Genetically encoded sensors have been extensively utilized for the analysis of biomarkers. However, their applications remain limited by the scarcity of analyte-binding proteins or peptides and the challenges associated with translating analyte recognition into a quantifiable signal. Herein, we report a novel class of activable chimeric sensors that harness bioluminescent resonance energy transfer (BRET) between nanoluciferase proteins and fluorescent small-molecule probes. We demonstrated that the responsiveness of these hybrid sensors can be significantly enhanced by modulating the electrostatic surface potential of the fusion protein in the vicinity of the recognition site. As proof of concept, we engineered two chimeric bioluminescent sensors, Bp7-pH and Bp7-NO, for the detection of pH and nitric oxide (NO), respectively. Both sensors exhibited strong BRET signal modulation upon exposure to their target analytes, leading to distinct and quantifiable shifts in luminescence color. Importantly, we successfully applied these sensors for field-deployable imaging of pH and NO dynamics in a murine wound infection model. Given the vast array of existing fluorescent molecular sensors, the detection targets of these chimeric bioluminescent sensors can be easily modified by altering the recognition moieties. Collectively, this work establishes a generalizable and tunable design strategy for developing highly sensitive chimeric bioluminescent sensors, with broad applicability in physiological and pathological monitoring. (c) 2026 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

BACKGROUND:Excessive accumulation of microplastics (MPs) inside the body can induce serious diseases, and the potential pathological mechanism was related to endoplasmic reticulum (ER) stress induced by the surge of intracellular reactive oxygen species (ROS). However, until now, the invasion of MPs triggering this unique process remains elusive. RESULTS:Herein, peroxynitrite (ONOO-), was selected as the model target, and an ER-targeted fluorogenic probe (ER-NA-PB) was constructed for real-time visualizing the fluctuation of ONOO- during cellular ER stimulated by MPs. ER-NA-PB exhibited excellent sensitivity (15.2 nM) and selectivity toward ONOO- in aqueous solution, and the exogenous and endogenous ONOO- fluctuation in ER also can be visualized by this potently fluorescent tracing tool. In addition, ER-NA-PB revealed that the content of ONOO- in ER during stimulation of MPs was elevated with increase of MPs concentration and prolongation of stimulation time. SIGNIFICANCE:For the first time, the fluctuation of active molecule in embryonic tissue was revealed by fluorescence probe, and this research provides a promising tool for deeper studying the pathological mechanisms underlying relevant diseases caused by MPs pollution inducing ER stress.

Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.

High signal-to-background ratio (SBR) monitoring of formaldehyde (FA) delivery in living systems is of great significance for the precise study of the physiological and pathological functions of FA. However, traditional FA donors mainly use fluorescent signals to monitor FA release, which hampers the high-performance bioimaging due to the undesirable background autofluorescence, leading to poor SBR and compromised imaging sensitivity. Herein, we present the first bioluminogenic donor B-FAD for esterase-activated FA release with high contrast bioluminescence imaging. By protecting the hydroxyl group of D-luciferin with acetoxymethyl ether group, B-FAD cannot be catalyzed by luciferase to emit light. In the presence of esterase, the ester bond of B-FAD breaks, producing FA and D-luciferin. B-FAD selectively and sensitively releases FA in the presence of esterase, accompanied by 34-fold bioluminescence enhancement. In addition, B-FAD has been successfully applied for FA delivery and bioluminescence imaging in living cells, with a SBR about 50 times higher than those obtained by fluorescence imaging. Therefore, in addition to providing an effective tool for imaging FA delivery in living cells, this work establishes a general approach for high contrast monitoring other reactive biological species release by easily changing the responsive unit on D-luciferin.

Enzymes are an important tool used for signal amplification in biosensing. However, traditional amplification methods based on enzymes are always dependent on their catalytic activities, so their signals fluctuate with the change of microenvironment (e.g., pH and temperature). In this work, we communicate an activity-independent enzyme-powered (AIEP) amplification strategy for biosensing to improve signal stability and fidelity. To verify this hypothesis, the monitoring of oxidative stress during drug-induced liver injury was carried out. Carboxylesterase (CEs), highly expressed in hepatic tissue, was selected as the amplification tool. A CEs configuration-matching fluorophore (CMF) was designed and screened, and a nanobeacon was fabricated by loading CMF within an O(2)( center dot--)responsive polymeric micelle. Since the degradation of the nanobeacon was triggered by O-2( center dot--), CMF was released to bind with CEs, and the fluorescence was lit by CEs-CMF configuration matching but not catalytic reaction. Results demonstrated that the oxidative stress during drug-induced liver injury could be successfully monitored, and the hepatoprotective effects of repair drugs could be evaluated by cell and in vivo imaging. This strategy is flexible for bioactive molecules by altering the responsive unit and generally accessible for pharmacological evaluation.

ATP,a small molecule with high intracellular concentration (mMlevel), provides a fuel to power signal amplification, which is meaningfulfor biosensing. However, traditional ATP-powered amplification isbased on ATP/aptamer recognition, which is susceptible to the complexbiological microenvironment (e.g., nuclease). In this work, we communicatea signaling manner termed as ATP-specific polyvalent hydrogen binding(APHB), which is mimetic to ATP/aptamer binding but can avoid interferencefrom biomolecules. The key in APHB is a functional fluorophore thatcan selectively bind with ATP via polyvalent hydrogen, and the fluorescencewas lighted with the changes of the molecular structure from flexibilityto rigidity. By designing, synthesizing, and screening a series ofcompounds, we successfully obtained an ATP-specific binding-lightedfluorophore (ABF). Experimental verification and a complex analoguedemonstrated that two melamine brackets in the ABF dominate the polyvalenthydrogen binding between the ABF and ATP. Then, to achieve amplificationbiosensing, fibroblast activation protein (FAP) in activated hepaticstellate cells was taken as a model target, and a nanobeacon consistingof an ABF, a quencher, and an FAP-activated polymer shell was constructed.Benefiting from the ATP-powered amplification, the FAP was sensitivelydetected and imaged, and the potential relationship between differentiationof hepatocytes and FAP concentration was first revealed, highlightingthe great potential of APHB-mediated signaling for intracellular sensing.

Fluorescent probes have emerged as powerful tools for the detection of different analytes by virtue of structural tenability. However, the requirement of an excitation source largely hinders their applicability in point-of-care detection, as well as causing autofluorescence interference in complex samples. Herein, based on bioluminescence resonance energy transfer (BRET), we developed a reaction-based ratiometric bioluminescent platform, which allows the excitation-free detection of analytes. The platform has a modular design consisting of a NanoLuc-HaloTag fusion as an energy donor, to which a synthetic fluorescent probe is bioorthogonally labeled as recognition moiety and energy acceptor. Once activated by the target, the fluorescent probe can be excited by NanoLuc to generate a remarkable BRET signal, resulting in obvious color changes of luminescence, which can be easily recorded and quantitatively analyzed by a smartphone. As a proof of concept, a fluorescent probe for HOCl was synthesized to construct the bioluminescent system. Results demonstrated the system showed a constant blue/red emission ratio which is independent to the signal intensity, allowing the quantification of HOCl concentration with high sensitivity (limit of detection (LOD) = 13 nM) and accuracy. Given the universality, this reaction-based bioluminescent platform holds great potential for point-of-care and quantitative detection of reactive species.

Mitophagy is the lysosome-dependent degradation of damaged and dysfunctional mitochondria, which is closely associated with H2O2-related redox imbalance and pathological processes. However, development of fast-responding and highly sensitive reversible fluorescent probes for monitoring of mitochondrial H2O2 dynamics is still lacking. Herein, we report a reversible fluorescent probe (M-HP) that enables real-time imaging of H2O2-related redox imbalance. In vitro studies demonstrated that M-HP had a rapid response and high sensitivity to the H2O2/GSH redox cycle, with a detection limit of 17 nM for H2O2. M-HP was applied to imaging of H2O2 fluctuation in living cells with excellent reversibility and mitochondrial targeting. M-HP reveals an increase in mitochondrial H2O2 under lipopolysaccharide stimulation and a decrease in H2O2 following the combined treatment with rapamycin. This suggests that the level of oxidative stress is significantly suppressed after the enhancement of mitophagy. The rationally designed M-HP offers a powerful tool for understanding redox imbalance during mitophagy.

As a highly effective tool with a superior signal-to-noise ratio, chemiluminescent probes find extensive utility in the in vivo detection of diverse biochemical aspects, including inflammation and the tumor microenvironment. However, the widespread adoption of chemiluminescence detection is hindered by the requirement of large precision instruments, thereby limiting its application scope. Consequently, further development and improvement of the chemiluminescence detection method are imperative. This study presents a novel chemiluminescent probe that enables precise detection of β-galactosidase (β-gal) both in vitro and in vivo by modifying the specific cleavage recognition groups of β-gal to the hydroxyl group of the dioxane-based chemiluminescent scaffold. In comparative tests against traditional chemical probes, this probe exhibited an exceptional signal-to-background ratio that significantly surpasses its counterparts. To enhance detection capabilities, a customized Point-of-Care Testing (POCT) instrument specifically designed for chemiluminescence detection was developed. This compact, user-friendly, and cost-effective instrument demonstrated comparable performance to large precision instruments. Notably, the structural characteristics of the probe, utilizing the 'hydroxyl deprotection' strategy, allow for convenient, real-time, and accurate detection of diverse targets by modifying the recognition groups tailored to specific targets, in conjunction with the POCT instrument. This research introduces a pioneering approach for generating optical signal tests in the laboratory using chemiluminescent probes, thereby unlocking the potential for rapid clinical detection and self-diagnosis of certain diseases in a home setting.

基于脱氧核酶（DNAzyme）的基因治疗作为一种新兴的肿瘤治疗手段，显示出广阔的应用前景和一定的临床实用价值。然而，单独的DNAzyme难以跨越细胞膜；采用脂质体、核酸自组装纳米结构等作为载体递送存在制备复杂、负载效率低等缺陷。此外，DNAzyme还需要较高浓度的金属离子作为辅酶因子，细胞中金属离子不足阻碍了其治疗效果。针对以上挑战，受金属-有机配位驱动自组装启发，本工作利用锰离子与核酸之间的配位作用，构筑了一类新型的多功能B-Mn-DNAzyme纳米颗粒用于肿瘤细胞的协同杀伤。采用一锅法制备得到的Mn-DNAzyme纳米颗粒具有80.6%的DNAzyme负载率，且在磷酸盐缓冲液中能降解释放锰离子和DNAzyme。释放的DNAzyme以锰离子为辅酶因子，催化裂解mRNA，下调疾病相关蛋白达到基因沉默效果。锰离子可与内源性过氧化氢发生类芬顿反应产生羟基自由基杀伤肿瘤细胞，实现化学动力学治疗。通过进一步包覆肿瘤细胞膜得到的B-Mn-DNAzyme能高效递送DNAzyme至肿瘤细胞，实现肿瘤细胞基因沉默和化学动力学的协同杀伤，为肿瘤治疗提供有效。

To achieve the goals of scientific research on back-feeding teaching and teaching innovation to promote personnel training, reforming traditional chemical experiments is necessary.Based on previous scientific research achievements of the research team, a comprehensive chemical experiment was designed to synthesize and characterize fluorescent copper nanoclusters using DNA molecules as templates, and the conditions for stable synthesis of fluorescent copper nanoclusters were obtained.The experiment included the synthesis of nanoclusters, use of basic spectral analysis instruments, and spectral data processing.The experiment integrates the frontier knowledge of analytical chemistry, biochemistry, and nanoscience, among others.The experimental conditions were simple and mild, and the experimental duration was reasonable, meeting the requirements of experimental teaching, stimulating students' interest in scientific research, enhancing their comprehensive ability, and promoting experimental teaching.

The development of amplification strategies is one of the central challenges for detection of low-abundance targets. One-to-many (1:M) amplification strategies in which one target lights many signal probes, has improved the detection sensitivity in bulk solution, but with discounted contrast in cell imaging, because the lighted probes are dissociative and dispersible. In this work, a one-to-large (1:L) signaling mechanism, in which the lighted probes were orderly connected to each other, was conceptually proposed to enhance the contrast in cell imaging by avoiding signal dispersion in amplification. Accordingly, target-triggered hairpin-free chain-branching assembly (HFCBA) holds great potential to implement the 1:L mechanism, but using it in cell imaging has yet to be demonstrated. As a proof of concept, a group of probes were first programmed to implement miRNA-21-triggered HFCBA. After transfection of probes, gradually-growing signal flares in cells were monitored along with the growth of DNA dendrimers; and the in situ fluorescence accumulation in HFCBA resulted in highly-enhanced contrast to the surrounding by avoiding signal dispersion in amplification. The contrast-enhanced imaging with signal amplification is significant for biological analysis and molecular medicine. We expect the 1:L mechanism will provide a new thought for high-performance imaging of biomarkers in cells.

In situ signal amplification strategy is an effective mean to detect low-abundance of endogenous targets. However,most of signal amplification strategies rely on exogenous substances,which inevitably change the intracel-lular microenvironment and causing certain interference to the body. For this problem,it has been reported that using endogenous substances,such as metal ions,nucleic acids and proteases,can achieve in situ fluorescence signal amplification. Fluorescence imaging of different biomarkers is of great significance for the imaging of low-abundance target molecules. In this review,we summarize the related researches on endogenous substance-assisted signal ampli-fication and the application of cell imaging. Firstly,we introduce endogenous nucleic acid,enzyme,protein,ATP and metal ion-assisted signal amplification strategies and discuss their signal amplification mechanisms. Then,the research progress of endogenous substance-assisted signal amplification probes in the detection and imaging of low-abundance substances was summarized. Finally,we look forward to the advantages and application prospects of this strategy in cell imaging.

过氧化氢(H2O2)是一种重要的活性氧物种,广泛存在于生物体内及食品、药品生产过程中.生物体内H2O2作为第二信使,参与细胞的生长、增值、分化、迁移等生理过程.然而,异常浓度的H2O2会对机体造成损害,导致各种疾病的发生.H2O2作为漂白剂、消毒剂等已被广泛用于食品生产领域,由此也带来潜在危害.因此,开发高效的H2O2检测手段对于生命健康具有重要意义.荧光探针因具有结构可调,快速原位检测及良好的生物相容性等优势而被广泛用于复杂体系中H2O2的实时检测.在这篇综述中,重点介绍H2O2荧光探针的设计思路及其在生物、食品检测中的应用进展,并探讨该领域面临的挑战与发展方向.

Epilepsy is a neurological brain disease, and its recurrent seizures are related to the reductive substance-powered antioxidant defense system (ADS). However, until now, there has been no report on the study of in situ antioxidant fluctuation during epilepsy of varying severity. In this work, hydrogen sulfide (H2S) was selected as the model target, a H2S-responsive near-infrared fluorophore was designed and synthesized, and an amphiphilic molecule was synthesized and modified with angiopep-2 peptide at its hydrophilic terminus. A nanobeacon termed as BFPP was prepared by the formation of micelles with the package of the fluorophore. The nanobeacon was sensitive to H2S, with a low detection limit of 17 nM. The H2S fluctuation in cells can be monitored by fluorescence imaging. In addition, angiopep-2 peptide at the surface of BFPP helps it cross the blood-brain barrier, and near-infrared fluorescence improves in vivo imaging. BFPP revealed that H2S was at a moderate level in the normal brain, but its level was obviously elevated during mild epilepsy because of the activation of the ADS while significantly suppressed during severe epilepsy due to neuronal damage. This approach is generally accessible for other targets by altering the responsive fluorophore, with significance for in situ analysis of brain pathology.

For sensing low abundance of biomarkers, utilizing nanocarriers to load dyes is an efficient method to amplify the detected signal. However, the non-specific leak of the internal dyes in this approach is accompanied by false positive signals, resulting in inaccurate signal acquirement. To address this issue, in this work, we reported a novel signal amplification strategy with dye as a scaffold to construct a self-immolative dye-doped polymeric probe (SDPP). In our proposed approach, the dyes were covalently integrated into the main chain of a polymer, which can avoid the non-specific leak of the dye when used in a rigorous biological environment, thus evading the false positive signal. As a prototype of this concept, a SDPP, which responds to hydroxyl radicals (•OH), was rationally fabricated. Upon being activated by •OH, SDPP will liberate the dye through a self-immolative reaction to bind with protein for amplifying the fluorescence signal. Compared with a dye-loaded nanoprobe, SDPP can precisely track intracellular basal •OH levels and visualize the •OH associated with myocarditis in vivo. More importantly, the attempt in this work not only provides an effective molecular tool to investigate the role of •OH in cardiopathy, but also puts forward a new direction to current signal-amplifying strategies for precisely and reliably acquiring the intracellular molecular information.

The aim of this study was to compare the effects between calcium channel blockers and diuretics when used in combination with angiotensin II receptor blocker on aortic systolic blood pressure (BP) and brachial ambulatory systolic BP. We conducted a prospective, randomized, open-label, blinded end point study in 207 hypertensive patients (mean age: 68.4 years). Patients received olmesartan monotherapy for 12 weeks, followed by additional use of azelnidipine (n=103) or hydrochlorothiazide (n=104) for 24 weeks after randomization. The central BP by radial artery tonometry, aortic pulse wave velocity, and ambulatory BP were assessed at baseline and 24 weeks later. After adjustment for baseline covariates, the extent of the reduction in central systolic BP in the olmesartan/azelnidipine group was significantly greater than that in the olmesartan/hydrochlorothiazide group (the between-group difference was 5.2 mm Hg; 95% CI: 0.3 to 10.2 mm Hg; P =0.039), whereas the difference in the reduction in brachial systolic BP between the groups was not significant (2.6 mm Hg; 95% CI: −2.2 to 7.5 mm Hg; P =0.29). The aortic pulse wave velocity showed a significantly greater reduction for the olmesartan/azelnidipine combination than for the olmesartan/hydrochlorothiazide combination (0.8 m/s; 95% CI: 0.5 to 1.1 m/s; P <0.001) after adjustment for covariates. The extent of the reduction in brachial ambulatory systolic BP was similar between the groups. These data showed that the combination of olmesartan (20.0 mg) and azelnidipine (16.0 mg) had a more beneficial effect on central systolic BP and arterial stiffness than the combination of olmesartan (20.0 mg) and hydrochlorothiazide (12.5 mg), despite the lack of a significant difference in brachial systolic BP reduction between the 2 treatments.

The novel theranostic nanosystems based on two-photon fluorescence can achieve higher spatial resolution of deep tissue imaging for simultaneous diagnosis and therapy of a variety of cancers. Herein, we have designed and prepared FRET-based two-photon mesoporous silica nanoparticles (MTP-MSNs) for single-excitation multiplexed intracellular imaging and targeted cancer therapy for the first time. This nanosystem includes two constituents, containing (1) multicolor two-photon mesoporous silica nanoparticles and (2) cancer cell-targeting aptamers that act as gatekeepers for MTP-MSNs. After incubation with cancer cells, the Dox-loaded and aptamer-capped MTP-MSNs could be internalized into the cells, opening the pores and releasing the drug. Furthermore, using two-photon multicolor fluorescence, MTP-MSNs could serve as good contrast agents for multicolor two-photon intracellular imaging with increased imaging depth and improved spatial localization of tissue. In sum, these multicolor MTP-MSNs provide a promising system for traceable targeted cancer therapy with further applications in multiplex intracellular imaging and the screening of drug.

Long-term specific tracing of the fibroblast activation protein (FAP) has been of great importance because it is heavily expressed by stromal fibroblasts of multiple diseases, and several disorders associated with FAP are chronical. Bioluminescence (BL) imaging has its advantages to detect FAP in vivo since no external excitation is required, but the current FAP-responsive BL probe was constructed by covalently masking the firefly luciferase substrate and easily secreted out from the animal, resulting in transient BL imaging of FAP. To circumvent this problem, a peptide-linked amphiphilic block copolymer-based probe (PABC) was developed and applied to the long-lasting BL image of FAP in vivo. For this purpose, an amphiphilic block copolymer containing an FAP-responsive peptide was fabricated to self-assemble into micelles, which act as a depot to load amounts of d-luciferin for constructing the BL probe. Upon reaction with FAP, the micelle would be destroyed to release the internal d-luciferin for BL emission by a luciferase-catalyzed reaction. By virtue of the high loading capability of micelles, the FAP was determined from 0.5 to 10 ng/mL with a detection limit of 0.105 ng/mL, and the high sensitivity makes the PABC capable of distinguishing cancer cells from normal ones. Importantly, compared with free d-luciferin, PABC can be used to persistently image the FAP in living cells and in vivo. This characteristic of long-lasting specific tracing of the FAP makes us envision that this BL probe could be used for screening of FAP inhibitors and diagnosing various FAP-related diseases in future.

大脑是中枢神经系统的主要组成部分,具有极为复杂的结构和功能.大脑中的生物活性分子,包括活性氧、活性硫、蛋白质等,在大脑生理病理过程中发挥着重要作用.实时、原位检测脑内生物活性分子对于脑疾病(包括脑血管疾病、神经退行性疾病和脑肿瘤等)的预防、早期诊断及有效治疗具有重要意义.小分子荧光探针因具有结构可调,良好的生物相容性及快速原位成像等优势而被广泛用于生命系统中活性分子的实时检测.在这篇综述中,重点介绍用于脑原位成像的小分子荧光探针的设计思路及应用进展,并探讨该领域面临的挑战与发展方向.