extracellular pH fluctuations and specifically recognize cancer cells for precise drug delivery. Here, we engineered a tetrahedral framework nucleic acid-based logic nanoprobe (isgc8-tFNA) on live cell membranes for simultaneously monitoring extracellular pH and targeted drug delivery. Isgc8-tFNA was anchored stably on the cell surface through three cholesterol molecules inserting into the bilayer of the cell membrane. Once responding to the acidic tumor microenvironment, isgc8-tFNA formed an i-motif structure, leading to turn-on FRET signals for monitoring changes of extracellular pH. The nanoprobe exhibited a narrow pH-response window and excellent reversibility. Moreover, the nanoprobe could execute logic identification on the cell surface for precise drug delivery. Only if both in the acidic microenvironment and aptamer-targeting marker are present on the cell surface, the sgc8-ASO-chimera strand, carrying an antisense oligonucleotide drug, was released from the nanoprobe and entered into targeted cancer cells for gene silence. Additionally, the in situ drug release facilitated the uptake of drugs mediated by the interaction between sgc8 aptamer and membrane proteins, resulting in enhanced inhibition of cancer cell migration and proliferation. This logic nanoprobe will provide inspiration for designing smart devices for diagnosis of pH-related diseases and targeted drug delivery.

Developing chemiluminescence probe with a slow kinetic profile, even a constant emission within analytical time, would improve the analytical sensitivity, but still remains challenging. This work reports a novel strategy to afford long-lasting in vivo imaging by developing a self-assembled chemiluminophore HPQCL-Cl via the introduction of the hydrogen-bond-driven self-assembled dye HPQ to Schaap's dioxetane. Compared with classical chemiluminophore HCL, self-assembled HPQCL-Cl was isolated from the physiological environment, thereby lowering its deprotonation and prolonging its half-life. Based on HPQCL-Cl, the long-lasting in vivo imaging of 9L-lacz tumor was achieved by developing a β-gal-responsive probe. Its signals remained constant (<5% change) for about 20 min, which may provide a wide time window for the determination of β-gal. This probe also showed high tumor-to-normal tissue ratio throughout tumor resection, highlighting its potential in image-guided clinical surgery.

Accurately quantifying microRNA levels in vivo is of great importance for cancer staging and prognosis. However, the low abundance of microRNAs and interference from the complex tumor microenvironment usually limit the real-time quantification of microRNAs in vivo. Herein, for the first time, we develop an ultrasensitive microRNA (miR)-21 activated ratiometric nanoprobe for quantification of the miR-21 concentration in vivo without signal amplification as well as dynamic tracking of its distribution. The core–satellite nanoprobe by miR-21 triggered in situ self-assembly was built on nanogapped gold nanoparticles (AuNNP probe) and gold nanoparticles (AuNP probe). The AuNP probe generated a photoacoustic (PA) signal and ratiometric SERS signal with the variation of miR-21, whereas the AuNNP probe served as an internal standard, enabling ratiometric SERS imaging of miR-21. The absolute concentration of miR-21 in MCF-7 tumor-bearing mice was quantified to be 83.8 ± 24.6 pM via PA and ratiometric SERS imaging. Our strategy provides a powerful approach for the quantitative detection of microRNAs in vivo, providing a reference for the clinical treatment of cancer.

Helicobacter pylori is closely linked to many gastric diseases such as gastric ulcers and duodenal ulcers. Therefore, biosensing H. pylori has attracted wide attention from both scientists and clinicians. Here, we proposed an electrochemiluminescence (ECL)-based platform that could sensitively detect H. pylori DNA. In this platform, a novel target-cycling synchronized rolling circle amplification was used for signal amplification. Silver nanoclusters (Ag NCs) were synthesized on the circle DNA products, embedding them with the ability to catalyze the electrochemical reduction of K2 S2 O8 , in turn resulting in rapid consumption of the ECL co-reactant near the working electrode, and leading to a decrease in the ECL emission intensity. In addition to its excellent stability and selectivity, the proposed strategy had a low detection limit of 10 pM, an indication that it can be beneficially applied to test biosamples. Furthermore, a biosensing chip was designed to improve the throughput and shed new light on large-scale clinical biosensing applications.

As a crucial indicator in food and water safety testing, the detection of Escherichia coli plays a significant role in maintaining environmental sanitation and promoting public health. Herein, based on the electrochemical activity characteristics of E. coli, we established an enhanced electrochemiluminescence aptasensor for E. coli analysis. This study presents a new method for accurate identification by utilizing a double aptamer recognition system. Specifically, a nano-cadmium sulfide (CdS) modified aptamer was used for primary labeling, while a second aptamer was immobilized on a graphene/chitosan composite electrode for re-capture. The use of two aptamers improves the accuracy of the identification process. Furthermore, the application of an electrode potential facilitates continuous electron transfer between the electrode and electrochemically active microorganisms, resulting in an enhanced electroluminescence signal in relation to the metabolic status. This strategy possesses better sensitivity, accuracy, and stability, demonstrating its potential for E. coli analysis.

Small molecule aptamers discovered by traditional selection methods usually lack conformational changes upon target binding. This limits the use of aptamers as molecular probes for small molecule detection and regulatory elements of genetic circuits. Here, we report a new method called capture and in vitro transcription-systematic evolution of ligands by exponential enrichment (CIVT-SELEX) to select DNA aptamers that can not only bind to small molecule ligands but also undergo significant conformational changes. Through this method, we select a structure-switching aptamer of uridine-5′-diphosphate (UDP). Taking advantage of its conformational changes, we first construct a UDP-responsive transcriptional switch by inserting the aptamer in a genetic circuit and demonstrate that it can respond to the addition of UDP and regulate the transcription of downstream genes. We also build a UDP aptamer-based biosensor that can be used for active glycosyltransferase screening. We believe this method can provide a universal platform for selecting small molecule aptamers with conformational changes and expand the use of aptamers in small molecule detection and genetic regulation.