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Heat shock seriously affects the normal functioning of an organism and can lead to damage and even death in severe cases. To prevent or treat heat shock-related diseases, we require a better understanding of the mechanism of thermocytotoxicity. Here, we designed a functionalized dual-response fluorescent probe ( HCy-SO 2 -HClO ) that could individually or simultaneously detect hypochlorous acid (HClO) and sulfur dioxide (SO 2 ) without interfering with each other and achieved the simultaneous tracing of both during the heat shock process for the first time. The introduction of the sulfonate group greatly increased the water solubility of the probe. Furthermore, the probe could effectively accumulate in the mitochondrial region. HCy-SO 2 -HClO could respond to HClO and SO 2 within 10 s and 20 min, respectively, realizing a double whammy detection of both on the time scale. HCy-SO 2 -HClO exhibited high specificity and sensitivity for HClO and SO 2 . The highly biocompatible probe HCy-SO 2 -HClO successfully achieved the detection of endogenous and exogenous SO 2 and HClO in living cells and in zebrafish. Moreover, the simultaneous detection of HClO and SO 2 in heat shock cells and mouse intestines was realized for the first time. This probe has achieved the detection of dual markers, which enhanced the accuracy and precision of disease detection and could serve as an effective research tool to prevent heat stroke-related diseases.

Mitochondria play a pivotal role in maintaining normal physiological functions. Mitochondrial autophagy, namely, mitophagy, is a selective catabolic disposal of impaired mitochondria through an autophagic mechanism during episodes of mitochondrial harm. This selective removal, e.g., mitophagy, is essential for mitochondrial quality control and is closely related to the pathogenesis of many diseases. The abnormal buildup of defective mitochondria in vivo was used as a target to prevent the development of cancer. The mitochondrial autophagy process of disease-related cells is usually accompanied by a decrease in polarity and pH, and the fluorescence sensing effects caused by these two factors are usually contradictory. Here, we propose a reinventing strategy to develop a dual-channel and dual-responsive fluorescent probe HDTVB that is capable of tracking mitochondrial autophagy by monitoring fluctuations in mitochondrial pH and polarity. Based on the aggregation-induced emission (AIE) moiety and hemicarpine moiety push-pull system with activated near-infrared (NIR) emission and pH-activatable cyclization reaction, HDTVB was able to differentiate tumors from normal sites via polarity- and acidity-triggered structural changes of the probe in the course of mitochondrial autophagy. HDTVB is expected to be applied to clinical diagnosis and tumor excision guided by fluorescence, offering a new route in physiological and biochemical research.

There is a potential synergistic effect between nonsteroidal anti-inflammatory drugs and hydrogen sulfide (H 2 S), but direct evidence for the study is lacking. With a single fluorescence detection method, it is difficult to accurately confirm the effectiveness of the synergistic effect. In this study, the fluorescent probe and the nonsteroidal anti-inflammatory drug naproxen were combined via different self-immolative spacer groups to obtain a diagnostic and therapeutic integrated fluorescent probe Nap-NP-NSB , which can release H 2 S. The quantitative release of H 2 S by Nap-NP-NSB was evaluated in vitro and in cells, and the synergistic effect of H 2 S and naproxen was confirmed by monitoring the treatment process of cellular inflammation and oxidative damage of gastric mucosa cells. Finally, in vivo fluorescence imaging and mass spectrometry imaging of the liver and stomach tissues and their sections were performed in the mouse model of acute hepatitis. The dual-modal detection method not only confirmed that Nap-NP-NSB had better anti-inflammatory activity and less gastric mucosal damage, but also enabled a more accurate visualization of the drug synergistic effect of naproxen and H 2 S. This work provides a dual visualization imaging method combining fluorescence and mass spectrometry imaging and develops a new idea for studying drug synergies based on self-immolative structures.

Drug-induced liver injury (DILI) is a major hepatic dysfunction commonly caused by hepatotoxic drug overdose, resulting in a considerable number of fatalities worldwide. Recent studies have highlighted the regulatory and hepatoprotective effects of carbon monoxide (CO) during the liver injury process. However, precisely tracking the dynamic changes in the composition of CO in DILI is still a great challenge. In this work, leveraging the innovative "quencher-insertion" strategy, a unique endoplasmic reticulum (ER)-targetable lanthanide complex-based luminescence probe, ER-ANBTTA-Eu 3+ /Tb 3+ , has been developed for the selective and accurate monitoring of CO fluxes in live cells and laboratory animals. The new probe is composed of three covalently linked functional moieties: the terpyridine polyacid-Eu 3+ /Tb 3+ -mixed chelates as the long-lived luminophore, a p -toluenesulfonamide moiety as the ER-anchoring motif, and an allyloxy-nitrobenzyl ether moiety as the CO-specific recognition unit. Upon reaction with CO in the presence of Pd 2+ ions, the Tsuji-Trost reaction leads to the cleavage of the allyloxy-nitrobenzyl group from the Eu 3+ /Tb 3+ -mixed chelates, which results in the restoration of Tb 3+ emission at 538 nm and the attenuation of Eu 3+ emission at 688 nm, leading to a dramatic increase of the I 538 / I 688 ratio. In addition to the exceptional response sensitivity and selectivity toward CO, ER-ANBTTA-Eu 3+ /Tb 3+ also exhibits the outstanding ER-locating capability, which allows the probe to be used for imaging of CO in the ER of live cells. Using this probe, combined with the time-gated luminescence imaging mode, the exogenous and endogenous CO in ER of live cells were monitored without the interference of background autofluorescence. Moreover, the upregulation of hepatic CO in DILI mice was successfully visualized. The results suggested the potential of ER-ANBTTA-Eu 3+ /Tb 3+ for deeply exploring the functions of CO in DILI pathogenesis.

The visualization and spatiotemporal monitoring of endogenous esterase activity are crucial for clinical diagnostics and treatment of liver diseases. Our research adopts a novel substrate hydrolysis-enzymatic activity (SHEA) approach using dicyanoisophorone-based fluorogenic ester substrates DCIP-R (R = R1-R6 ) to evaluate esterase preferences on diverse substrate libraries. Esterase-mediated hydrolysis yielded fluorescent DCIP-OH with a nanomolar detection limit in vitro . These probes effectively monitor ester hydrolysis kinetics with a turnover number of 4.73 s -1 and catalytic efficiency ( k cat / K m ) of 10 6 M -1 s -1 ( DCIP-R1 ). Comparative studies utilizing two-photon imaging have indicated that substrates containing alkyl groups ( DCIP-R1 ) as recognition elements exhibit enhanced enzymatic cleavage compared to those containing phenyl substitution on alkyl chains ( DCIP-R4 ). Time-dependent variations in endogenous esterase levels were tracked in healthy and liver tumor models, especially in diethylnitrosamine (DEN)-induced tumors and HepG2-transplanted liver tumors. Overall, fluorescence signal quantifications demonstrated the excellent proficiency of DCIP-R1 in detecting esterase activity both in vitro and in vivo , showing promising potential for biomedical applications.

To develop highly effective molecular tools for intravital imaging of hypochlorous acid (HOCl), in this study, we initially designed two-photon hybrid fluorophores, SDP and P-SDP , by conjugating the classical dye 2-(2'-hydroxyphenyl)benzothiazole with the two-photon hydroxylphenyl-butadienylpyridinium fluorophore. The designed fluorophores exhibit a synergistic interaction between excited-state intramolecular proton transfer and intramolecular charge transfer mechanisms, enabling near-infrared (NIR) emission and significant Stokes shifts. Subsequently, using these fluorophores, we developed two HOCl fluorescent probes, SDP-SN and P-SDP-SN , by further incorporating N , N -dimethylthiocarbamate as a specific recognition group for HOCl. Toward HOCl, both SDP-SN and P-SDP-SN demonstrate an ultrafast response (less than 3 s), NIR emission at wavelengths of 714 and 682 nm, and remarkable Stokes shifts of 303 and 271 nm, respectively. Leveraging these advantages in conjunction with their ability to cross the blood-brain barrier, the probes find successful application in two-photon cellular and intravital imaging of HOCl. This includes visualizing endogenous generation of HOCl in cellular models related to inflammation, hyperglycemia, and ferroptosis, as well as mapping in vivo generation of HOCl within the brain and abdominal cavity using a murine model of systemic inflammation.

Allostery is a phenomenon where the binding of a ligand at one allosteric site influences the affinity for another ligand at an active site. Different from orthosteric regulation, it allows for more precise control of biomolecular activity and enhances the stability of the molecules. Inspired by allosteric regulation of natural molecules, we present a Y-shaped allosteric DNA nanodevice, termed YssAP, that was pH-responsive and functionalized with the AS1411 aptamer for accurate fluorescence imaging of human apurinic/apyrimidinic endonuclease (APE1) activity in tumor cells. With rational design, YssAP could not be cut by APE1, and Cy5 was in the proximity of BHQ2, leading to suppressed signal emission. In contrast, since acidic pH acted as an allosteric effector, YssAP underwent a conformational change into an activated DNA probe (YdsAP) at acidic extracellular pH. After entering the tumor cell via the specific recognition of AS1411 aptamer, the overexpressed APE1 in the tumor cell cut the AP site on YdsAP. Cy5 moved far away from BHQ2, resulting in a strong signal output. Compared with the direct construction of the APE1 substrate, allosteric DNA nanodevices have more accurate imaging effects, which can be precisely adjusted by changing the switching state. We anticipate that this strategy will be applied in the screening of APE1 inhibitors and precise tumor diagnosis.

Maintaining tissue homeostasis necessitates the coordinated efforts of various cell types to regulate inflammation. Endoplasmic reticulum (ER) stress, a hallmark of inflammation, exacerbates tissue pathology in various human diseases. Glutathione (GSH), a pivotal regulator of cellular redox balance, controls disulfide bond formation in the ER, thereby shielding cells from oxidative stress. In this study, we developed a two-photon fluorescent probe, ER-GSH, with specific ER targeting and demonstrated its high sensitivity and rapid response to GSH. Experiments conducted on BV2 cells and a mice model of neuroinflammation induced by scrap leather revealed that inflammatory reactions led to ER stress and a substantial reduction in GSH levels. Notably, the anti-inflammatory drug NS-398 effectively inhibited cell inflammation and ER stress by maintaining GSH levels. These findings underscore the potential therapeutic significance of modulating GSH levels to alleviate the impact of neuroinflammation.

Tracking the dynamic distribution of native proteins in living cells or tissues in real time is essential for understanding the functional mechanisms involved in their physiological or pathological processes. The β3-adrenergic receptor (β3-AR) has important biological functions. It is expected to be a diagnostic indicator for aging, yet current probes for β-ARs are unable to dynamically monitor β3-AR in real time, which impedes the progress of β3-AR research. Here, we developed a ligand-directed anchoring probe, β3-ARP , that precisely covalently anchors with native β3-AR in living cells, for the diagnosis of senescence. The ligand-directed anchoring probe selectively recognizes and traces β3-AR and can achieve dynamic observation and monitoring of β3-AR in living cells, including the interaction between lipid droplets and mitochondria. Moreover, we were able to directly probe the distribution of β3-AR in various organs of aging mice in situ and track the location of native β3-AR in different types of primary neuronal cells using β3-ARP and two-photon imaging, which revealed the aberrant accumulation of lipid droplets and the distribution of β3-AR in neurological diseases. This research has resulted in a new covalently anchoring fluorescent probe, β3-ARP , which could serve as a powerful tool to explore the link between β3-AR and age-related diseases.

Here we report the application of chemometric analysis for modeling absorbance spectroscopy and fluorescence emission data from a resazurin-based assay targeting low-level bacterial detection in biofluids. Bacteria spiked samples were incubated with resazurin and absorbance and fluorescence data were collected at 30 min intervals. The absorbance data was subjected to Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) and compared with the univariate fluorescence spectroscopy approach. The analysis demonstrated the multidimensional nature of the absorbance data, highlighting the appearance of the resorufin peak at the 2 h time point with a low bacterial inoculum of 0.01 CFU mL -1 across all the samples tested-water, urine and serum. The PLSR models supported the PCA data and exhibited strong predictive capabilities for water ( R C 2 = 0.937, R CV 2 = 0.934), urine ( R C 2 = 0.899, R CV 2 = 0.880) and serum ( R C 2 = 0.985, R CV 2 = 0.967). Conversely, fluorescence is contingent upon resorufin existence, necessitating a prolonged waiting period postincubation with resazurin to verify the presence of bacteria, especially when contamination levels are low. Given the substantial global impact of bacteria-related infections, this method detects bacteria at low concentrations precisely and rapidly, improving efficiency and adaptability for point-of-care settings, promising swift diagnosis of bacterial infections, environmental monitoring, or food-quality control.

The colorimetric point-of-care test (POCT) offers a rapid and efficient method for detecting specific targets in real samples. However, traditional colorimetric methods often rely on complex signal amplification techniques or electronic devices to enhance detection sensitivity, which can inadvertently increase both cost and time, thus contradicting the fundamental goals of visual detection methods. Here, we presented a distance-based fluorescent immunosensor that utilized a gas-producing nanozyme for continuous gas production reaction as a signal. Specifically, the SOM-ZIF-8@Pt nanozyme catalyzed the production of O 2 from H 2 O 2 to cause an obvious increase in the pressure within a sealed chamber, thus driving the production of H 2 S to quench the fluorescence of CsPbBr 3 on the walls of the capillaries. Based on the competitive immunoassay, the fluorescence quenching lengths were relative with the concentration of aminopyrine in the range from 0.2 to 20 ng/L; thus, the fluorescent POCT-based homemade device was realized through the amplification of distance-based signals facilitated by the continuous gas production reaction. This strategy provides an effective way to realize POCT assays in resource-limited areas by transforming pressure variations into directly observable signals. Furthermore, distinguished by its high sensitivity, ease of operation, and portability, it also represents a significant advancement in biomedical diagnostics, particularly within home healthcare and clinical POCT.

We herein rationally designed a target-recyclable AIE-biosensor based on a split G-quadruplex for label-free detection of miRNA in acute kidney injury. Initially, the PG was in an "OFF" state, and the two split segments (G4-a and G4-b) of G4 were tethered at the two terminals of P1 and far away from each other due to the rigid duplex structure formed by the partially complementary intermediate sequences of P2 and P1, bringing MG with quenched fluorescence. In the presence of target, the 5'-PO 4 P2 was displaced from PG probe and competitively hybridized with target, which led to G4-a and G4-b tending to form an intact intermolecular G-quadruplex, providing sites for MG intercalation, thus generating an activated "ON" fluorescence signal due to the restriction of intermolecular motion. Successively, relying on the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of targets will be liberated, accompanied by forming more G-quadruplex and binding more MG, resulting in a strong fluorescence signal, further realizing the sensitive detection of the targets. As a proof of concept, miRNA-21 was chosen as the model target. Endowing with the precise target recognition and efficient cleavage activity of λ-Exo, the AIE-biosensor exhibited excellent detection sensitivity and specificity, which could quantitatively detect miRNA-21 down to 10.36 fM with a single mismatch specificity. The results revealed that the distinctive attributes of noninvasive, simple, and efficient in this G-quadruplex-based AIE-biosensor offered promising prospects for extensive applications in AKI screening and early clinical diagnosis.

In this work, a mitochondria-targeted NIR ratiometric and colorimetric fluorescent probe 1 was tactfully designed and synthesized by a novel design strategy of modifying chromene to pyridine for the first time. 1 exhibited a maximum absorption peak at 508 nm and a maximum fluorescence emission peak at 650 nm. Under the stimulus of biothiols (cysteine (Cys), homocysteine (Hcy), and glutathione (GSH)), the maximum absorption and fluorescence emission peaks of 1 blue-shifted to 448 and 541 nm, respectively, along with color changes from red to yellow under visible light and from red to green under a 365 nm ultraviolet (UV) lamp, which can be ascribed to the click reaction of biothiols with the α,β-unsaturated ketone of the chromene moiety with pyran ring-opening, phenol formation, and 1,6-elimination of the p -hydroxybenzyl moiety. 1 detected biothiols (Cys, GSH, and Hcy) with high sensitivity (LODs of 29, 23, and 16 nM for Cys, GSH, and Hcy, respectively), excellent selectivity, and fast response. Moreover, 1 can target mitochondria and image the fluctuation of intracellular biothiols by dual-emission channels.

The differentiation and maturation of osteoblasts are essential for bone formation. Zn 2+ plays a crucial role in cell differentiation and is involved in osteogenic differentiation. The concentration and distribution of Zn 2+ in the nucleus and cytoplasm indicate the differentiation states of osteoblasts. However, there is an absence of a real-time method for monitoring the dynamic fluctuations of endogenous Zn 2+ within the nucleus. Here, a novel Zn 2+ fluorescent probe ( NTAD-N1 ) with nuclear membrane permeability was designed and developed, allowing for distribution throughout the entire cell, including the nucleus. The NTAD-N1 probe successfully showed the dynamic distribution and concentration changes of Zn 2+ in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during the 21-day differentiation period. The results showed that free Zn 2+ increased significantly during differentiation of osteoblasts (2-21 days). Importantly, after 4 days of differentiation, osteoblasts are mainly distributed in the nucleus, which is confirmed by metallothionein expression. Subsequently, the level of free Zn 2+ in the cytoplasm remained at a high level, which promoted the increase in alkaline phosphatase activity and inhibited the activity of cis-aconitase in the tricarboxylic acid cycle, resulting in the accumulation of citric acid. This series of events promotes the formation of mineralized nodules. In the process of osteoblast differentiation, the detection time of Zn 2+ (≤7 days) is ahead of the late marker of alkaline phosphatase (14 days) and mineralized nodules (14-21 days). This indicates that Zn 2+ can be used as a biomarker and an intervention point for early differentiation of osteoblasts.

Cystatin C (Cys C), a crucial renal disease marker for chronic kidney disease (CKD), plays a vital role in early diagnosis and treatment guidance. However, most current methods for detecting Cys C rely on a single signal and find it difficult to perform noninvasive and portable diagnosis. Here, we developed a ratiometric fluorescent carbon dot (CD) detection system for point-of-care testing (POCT) of Cys C through fluorescence resonance energy transfer (FRET). The detection is based on the hydrolysis effect of papain on a bovine serum albumin (BSA) scaffold and the specific inhibitory effect of Cys C on papain, endowing high-resolution color variance. Moreover, a low-cost, portable, yet reliable smartphone-assisted miniaturized device for real-time quantitative POCT of Cys C has been developed with a limit of detection (LOD) as low as 0.4 μg/mL. This sensing platform can effectively differentiate patients from healthy volunteers, which facilitates self-screening for healthy individuals and home monitoring for CKD patients.

Visualizing the localization and distribution of lipids within arteries is crucial for studying atherosclerosis. However, existing lipid-specific probes face challenges such as strong hydrophobicity and nonspecific staining of lipophilic organelles or tissues, making them impractical for the precise identification of atherosclerotic plaques. To address this issue, we design a synergistically activated probe, Cbz-Lys-Lys-TPEB, which responds to cathepsin B (CTB) and H 2 O 2 for the in situ generation of aggregation-induced emission luminogens (AIEgens). This enables specific staining of lipids within arteries and precise imaging of atherosclerotic plaques. The probe combines a tetraphenylethene building block with a hydrophilic peptide sequence (Cbz-Lys-Lys) and phenylboric acid module, providing excellent water solubility and fluorescence quenching in a molecular dispersion state. Upon interaction with H 2 O 2 and CTB within plaques, the hydrophilic Cbz-Lys-Lys-TPEB probe is specifically cleaved and converted into hydrophobic AIEgens, leading to rapid aggregation and significant fluorescence enhancement. Interestingly, the in situ-liberated AIEgens display distinct lipid binding ability, effectively tracking the location and distribution of lipids in plaques. This synergistic target-activated AIEgen liberation strategy demonstrates significant feasibility for the reliable and accurate identification of atherosclerotic plaques, holding tremendous potential for clinical diagnosis and risk stratification of atherosclerosis.

Monitoring peroxynitrite (ONOO - ) fluctuations is particularly important for assessing pathological progression and oxidative damage due to their crucial role in maintaining the redox balance of organisms. However, due to the lack of efficient tools for differentially monitoring ONOO - fluctuations at different concentration ranges in vivo, the precise detection of endogenous ONOO - fluctuations under pathological conditions in living systems remains challenging. Herein, we rationally designed a double-enhanced emission cascade activatable near-infrared (NIR) fluorescent probe ( B-TCF ) for the measurement of ONOO - , which consists of a borate ester response group and a malononitrile hemicyanine fluorophore. Especially, after sequential oxidative hydrolysis of the borate ester group and xanthene skeleton, B-TCF exhibited a sequentially double-enhanced NIR emission response at 776 and 625 nm for different ONOO - concentration ranges. Moreover, B-TCF revealed excellent and promising performance for ONOO - in terms of high selectivity, sensitivity, and reaction rate ( k = 28.2 M -1 s -1 ). Motivated by the two-step emission signal enhancement and large wavelength shift in the NIR region, B-TCF enabled discriminative imaging of ONOO - with the low and high concentrations in living cells. Importantly, B-TCF was successfully applied for assessing the pathological progression of isoniazid and acetaminophen-induced liver damage in vivo by detecting the endogenous different ONOO - levels. Overall, this study not only demonstrates the first double-enhanced emission cascade activatable NIR fluorescent probe for measuring the dynamic variation of ONOO - in related diseases but also shows great potential as an effective molecular tool for evaluating the various stages of drug-induced liver damage.

Cardiovascular disease, a chronic and progressive arterial wall disease, is increasingly recognized for its clinical significance. Aminopeptidases N (APN), crucial in the pathophysiological processes of vulnerable plaque, have been linked to endothelial dysfunction, oxidative stress, and plaque formation, thus highlighting their potential as biomarkers for disease progression. However, current detection methods for APN in body fluids and in vivo have limitations, including insufficient sensitivity and specificity, time delays, and the inability to directly reflect enzyme activity in plaques. To address these challenges, we developed an optical probe, HD-APN , for in vivo imaging of aminopeptidases, providing a potential implementation in cardiovascular disease. Our work demonstrated the applicability of HD-APN for specific monitoring of aminopeptidase levels in plaques and serum, shedding light on its potential for further research in cardiovascular disease.

Solvatochromic fluorescent probes are crucial molecular tools to investigate and aggregate proteins' fold, visualize fine structures in biomembranes, and label different organelles in dual emission colors. However, solvatochromic fluorogens often displayed a weak emission at high polarity, hindering their bioimaging applications. To resolve this problem, herein, we propose an intramolecular charge transfer (ICT) inhibition strategy. The probe was designed with a single electronic donor and two acceptors in order to split and inhibit the ICT procedure. As a result, the probe displayed an intense emission at both low and high polarities and showed a large emission shift (84 nm) upon polarity change. Using the probe, we successfully imaged lipid droplets and the endoplasmic reticulum in different fluorescence colors. Moreover, the different degrees of lipid accumulation by oleic acid, stearic acid, and cholesterol (oleic acid > stearic acid > cholesterol) have been revealed. The lipid accumulation induced by the three lipids could be rapidly consumed under lipid-less conditions, and the lipids with stearic acid were the most difficult to be consumed. The biological results could facilitate the understanding and treatment of lipid accumulation and obesity. Furthermore, utilizing the polarity increase of diethylamine after the reaction with CO 2 , the ratiometric detection of CO 2 has been achieved for the first time with the probe.

Real-time monitoring of chemotherapy-induced senescence (CIS) in cancer remains a challenging task that would lead to new insights into the adaptive mechanisms of cancer therapy and provide guidance for cancer management. Here, we designed a tailor-made nanoprobe capable of imaging CIS in a sequential activation and self-amplified manner by reversing senescence-related impaired ferritinophagy. It contains an amphipathic polymer as a spatially responsive vehicle, a Fe 2+ -activable dye as the reporter, and an autophagy inducer as the signal amplifier. Owing to metabolic changes, the nanoprobe preferentially enriches in senescent cancer cells, leading to in situ activation and fluorescence switching of the reporter by labile Fe 2+ . Meanwhile, the inducer restores ferritinophagy and promotes autophagic degradation of accumulated ferritin, facilitating conversion of ferritin-bound iron into Fe 2+ for amplified imaging in senescent cancer cells yet keeping inert in nonsenescent cells. Of note, the accumulation and activation of the nanoprobe and sustained ferritin degradation occur at the same subcellular location, thus minimizing the diffusion process-induced nonspecific responses. The feasibility of this strategy is successfully demonstrated in both living cells and animal models. This work offers a new way for therapeutic evaluation and a basic understanding of the roles of senescence in cancer treatment.

Mitochondrial DNA G-quadruplexes (mtDNA G4s) play potential regulatory roles in mitochondrial functions. Fluorescent probes for imaging mtDNA G4s may provide useful information to unveil their regulating dynamics and functions. However, the existing probes for mtDNA G4s still exhibit short absorption and emission wavelengths and limited sensitivity. Here, we develop a new isaindigotone-derived near-infrared (NIR) fluorogenic probe for imaging mtDNA G4s in live cells and in vivo. Different fluorescent probes are engineered by conjugating the isaindigotone scaffold with varying electron-donating groups. It is shown that the probe ISAP using dimethylaminophenyl as the electron-donating group exhibits near-infrared absorption/emission and a high fluorescence activation fold in response to G4s. Molecular docking simulations reveal that ISAP binds to c -Myc G4 via multiple π-π stacking and hydrogen-bond interaction. Cellular studies show that ISAP exhibits an excellent mitochondrial targeting ability and allows specific imaging of mtDNA G4s. We further employed ISAP to image the dynamics of mtDNA G4s under glycolysis and oxidative stresses in live cells. Its capability to mtDNA G4s in vivo is showcased using a tumor-bearing mice model. This probe may serve as a useful tool to image mtDNA G4s and interrogate their biological roles in living systems.

Mitochondrial autophagy, known as mitophagy, is a vital cellular process that involves the selective degradation of damaged or dysfunctional mitochondria through autophagy, which is critical to the functional integrity of the entire mitochondrial network and determines the survival and death of cells. An abnormal pH may lead to an imbalance in mitochondrial homeostasis and the occurrence of mitochondrial autophagic acidification and dysfunction. SCN - is also an important anion in cellular metabolism, and its abnormal concentration may lead to mitochondrial damage. However, so far, there are few reports on the simultaneous realization of pH and SCN - detection in mitochondria. Therefore, to complement the blank in this area, we developed the polysiloxane-based fluorescent probe P0-CMN that is capable of simultaneously visualizing pH and SCN - fluctuation levels in mitochondria. The probe P0-CMN has the desired mitochondrial-targeting properties and sensitivity to pH and SCN - . It is able to simultaneously monitor pH and SCN - changes in mitochondria in a dual-channel mode. In addition, probe P0-CMN can visualize pH changes during mitochondrial autophagy. This work provides an effective strategy for the design of dual-responsive fluorescent probes and further broadens the application of polysiloxane fluorescent materials.

Herein, a novel functional DNA structure, acid-stimulated self-assembly DNA nanonetwork (ASDN), was proposed for miRNA-221 sensitive detection and high-resolution living cancer cell imaging. Significantly, the self-assembly of ASDN only occurred in the acidic extracellular environment of cancer cells, which could be endocytosed by cancer cells to eliminate the interference of noncancer cells and deliver the ASDN into cancer cells. Subsequently, endogenous miRNA-221 could trigger the catalytic hairpin assembly within ASDN, resulting in the separation of the fluorophore Cy5 and the quencher BHQ2 to recover the substantial Cy5 fluorescence signals, thus achieving signal amplification for sensitive detection of miRNA-221 with a detection limit of 5.5 pM, as well as facilitating high-resolution and low-background imaging of miRNA-221 in cancer cells. In consequence, this strategy provides an innovative DNA nanonetwork to distinguish cancer cells from other cells for sensitive detection of biomarkers, offering a meaningful reference for the application of DNA nanostructure self-assembly technology in relevant fundamental research and disease diagnosis.

Lysosomes are multifunctional organelles vital for cellular homeostasis with distinct subpopulations characterized by varying levels of Cl - , Ca 2+ , and H + . In situ visualization of these parameters is crucial for lysosomal research, yet developing probes that can simultaneously detect multiple ions remains challenging. Herein, we developed a lysosome-targeting ternary recognition ratiometric fluorescent probe based on tetrahedral DNA nanostructures (TDNs) to analyze lysosome subpopulations by Cl - , Ca 2+ , and pH. The TDN probe is assembled from four single-stranded DNAs, each end-modified with responsive fluorophores (Pr-Cl for Cl - , Pr-Ca for Ca 2+ , and Pr-pH for pH) or a reference fluorophore (Cy5). The fluorophores are integrated at the vertices of the rigid TDN to minimize mutual interference, and their fixed stoichiometry establishes a robust ternary recognition ratiometric fluorescence sensor for in situ resolution of lysosome subpopulations in living cells. Accordingly, a rise in lysosome subpopulations 2/6 characterized by low [Cl - ], medium/high [Ca 2+ ], and high pH was observed in the Niemann-Pick disease model cells but seldom observed in the control group. Conversely, there was a marked decline in the fraction of subpopulations 1/4/5 characterized by high [Cl - ], medium to low [Ca 2+ ], and pH. These changes were substantially reversed upon treatment. The probe holds great promise for studying lysosome subpopulations and the diagnosis and treatment of related diseases.

Hepatic fibrosis is a common chronic liver disease, and its severe progression can culminate in cirrhosis and hepatocellular carcinoma (HCC). Precise diagnosis and staging of hepatic fibrosis are essential to prevent liver cirrhosis and HCC. Simultaneous detection of multiplex collagen biomarkers within liver tissue is crucial for staging hepatic fibrosis. We herein for the first time constructed multiplex collagen fingerprinting for the staging of hepatic fibrosis using high-precision fluorescence-guided surface-enhanced Raman scattering (SERS) imaging. SERS/fluorescent probes, collectively referred to as SF, comprising silver nanoparticles (Ag NPs), Raman reporters, and FAM-labeled collagen targeting peptides. These probes exhibit exceptional aqueous dispersion and stability, attributed to the increased number of Asp residues in CTP. Meanwhile, SF probes, namely SF-I, SF-IV, and SF-D have demonstrated specific targeting of type I, type IV, and denatured collagen, respectively, within hepatic fibrotic tissues. The results from fluorescence-guided SERS imaging underscore the method's capacity for typing, localization, and quantification of collagen, thus providing novel insights into collagen's role in the development of hepatic fibrosis. The collagen fingerprinting strategy offers a potent toolkit for the multifaceted profiling of collagen superfamilies, holding significant implications for the precise staging of hepatic fibrosis.

Keloids represent pathologic conditions characterized by the presence of hyalinized collagen bundles and chronic inflammatory reactions. Recently, increased ROS production and disrupted apoptosis mechanisms in keloids have been reported, although the detailed mechanisms remain unclear. Herein, we developed a specific fluorescence probe, Pro-NBS , to investigate ClO - levels in keloids. The probe demonstrated high specificity for ClO - over other ROS and exhibited a strong linear detection relationship. Based on its performance, we focused on the TGF-β pathway in the development of keloids. ROS upregulation was observed in keloid-derived fibroblasts. Using ClO - as an intrinsic overexpression marker, our probe effectively distinguished between normal fibroblasts and keloid-derived fibroblasts both in vitro and in vivo. Furthermore, Pro-NBS showed potential for monitoring the progression and evaluating the systematic therapy of abnormal scarring or keloids.

Given the promising prospect of aggregation-induced emission luminogens (AIEgens) in fluorescence assays, it is interesting and significant to endow AIEgens with molecular recognition capability (such as enzyme-like activity). Here, an AIE nanomaterial with intrinsic enzyme-like activity (named as "AIEzyme") is designed and synthesized via a facile coordination polymerization of Zr 4+ and AIE ligands. AIEzyme possesses enhanced and stable fluorescence in different solvents because of the AIE effect of ligands in the rigid structure of a coordination polymer. On the other hand, the organophosphorus hydrolase (OPH)-mimicking activity of AIEzyme exhibits excellent affinity and specific activity. Interestingly, the OPH-like activity can quench the inherent fluorescence of AIEzyme by the hydrolysate of a typical organophosphorus nerve agent (OPNA), diethyl-4-nitrophenylphosphate. Due to the sensitive activity-induced quenching effect for AIE, the self-reporting fluorescence assay method based on AIEzyme was established, which shows ultrahigh sensitivity, high selectivity, good storage stability, and acceptable reliability for a real sample assay. Moreover, the simultaneous colorimetric method broadens the detection range and the application scenarios. The proposed assay method avoided the interference of O 2 during detection because the OPH-like activity does not derive from the generation of ROS. As a bonus, AIEzyme can also be used for the degradation of OPNAs by OPH-like activity, and the process can be self-monitored by AIE quenching. This work would provide a new opportunity for expanding the application of AIEgens and artificial enzymes by endowing AIEgens with enzyme-like activity.

A luminescent nanoprobe capable of orthogonal sensing of two independent events is highly significant for unbiased disease-related detection such as the detection of senescent cells. Moreover, it is invaluable that the nanoprobe possesses a two-photon excitable characteristic that is highly suitable for imaging living cells and tissues. Herein, we present a two-photon-excitable multiluminescent orthogonal-sensing nanoprobe (OS nanoprobe) capable of detecting both pH elevation and β-galactosidase (β-gal) overexpression in senescent cells. In the design, Zr-based dual-emissive metal-organic frameworks prepared from two mixed amino linkers, referred to as NH 2 -MU, were used as the component for the ratiometric sensing of pH; additionally, fluorogenic resorufin-β-d-galactopyranoside, linked to the NH 2 -MU framework, enables β-gal detection. In the OS nanoprobe, the signals for pH and β-gal sensing remain independent while maintaining high colocalization. The two-photon excitable organic linkers of NH 2 -MU impart the OS nanoprobe with a bioimaging capability, allowing for the differentiation of senescent human foreskin fibroblast (HFF) cells from younger HFF cells or LacZ positive cells with the 800 nm laser excitation. This study marks the first instance of achieving the multiplexed orthogonal fluorescent sensing of cellular senescence using a two-photon excitation strategy, suggesting the potential of using versatile metal-organic framework (MOFs)-based fluorophores to realize the orthogonal multiplexing of disease-related biomarkers through multiphoton excitation.

The construction of far-red fluorescent molecular rotors (FMRs) is an imperative task for developing nucleic acid stains that have superior compatibility with cellular systems and complex matrices. A typical strategy relies on the methine extension of asymmetric cyanines, which unfortunately fails to produce sensitive rotor character. To break free from this paradigm, we have synthesized far-red hemicyanines using a dimethylamino thieno[3,2- b ]thiophene donor. The resultant probes, designated as ATh 2 Ind and ATh 2 Btz, possess excitation maxima (λ max ) of >600 nm and have been rigorously characterized by NMR, electrochemistry, and computational methods. The dyes possess alternating charge patterns like indodicarbocyanine (Cy5), but with twisted intramolecular charge transfer (TICT) rotational barriers at 60°, akin to the classical FMR thiazole orange (TO1). ATh 2 Btz also displays cyanine characteristics, enhancing its response upon binding to nucleic acids and allowing for efficient staining of cellular nuclei. When binding to the DNA aptamer for quinine (MN4), ATh 2 Btz exhibits a K d of 17 nM, a 660-fold light-up response, brightness (Φ fl x ε max ) of ∼37,000 M -1 cm -1 , and λ ex /λ em of 655/677 nm. The resulting far-red DNA-based MN4-ATh 2 Btz platform has been termed "pomegranate."

In situ visualization of microRNA (miRNA) in cancer cells and diseased tissues is essential for advancing our comprehension of the onset and progression of associated diseases. Two-photon (TP) imaging, as an imaging technology with high spatiotemporal resolution, deep tissue penetration, and accurate target quantification, has distinctive advantages over single-photon imaging and has attracted increasing attention. Extensive research has been conducted on two-photon dye-doped silica nanoparticles, which exhibit a large two-photon absorption (TPA) cross-section, high fluorescence quantum yield, and excellent biocompatibility. However, the low abundance of RNA in tumor cells leads to insufficient signal output. Based on functional nucleic acid, a catalyzed hairpin self-assembly (CHA) signal amplification strategy, which has simplicity, robustness, and nonenzymatic characteristics, can achieve the amplification of DNA or RNA signals. Here, a two-photon silica nanoamplifier (TP-SNA) utilizing TP dye-doped silica nanoparticles (SiNPs) and functional nucleic acid was constructed, employing triggering catalyzed hairpin self-assembly and fluorescence resonance energy transfer (FRET) for highly sensitive detection and precise TP imaging of endogenous miRNAs in tumor cells and tissues at varying depths. The TP-SNA demonstrated the capability to detect miR-203 with a detection limit of 33 pM. The maximum two-photon tissue penetration depth of the two-photon nanoamplifier was 210 μm. The two-photon nanoamplifier developed in this study makes full use of the advantages of accurate TP ratiometric bioimaging and the CHA signal amplification strategy, which shows good application value for future transformation into clinical diagnosis.

Diagnosing cancer in its early stages can play an important role in prolonging the lifespan of patients, which demands the use of powerful tools to detect biomarkers accurately. However, since most fluorescent probes described for cancer diagnosis are limited to recognizing a single molecule, achieving the high accuracy criteria remains difficult. Here, sensor 1 is constructed for the sequential detection of D , ONOO - , and viscosity. Initially, sensor 1 detected D and underwent an intramolecular charge transfer mechanism, resulting in the formation of 2 and fluorescence quenching at 587 nm. Subsequently, the intermediate ( 2 ) monitored ONOO - and reproduced sensor 1 reversibly with fluorescence enhancement at 496 nm, showing concentration-related quantitative analysis. Similar sensing processes were observed in monitoring ONOO - and viscosity by synthetically developed sensor 2 . The proposed mechanisms of sensors 1 and 2 are verified through various characterizations ( 1 H NMR, HR-MS, and HPLC) and DFT calculations. Investigations on endogenous ONOO - and mitochondrial viscosity in cancer (HeLa) and normal (NCM460) cells were conducted to distinguish cancerous cells from normal cells. We anticipated that sensor 2 could effectively serve as a reliable bioanalytical reagent for cancer diagnosis at an earlier stage through sequential detection of two cancer markers, ONOO - and mitochondrial viscosity, in living cells. Importantly, sensor 2 has been employed for imaging ONOO - in normal and liver injury mouse models and tissues, achieving outstanding results.

Axl is an important receptor tyrosine protein kinase that plays a key role in the development and progression of various diseases, such as cancer and inflammation. Developing a highly sensitive Axl detection method can help improve accuracy, better address-specific clinical needs, and guide personalized treatment. In this study, a CHA-CRISPR/Cas13 fluorescence probe was established using Axl-specific aptamers as a mediator to displace the polynucleotide chain (TA). Through TA construction, an entropy-driven nucleotide catalytic hairpin assembly system was created to cyclically release RNA that activates clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13 activity, triggering its cleavage activity. The activated CRISPR/Cas13 system cleaves the reporter labeled with BHQ1 and FAM at both ends, leading to the recovery of FAM fluorescence. Based on the optimization design using the free energy (△ G ) and secondary structure software simulation results of the nucleic acid sequence, the fluorescence intensity of the probe is proportional to the concentration of Axl. Results showed a good linear relationship between fluorescence intensity increment and log C Axl ( C Axl in the range of 3.33-667 pM, r = 0.9907). The probe exhibited ultrahigh sensitivity with a detection limit of 0.84 pM. It was successfully applied in the detection of human serum samples, showing a higher Axl level in cervical cancer patients compared to breast cancer patients. The probe was also successfully applied in the imaging of various tumor cells, consistent with serum detection results. In conclusion, this probe represents an effective new method for detecting Axl, demonstrating outstanding specificity and sensitivity. It provides technological support for tumor diagnosis and shows the potential for detecting circulating tumor cells in blood through cell imaging.

Cisplatin is a common chemotherapy drug for multiple solid tumors; however, due to the nitrification of peroxynitrite (ONOO - ), a series of side effects seriously affect its dose and efficacy. Considering that the reactivity of ONOO - is significantly affected by pH in vitro, revealing their roles in living cells contributes to understanding the side-effect process induced by cisplatin. Herein, we present a near-infrared fluorescent logic gate for sensing pH/ONOO - , which can accurately discriminate four scenarios (no analyte, analyte H + alone, analyte ONOO - alone, and H + + ONOO - ) and which one comes first. With this probe, the significant roles of pH and ONOO - in cisplatin treatment are disclosed, in which the cell account shows a dramatic reduction accompanied by decreased pH and upregulated ONOO - levels. By artificially recovering the pH, the ONOO - content and cell account can maintain a stable state, possibly due to the protection from acidification and nitration. This work provides an ideal pH/ONOO - logical sensor for revealing their potential roles under cisplatin, which is expected to proffer new insights into more related diseases and drug research.

As digital data undergo explosive growth, deoxyribonucleic acid (DNA) has emerged as a promising storage medium due to its high density, longevity, and ease of replication, offering vast potential in data storage solutions. This study focuses on the protection and retrieval of data during the DNA storage process, developing a technique that employs flow cytometry sorting (FCS) to segregate multicolored fluorescent DNA microparticles encoded with data and facilitating efficient random access. Moreover, the encapsulated fluorescent DNA microparticles, formed through layer-by-layer self-assembly, preserve structural and sequence integrity even under harsh conditions while also supporting a high-density DNA payload. Experimental results have shown that the encoded data can still be successfully recovered from encapsulated DNA microparticles following de-encapsulation. We also successfully demonstrated the automated encapsulation process of fluorescent DNA microparticles using a microfluidic chip. This research provides an innovative approach to the long-term stability and random readability of DNA data storage.

The noncanonical conformations within the genomes of viral pathogens is of significant diagnostic value, due to their unique secondary structures and interactions with specific fluorogenic molecules. In particular, adaptation of the G-quadruplex (GQ) conformation by the specific gene sequence leads to distinct topological features, resulting in unique binding sites that are crucial for the selective recognition of human immunodeficiency virus (HIV) by small molecules. Leveraging the selective fluorescence response of a benzobisthiazole-based fluorogenic probe to the LTR-III GQ target, we developed a GQ-based diagnostic platform for HIV detection. The successful fluorescence recognition of an amplified 176-nucleotide genomic segment harboring the LTR-III GQ, facilitated by pH-controlled GQ-targeted reliable conformational polymorphism (GQ-RCP), validates this method as an effective GQ-topology-targeted diagnostic tool for HIV.

The multimode assay based on multiple response mechanisms has received great attention to effectively improve the accuracy of a sensing platform. However, multifunctional sensing materials for simultaneously satisfying the multiple-mode detections are still in shortage due to the incompatibility of the signal transduction mechanisms in different modes. Here, taking human papillomavirus 16 (HPV-16) DNA (TDNA) as the model due to its important role in cervical cancer, a novel multifunctional material, ethyl violet (EV)@NH 2 -MIL-88B(Fe) (ENM) hybrids, have been successfully prepared, which could simultaneously satisfy CRISPR-Cas12a-assisted photoelectrochemical (PEC)-fluorescent (FL)-colorimetric (CL) triple-mode detection of TDNA. Based on the TDNA-induced trans-cleavage ability of CRISPR-Cas12a and efficient separation of magnetic beads, ENM was obtained from the single-stranded DNA-surrounded streptavidin-modified magnetic beads-ENM (SMB-ssDNA-ENM) and decomposed by pyrophosphate to get free EV, 2-aminoterephthalic acid (NH 2 -BDC), and Fe 3+ . Thus, TDNA was sensitively detected based on the EV-enhanced PEC signal of SnS 2 nanosheets (PEC mode), fluorescent signal of NH 2 -BDC (FL mode), and characteristic absorption peak at about 720 nm of Fe 3+ -induced Prussian blue (PB) (CL mode). The designed PEC-FL-CL triple-mode biosensing platform had good performance for the detection of TDNA with a wide linear range (0.1 fM-100 nM) and ultralow detection limits (0.07 fM for PEC, 0.03 fM for FL and 0.09 fM for CL). Additionally, the developed PEC-FL-CL triple-mode biosensing platform has great potential for applications in early disease diagnosis and bioanalysis, as it can be easily extended to other DNA assays through modification of the crRNA sequence within the CRISPR-Cas12a system.

The current limitations of single-molecule localization microscopy (SMLM) in deep tissue imaging, primarily due to depth-dependent aberrations caused by refractive index (RI) mismatch, present a significant challenge in achieving high-resolution images at greater depths. To extend the imaging depth, we optimized the imaging buffer of SMLM with the RI matched to that of the objective immersion medium and systematically evaluated five different RI-matched buffers, focusing on their impact on the blinking behavior of red-absorbing dyes and the quality of reconstructed super-resolution images. Particularly, we found that clear unobstructed brain imaging cocktails-based imaging buffer could match the RI of oil and was able to clear the tissue samples. With the help of the RI-matched imaging buffers, we showed high-quality dual-color 3D SMLM images with imaging depths ranging from a few micrometers to tens of micrometers in both cultured cells and sectioned tissue samples. This advancement offers a practical and accessible method for high-resolution imaging at greater depths without the need for specialized optical equipment or expertise.

The advancement of acetylcholinesterase (AChE) activity and its inhibitor assays is crucial for clinical diagnosis, drug screening, and environmental monitoring. A nanozyme-mediated cascade reaction system could offer promising prospects for a wide range of applications in such biosensing; however, the creation of nanozyme catalysts with diverse functionalities remains a significant challenge. Herein, we have proposed a multifunctional iron-doped polymer dots (Fe-PDs) nanozyme possessing excellent fluorescence and peroxidase (POD)-mimicking activity. Notably, the Fe-PDs nanozyme is capable of catalyzing H 2 O 2 to produce a series of reactive oxygen species, which can simultaneously quench the fluorescence of Fe-PDs and induce a chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB), enabling the dual-mode detection of H 2 O 2 through both fluorescence turn-off and absorbance turn-on signals. Furthermore, by integrating acetylcholine (ACh) and choline oxidase (ChOx), we have developed a three-enzyme (AChE-ChOx-POD) cascade-based fluorometric and colorimetric dual-mode sensing platform for monitoring AChE activity and its inhibitors. The sensitive and convenient dual-mode sensor has achieved low limits of detection with 0.5 mU/mL (fluorometry) and 0.014 mU/mL (colorimetry) for AChE, respectively, which are superior to the traditional Ellman's assay. More significantly, this sensor can also be extended to detect the reversible and irreversible inhibitors of AChE, such as tacrine (IC 50 = 23.3 nM) and carbaryl (LOD = 0.8 nM). We firmly believe that this innovative dual-mode nanozyme-involved multienzyme cascade system-based sensing strategy will stimulate further exploration and serve as a versatile and practical tool for biochemical sensing applications.

In this study, we present an innovative "click-to-release" strategy for the design of highly specific H 2 S n bioorthogonal probes that undergo a specific click reaction with H 2 S n and release fluorophores by a following rearrangement. A library of cyclooctyne derivatives was established and successfully demonstrated the availability of the release strategy. Then, a model probe CM-CT was synthesized, which can achieve effective fluorophore release (>80%) in the presence of a H 2 S n donor. To further validate the application of this class of probes, a new probe QN-RHO-CT based on Rhodamine 110 was developed. This probe showed good water solubility (>160 μM) and fast release kinetics and can achieve selective H 2 S n detection in living cells. We used this probe to study the process of H 2 S-mediated protein S-persulfidation and demonstrated that excess H 2 S would directly react with protein persulfides to generate H 2 S 2 and reduce the persulfides to thiols. Additionally, we elucidated the click-to-release mechanism in our design through a detailed mechanistic study, confirming the generation of the key intermediate α, β-unsaturated cyclooctanethione. This bioorthogonal click-to-release reaction provides a useful tool for investigating the function of H 2 S n and paves the way for biological studies on H 2 S n .

Developing NIR-IIb luminescence probes with rapid visualization and a high penetration depth is essential for diabetes research. Combining a sensitizing switch with lanthanide-doped nanoparticles (LnNPs) has been employed to fabricate the NIR-IIb probes. However, these probes mainly adopt heptamethine cyanine dye as the antenna, and the NIR-IIb signal is activated by inhibiting the photoinduced electron transfer (PET) of the dye. Due to limited recognition units, this strategy makes many biomolecules undetectable, such as cysteine (Cys), which is closely related to diabetes. Herein, in this article, hemicyanine dye, NFL-OH, was verified as a new antenna to sensitize NIR-IIb emission from LnNPs. Unlike traditional cyanine dyes, hemicyanine's fluorescence intensity can also be modulated by intramolecular charge transfer (ICT), thereby expanding the range of detectable targets for NIR-IIb probes based on sensitization mechanism. Through switching the hemicyanine-sensitized NIR-IIb emission, we successfully fabricated an NFL-Cys-LnNPs' nanoprobe, which can effectively monitor Cys concentration in the liver of diabetic mice during diabetes progression and evaluate the efficacy of diabetic drugs. Our work not only presents an excellent tool for Cys imaging but also introduces new concepts for designing NIR-IIb probes.

Accurate and rapid imaging of tumor cells is of vital importance for early cancer diagnosis and intervention. Aptamer-based fluorescence sensors have become a potent instrument for bioimaging, while false positives and on-target off-tumors linked to single-biomarker aptasensors compromise the specificity and sensitivity of cancer imaging. In this paper, we describe a sequential response aptasensor for precise cancer cell identification that is based on a DNA "AND" logic gate. Specifically, the sensor consists of three single-stranded DNA, including the P-strand that can sensitively respond to an acid environment, the L-strand containing the ATP aptamer sequence, and the R-strand for target cell anchoring. These DNA strands hybridize with one another to create a Y-shaped structure (named Y-ALGN). The aptamer in the R-strand is utilized to anchor the sensor to the target cell membrane primarily. Responding to the extracellular acidic environment of the tumor (input 1), the I-motif sequence forms a tetramer structure so that the P-strand is released from the Y-shaped structure and exposes the ATP binding sites in the L-strand. Extracellular ATP, as input 2, continuously operates the DNA aptasensor to complete the logic computation. Upon the sequential response of both protons and ATP molecules, the aptasensor is activated with restored fluorescence on a particular cancer cell membrane. Benefiting from the precise computation capacity of the "AND" logic gate, the Y-ALGN aptasensor can distinguish between MCF-7 cells and normal cells with high accuracy. As a simple and dual-stimuli-responsive strategy, this nanodevice would offer a fresh approach for accurately diagnosing tumor cells.

Cell-cell interactions are essential for the proper functioning of multicellular organisms. For example, T cells interact with antigen-presenting cells (APCs) through specific T-cell receptor (TCR)-antigen interactions during an immune response. Fluorescence-activated droplet sorting (FADS) is a high-throughput technique for efficiently screening cellular interaction events. Unfortunately, current droplet sorting instruments have significant limitations, most notably related to analytical throughput and complex operation. In contrast, commercial fluorescence-activated cell sorters offer superior speed, sensitivity, and multiplexing capabilities, although their use as droplet sorters is poorly defined and underutilized. Herein, we present a universally applicable and simple-to-implement workflow for generating double emulsions and performing multicolor cell sorting using a commercial FACS instrument. This workflow achieves a double emulsion detection rate exceeding 90%, enabling multicellular encapsulation and high-throughput immune cell activation sorting for the first time. We anticipate that the presented droplet sorting strategy will benefit cell biology laboratories by providing access to an advanced microfluidic toolbox with minimal effort and cost investment.

Bacteria can cause infectious diseases even at ultralow concentrations (<1 CFU/mL). It is important to rapidly identify bacterial contamination at ultralow concentrations. Herein, FITC-labeled gelatinase-sensitive nanoparticles (GNPs@FITCs) and NFM@GNP@FITCs are designed and fabricated as ultralow concentration bacteria detection platforms based on an enzymatic cascade reaction-amplifying strategy. Bacterial secretions could trigger the dissociation of GNPs@FITCs to release FITC, with gelatinase used as the model secretion. The detectable signal of ultralow concentration bacteria could be amplified effectively by the gelatinase-triggered cascade reaction. Bacterial concentration was evaluated by the change in fluorescence density. The results showed that the GNPs@FITCs and NFM@GNP@FITCs could be used for identifying bacterial contamination qualitatively, even when the bacterial contamination is lower than 1 CFU/mL. Moreover, the method has better timeliness and convenience, when compared with national standards. As solid films, NFM@GNP@FITCs have better long-term storage stability than GNPs@FITCs. The potential applications of GNPs@FITC and NFM@GNP@FITCs were proved by detecting pathogenic bacteria in food. All the results showed that the method has great potential for screening pathogenic bacterial contamination qualitatively.

Acute alcoholic liver injury (AALI) has become an important cause of liver disease worldwide, and there is an urgent need to develop noninvasive and sensitive methods to detect and evaluate AALI. We report herein three novel but readily available mitochondrial targeting fluorescence probes ( ICR , ICJ , and ICQ ) for AALI detection. These probes contain different electron-donating groups, among which ICQ exhibits NIR fluorescence (740 nm), a large Stokes shift (110 nm), and a sensitive response to viscosity (73-fold enhancement in fluorescence from water to glycerol), making it suitable for in vivo imaging. ICQ also exhibits an excellent ability to image mitochondrial viscosity changes in cells. More importantly, ICQ can target the liver selectively and image the viscosity changes in the liver noninvasively. Through establishing an AALI mouse model, ICQ was successfully applied to the in situ imaging changes in liver viscosity during the AALI process. The results showed a significant increase in liver viscosity in AALI mice, indicating that viscosity can serve as a marker for AALI, and ICQ is a promising noninvasive and sensitive tool for detecting and evaluating AALI.

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

Mitochondrial polarity is a critical indicator of numerous pathological and biological processes; thus, the development of fluorescent probes capable of targeting mitochondria and visually monitoring its polarity is of great significance. In this study, fluorescent probes were designed with a N, N-dialkylamino rhodol scaffold as the fluorophore sensitive to polarity environments, in which the alkyl chain length was adjusted rationally to obtain distinct polarity recognition modes. By integrating mitochondria targeting groups, three fluorogenic chemical probes ROML-1 , ROML-2 , and ROML-3 have been obtained, featuring the capability to target mitochondria and monitor its polarity precisely, dynamically and visually. The probes displayed a distinctive response to the alterations in polarity. ROML-1 and ROML-2 followed a turn-on pattern while ROML-3 was ratiometric. It has been demonstrated that the hypersensitivity to polarity and ratio fluorescence property of ROML-3 was attributed to methyl groups rather than ethyl or butyl groups. The introduction of short methyl chains made the dihedral angle between the dialkylamino substituent and fluorophore of ROML-3 (spirocyclic form) rotatable and enlarged the energy gap between the ground state and excited state, which has been validated by the results of density functional theory (DFT) calculations. Furthermore, ROML-3 was used to monitor mitochondrial polarity via confocal microscopy imaging, which revealed that compared to healthy cells the polarity of mitochondria in cancer cells was enhanced; meanwhile, the polarity of mitochondria in senescent cells was higher in contrast with young cells. The present probe ROML-3 has been proven to be an efficient tool to monitor mitochondrial polarity dynamics, which demonstrated potential significance in biomedical research and disease diagnosis.

Despite many luminescent advantages including outstanding absorption coefficient and high quantum yield, pyrene and its derivatives have been suffering from a dramatic aggregation-caused quenching (ACQ) effect. Although the dramatic ACQ effect of pyrene-based fluorophores has been restrained in pyrene-doped metal-organic frameworks (MOFs), the low loading of fluorescent (FL) units substantially impedes the improved luminescent behaviors. Herein, pyrene-based MOFs hydrogel was synthesized with a high loading of pyrene as the unique organic linker blocks instead of a dopant in MOFs. The gel matrix contributed to rigidifying the location of the FL emitters and achieving intensive FL emission and high luminescent stability and therefore efficiently overcoming the ACQ effect. Furthermore, the protonation of pyrene in the MOFs hydrogel remarkably decreased the luminescent intensity, which endowed the FL hydrogel with highly pH-responsive activity in the broad range (pH 4-10). Interestingly, glucose oxidase was immobilized into ZIF-8 as a highly efficient luminescent quencher, which contributed to catalyzing the form of gluconic acid and thus drastically quenching the FL signal of the MOFs hydrogel. Furthermore, the emitter-quencher pair of pyrene-based MOFs hydrogel and glucose oxidase was successfully employed to develop an ultrasensitive FL immunoassay platform for cardiac troponin I (as a model analyte). The limit of detection for cardiac troponin I was 5.2 pg/mL (3σ). The proof-of-principle study demonstrated the thrilling auxiliary effect of tailorable MOFs hydrogel on boosting the feasibility of aqueous insoluble FL chromophores for trace analysis.

The etiology and pathological complexity of acute kidney injury (AKI) pose great challenges for early diagnosis, typing, and personalized treatment. It is an important reason for poor prognosis and high mortality of AKI. In order to provide a relatively noninvasive diagnostic and typing method for AKI, we proposed the pathological changes of albumin permeability after glomerular injury and reabsorption efficiency after tubular injury as potential entry points. Thus, a renal tubule labeling fluorescent dye which features albumin concentration-related fluorescence intensity was used to fit these pathological changes. Utilizing this fluorescence assay, we realized urinary tract obstruction imaging as early as 12 h after morbidity. For glomerular and tubular injury discrimination, compared to a healthy control, membranous nephropathy as a representative glomerular injury resulted in enhanced fluorescence intensity of the kidney due to increased albumin penetration, while renal tubular injury caused insufficient dye reabsorption to exhibit weakened fluorescence intensity. The significant differences demonstrated the feasibility of this approach for fluorescence imaging-based AKI typing in vivo.

The study demonstrates the potential of an optical nose made by depositing an array of fluorescent nanomaterials on a paper substrate for the early detection of leukemia in adults. This is based on the fact that blood volatile organic compounds (VOCs) are useful leukemia biomarkers. The integrated design was miniaturized and comprised both sensing zones and a sample holding zone, which were installed on a small sheet of paper within a miniature cubic reaction chamber fabricated by using 3D printing technology. The sensing device, comprising seven fluorescent sensing elements, namely, metal nanoclusters, quantum dots, and carbon dots was capable of detecting VOCs in the blood headspace and providing a colorimetric signature that could discriminate between blood samples from healthy and cancerous individuals. A total of 70 new leukemia cases and 51 healthy controls aged 20-50 years were studied. The device required a 60 μL portion of the blood sample and reacted to blood VOCs after 3 h when kept at 50 °C. The imaging data from the device was processed by linear discriminant analysis, and the results confirmed efficient identification of patient samples from healthy samples with 100% accuracy. Overall, the array system is noninvasive (or minimally invasive), portable, fast, inexpensive, and requires only a small amount of blood sample.

Aminopeptidase N (APN/CD13) is a widely expressed transmembrane ectoenzyme that is crucial for maintaining normal physiological activities. It exhibits abnormal activity closely associated with hepatic fibrosis and nonalcoholic fatty liver disease (NAFLD). Therefore, there is a high demand for noninvasive detection of aminopeptidase N (APN) in the diagnosis and research of related diseases. Here, we developed a small molecule fluorescent probe, Hcy-APN, which is a fluorescent probe with high sensitivity and selectivity for the detection of APN. Furthermore, we synthesized the fluorescent nanoprobe Hcy-APN@MSN by self-assembling Hcy-APN and mesoporous silica nanoparticles in solution using a combination of molecular probe design and nanofunctionalization strategies. The detection limit of this probe was 1.5 ng/mL. Hcy-APN@MSN exhibits more stable spectral characteristics compared to Hcy-APN and is suitable for detecting APN activity in live cells and mice. Hcy-APN@MSN was utilized for in vivo and intracellular imaging of NAFLD and hepatic fibrosis at different stages, as well as for a systematic assessment of APN levels in the liver. The results confirm an elevation in the expression levels of APN in NAFLD and hepatic fibrosis models. Furthermore, we investigated the inhibitory effect of the APN inhibitor bestatin in nonalcoholic fatty liver and hepatic fibrosis disease models, confirming its regulatory effect on APN levels in cells and in vivo in both disease models. Therefore, this study may offer diagnostic possibilities for detecting NAFLD and hepatic fibrosis.