Portable multimodal platform with carbon nano-onions as colorimetric and fluorescent signal output for trypsin detection.

Multimodal biosensors with independent signaling pathways can self-calibrate and improve the reliability of disease biomarker detection. Herein, a colorimetric-fluorescent dual-mode paper-based biosensor with PAN/Fe(III)-CNOs (FPCs) as core components has been developed, which information is recognized by smartphone and naked eye. Using 1-(2-pyridylazo)-2-naphthol (PAN) as a mediator, Fe(III) is enriched on the surface of carbon nano-onions (CNOs), endowing FPCs with excellent mimetic enzyme activity and photothermal conversion ability, which allows it to output amplified colorimetric signals under laser irradiation. In addition, the complexation of PAN with Fe(III) broadens its absorption spectrum, which makes FPCs more suitable to be energy acceptors to quench fluorescence of polymer dots (Pdots), resulting in the changes of output fluorescent signal. Based on the above design, a portable colorimetric-fluorescent dual-mode biosensor is proposed for trypsin detection with Pdots as fluorescence sources and FPCs as fluorescence quenchers and nanoenzymes. This work provides a convenient way for constructing portable visual multimodal biosensors, which is expected to applied in various disease diagnosis.

A colorimetric sensor based on multiple elements doped carbon dot nanozyme for rapid detection of 1-naphthol in human urine samples.

The residual carbaryl in crops can cause serious damage to the human kidney and nervous system after entering the human body, which may be metabolized to 1-naphthol (1-NAP) and excreted through urine. 1-NAP is often used as the biomarker for carbaryl exposure, so the intake or leakage of carbaryl can be monitored by detecting the concentration of 1-NAP. Herein, Co, N, P ternary co-doped carbon dots (CoNP-CDs) derived from vitamin B12 were synthesized by a facile hydrothermal method. CoNP-CDs exhibited oxidase-like activity and excellent peroxidase-like activity, which was attributed to the Fenton-like reaction of Co2+/Co3+ and the presence of pyrrole N and P elements, which together provided multiple active sites for chromogenic substrates. Due to the dual enzyme-like activity of CoNP-CDs, hydroxyl radicals (OH) and superoxide radicals (O2-) were generated during the catalytic process, which could rapidly oxidize colorless 3,3',5,5'-tetramethyl benzidine (TMB) to blue oxidation products (oxTMB). The α-carbon in 1-NAP can be attacked by OH, and the catalytic oxidation process of TMB can be inhibited by the consumption of OH, so that the blue color of the solution became lighter. Based on this principle, a smartphone-assisted colorimetric sensing platform was constructed for the detection of 1-NAP, and which resulted in a linear range of 1.07-37.3 µM and a visual detection limit of 0.68 µM. Moreover, the colorimetric sensing system showed satisfactory recoveries in the detection of human urine samples. The colorimetric sensing system owned the advantages of fast response, strong selectivity and simple operation, and provided a potential strategy for the on-site detection of 1-NAP.

Design, synthesis and biological evaluation of marine naphthoquinone-naphthol derivatives as potential anticancer agents.

1'-Hydroxy-4',8,8'-trimethoxy-[2,2'-binaphthalene]-1,4-dione (compound 5), a secondary metabolite recently discovered in marine fungi, demonstrates promising cytotoxic and anticancer potential. However, knowledge regarding the anticancer activities and biological mechanisms of its derivatives remains limited. Herein, a series of novel naphthoquinone-naphthol derivatives were designed, synthesised, and evaluated for their anticancer activity against cancer cells of different origins. Among these, Compound 13, featuring an oxopropyl group at the ortho-position of quinone group, exhibited the most potent inhibitory effects on HCT116, PC9, and A549 cells, with IC50 values decreasing from 5.27 to 1.18 µM (4.5-fold increase), 6.98 to 0.57 µM (12-fold increase), and 5.88 to 2.25 µM (2.6-fold increase), respectively, compared to compound 5. Further mechanistic studies revealed that compound 13 significantly induced cell apoptosis by increasing the expression levels of cleaved caspase-3 and reducing Bcl-2 proteins through downregulating the EGFR/PI3K/Akt signalling pathway, leading to the inhibition of proliferation in HCT116 and PC9 cells. The present findings suggest this novel naphthoquinone-naphthol derivative may hold potential as an anticancer therapeutic lead.

Recoverable and reusable light-induced multi-arm azobenzenes-Fe3O4 hybrid sorbent for enrichment of phthalate plasticizer and utilized as a SALDI substrate for the detection of 2-naphthol.

The construction, structural identifications along with compositional properties, using TEM, FT-IR, and XPS spectroscopies, of an innovative light-induced multi-arm azobenzenes based Fe3O4 magnetic nanoparticles (Azo-Fe3O4 MNPs) are being reported. Such organic (light-sensitive dendrimers-like structure), inorganic (magnet core), hybrid material has been applied as an efficient recoverable/reusable extractive sorbent for the detection of phthalate plasticizers from acetate buffer solution. The extraction study was controlled within consecutive procedures via UV-light exposure to achieve pore-size control which then further subjected for the evaluation of the analytes' retention, separation, and release as well as the detection of the phthalate pollutants using GCMS-. Various experimental conditions, such as time of extraction, salt concentration, pH, and desorption time, were studied and adjusted. Additionally, the extraction repeatability (RSD from 0.46 % to 6.12%, n = 5) of the studied sorbent was comparable to other published work. The linear range extended from 6.25 to 100 µg L-¹ and detection limits (LOD) within the range of 41- 150 ng L-1 were achieved, demonstrating good linearity with values ranging from 0.9992 to 0.9892. The inter-batch and intra-batch RSD ranged from 0.46 % to 6.12 %, respectively. Additionally, it provides effective detection of 2-naphthol when used as a SALDI substrate.

Preparation of a New Hexafluorobutanol-Farnesol Based Supramolecular Solvent and Its Application in Microextraction of Sudan Dyes From Beverage and Water Samples.

In this work, a new supramolecular solvent (SUPRAS) was prepared for the first time using hexafluorobutanol (HFB) and farnesol (FO). FO acts as an amphiphile and HFB as a coacervation inducer and density regulator. The method of dispersive liquid-liquid microextraction followed by high-performance liquid chromatography, supported by a vortex technique, was established using the prepared SUPRAS for the determination of Sudan dyes in aqueous samples. The extraction parameters, including FO content, HFB content, vortex time, salt addition, and solution pH, were thoroughly investigated and optimized. Under the optimized conditions, the linearity range is 10-750 ng/mL for Sudan I, and 10-1000 ng/mL for Sudan III and Sudan IV, respectively. The limits of detection for Sudan dyes were in the range of 0.8-3.1 ng/mL. The inter- and intra-day relative standard deviations for Sudan dyes were in the range of 1.3%-4.1% and 0.2%-2.6%, respectively. Finally, the proposed method was applied to the determination of Sudan dyes in beverage and river water samples with recoveries ranging from 93.9% to 122.1%. Compared to conventional techniques for the extraction of Sudan dyes in real samples, the proposed method is simpler, faster, and more environmentally friendly.

Colorimetric Detection of Aqueous N-Nitrosodimethylamine via Photonitrosation of a Naphtholsulfonate Indicator.

N-Nitrosamines are contaminants found throughout the environment, including in drinking water, and many nitrosamines are likely potent carcinogens. Correspondingly, there is a need for rapid and cost-effective in-field detection methods that can provide timely information about their contamination levels in water. This study details a colorimetric assay for detecting aqueous N-nitrosodimethylamine (NDMA) by photochemical nitrosation of a commercial naphtholsulfonate, to offer an attractive alternative to traditional laboratory-based analysis. The resulting naphthoquinone-oxime coordinates to aqueous iron(II) ions to form a green complex, allowing for direct visual detection. Characterization via Mössbauer and electron paramagnetic resonance (EPR) spectroscopy, alongside single-crystal structure determination, provides comprehensive structure information on the iron indicator complex. Optimization of detection conditions, including UV irradiation and response times, led to an improved colorimetric detection method with a limit of detection of 0.66 ppm for NDMA. The practical applicability and selectivity of this colorimetric detection scheme make it a promising candidate for the development of field-deployable sensors for NDMA in environmental water samples.

Preparation and evaluation of a 1,1'-bi-2-naphthol-based chiral macrocycle bonded silica chiral stationary phase for high performance liquid chromatography.

Macrocycles play vital roles in supramolecular chemistry and chromatography. 1,1'-Bi-2-naphthol (BINOL)-based chiral polyimine macrocycles are an emerging class of chiral macrocycles that can be constructed by one-step aldehyde-amine condensation of BINOL derivatives with other building blocks. These macrocycles exhibit good characteristics, such as facile preparation, rigid cyclic structures, multiple chiral centers, and defined molecular cavities, that make them good candidates as new chiral recognition materials for chromatographic enantioseparations. In this study, a BINOL-based [2+2] chiral polyimine macrocycle was synthesized by one-step condensation of enantiopure (S)-2,2'-dihydroxy-[1,1'-binaphthalene]-3,3'-dicarboxaldehyde with (1R,2R)-1,2-diaminocyclohexane. The product was modified with 5-bromo-1-pentene and then attached to thiolated silica using click chemistry to construct a new chiral stationary phase (CSP). The enantioselectivity of the new CSP was explored by separating various racemates under normal phase (NP) and reversed phase (RP) high performance liquid chromatography (HPLC). Thirteen racemates and eight racemates were enantioseparated under the two separation modes, respectively, including chiral alcohols, phenols, esters, ketones, amines, and organic acids. Among them, nine racemates achieved baseline separation under NP-HPLC and seven racemates achieved baseline separation under RP-HPLC. High resolution separation was observed with benzoin (Rs = 5.10), epinephrine (Rs = 4.98), 3-benzyloxy-1,2-propanediol (Rs = 4.42), and 4,4'-dimethylbenzoin (Rs = 4.52) in NP-HPLC, and with 4-methylbenzhydrol (Rs = 4.72), benzoin ethyl ether (Rs = 3.79), 1-phenyl-1-pentanol (Rs = 3.68), and 1-(3-bromophenyl)ethanol (Rs = 3.60) in RP-HPLC. Interestingly, the CSP complemented Chiralcel OD-H, Chiralpak AD-H, and CYCLOBOND I 2000 RSP columns for resolution of these test racemates, separating several racemic compounds that could not be well separated by the three commercially available columns. The influences of injected sample amount on separation were also evaluated. It was found that the column exhibited excellent stability and reproducibility after hundreds of injections, and the relative standard deviations (n = 5) of the retention time and resolution were less than 0.49% and 0.69%, respectively. This study indicates that the BINOL-based chiral macrocycle has great potential for HPLC enantioseparation.

Development of a dispersive solid phase microextraction method based on the application of MgAl, NiAl, and CoAl-layered double hydroxides for the efficient removal of α- and ß-naphthol isomers from water samples by capillary electrophoresis.

The present work describes a quick, simple, and efficient method based on the use of layered double hydroxides (LDH) coupled to dispersive solid phase micro-extraction (DSPME) to remove α-naphthol (α-NAP) and ß-naphthol (ß-NAP) isomers from water samples. Three different LDHs (MgAl-LDH, NiAl-LDH, and CoAl-LDH) were used to study how the interlayer anion and molar ratio affected the removal performance. The critical factors in the DSPME procedure (pH, LDH amount, contact time) were optimized by the univariate method under the optimal conditions: pH, 4-8; LDH amount, 5 mg; and contact time, 2.5 min. The method can be successfully applied in real sample waters, removing NAP isomers even in ultra-trace concentrations. The large volume sample stacking (LVSS-CE) technique provides limits of detections (LODs) of 5.52 µg/L and 6.36 µg/L for α-naphthol and ß-naphthol, respectively. The methodology's precision was evaluated on intra- and inter-day repeatability, with %RSD less than 10% in all cases. The MgAl/Cl--LDH selectivity was tested in the presence of phenol and bisphenol A, with a removal rate of >92.80%. The elution tests suggest that the LDH MgAl/Cl--LDH could be suitable for pre-concentration of α-naphthol and ß-naphthol in future works.

Geosmin, a Food- and Water-Deteriorating Sesquiterpenoid and Ambivalent Semiochemical, Activates Evolutionary Conserved Receptor OR11A1.

Geosmin, a ubiquitous volatile sesquiterpenoid of microbiological origin, is causative for deteriorating the quality of many foods, beverages, and drinking water, by eliciting an undesirable "earthy/musty" off-flavor. Moreover, and across species from worm to human, geosmin is a volatile, chemosensory trigger of both avoidance and attraction behaviors, suggesting its role as semiochemical. Volatiles typically are detected by chemosensory receptors of the nose, which have evolved to best detect ecologically relevant food-related odorants and semiochemicals. An insect receptor for geosmin was recently identified in flies. A human geosmin-selective receptor, however, has been elusive. Here, we report on the identification and characterization of a human odorant receptor for geosmin, with its function being conserved in orthologs across six mammalian species. Notably, the receptor from the desert-dwelling kangaroo rat showed a more than 100-fold higher sensitivity compared to its human ortholog and detected geosmin at low nmol/L concentrations in extracts from geosmin-producing actinomycetes.

Enantioselective synthesis of all stereoisomers of geosmin and of biosynthetically related natural products.

Synthetic routes to geosmin and its enantiomer are well established, but the enantioselective synthesis of stereoisomers of geosmin is unknown. Here a stereoselective synthesis of all stereoisomers of geosmin is reported, yielding all compounds in high enantiomeric purity. Furthermore, the stereoselective synthesis of a geosmin derivative isolated from a mangrove associated streptomycete was performed, establishing the absolute configuration of the natural product. Finally, a new side product of the geosmin synthase from Streptomyces ambofaciens was isolated and its structure was elucidated by NMR spectroscopy. The absolute configuration of this new compound was determined through a stereoselective synthesis.

Digital multimeter-based portable photoelectrochemical immunoassay with enzyme-catalyzed precipitation for screening carbohydrate antigen 125.

The degree of the carbohydrate antigen 125 (CA-125) level in serum is positively correlated with the severity of ovarian cancer. In this study, a facile photoelectrochemical (PEC) immunoassay was devised for sensitive detection of CA-125 employing enzyme-catalyzed precipitation to weaken the photocurrent of hollow porous In2O3 nanotubes incorporating CdS nanoparticles. Upon the addition of the target analyte, horseradish peroxidase (HRP) enriches as a result of the formation of the sandwich immunocomplex, which can catalyze the conversion of 4-chloro1-naphthol (4-CN) to benzo-4-chlorohexadienone (4-CD) employing H2O2 as a cofactor. The as-produced insoluble precipitate acts as an obstacle to hinder the absorption of visible light by photoactive materials, thereby resulting in a decrease in photocurrent. Moreover, the weakened signal can be easily read out by a digital multimeter (DMM), advancing the convenience of the detection system. The preliminary analysis data indicate that the PEC immunoassay shows an efficient response to CA-125 levels ranging from 0.1 to 100 U mL-1 with a limit of detection (LOD) as low as 0.046 U mL-1 (S/N = 3). Most importantly, the proposed portable method has shown satisfactory performance in terms of selectivity, reproducibility, stability, and analysis in complex biological matrices.

Photoelectrochemical immunoassay of cardiac troponin I based on ZnTCPP/CdIn2S4 type-II heterojunction and co-catalyzed precipitation biolabeling.

CdIn2S4 and zinc tetrakis(4-carboxyphenyl)porphyrin (ZnTCPP) were synthesized by hydrothermal method, and an organic dye-sensitized inorganic semiconductor ZnTCPP/CdIn2S4 type II heterojunction was constructed on a fluorine-doped tin oxide (FTO) substrate electrode. A sandwich immunostructure for signal-attenuation photoelectrochemical (PEC) detection of cardiac troponin I (cTnI) was constructed using the ZnTCPP/CdIn2S4/FTO photoanode and a horseradish peroxidase (HRP)-ZnFe2O4-Ab2-bovine serum albumin (BSA) immunolabeling complex. The bioenzyme HRP and the HRP-like nanozyme ZnFe2O4 can co-catalyze the oxidation of 4-chloro-1-naphthol (4-CN) by H2O2 to produce an insoluble precipitate on the photoanode, thus notably reducing the anodic photocurrent for quantitative determination of cTnI. Under the optimal conditions, the photocurrent at 0 V vs. SCE in 0.1 M phosphate buffer solution (pH 7.40) containing 0.1 M ascorbic acid was linear with the logarithm of cTnI concentration from 500 fg mL-1 to 50.0 ng mL-1, and the limit of detection (LOD, S/N = 3) is 0.15 pg mL-1. Spiked recoveries were 95.1% ~ 104% for assay of cTnI in human serum samples.

An enantioselective fluorescent probe for detecting arginine and glutamic acids.

Amino acids are important chiral compounds in the human body, and are important basic components that make up the human body and play an important role in the human body. Among them, different enantiomers of an amino acid may have different roles, and different types of amino acids can be interconverted. However, the content of D-amino acids is much lower than that of L-amino acids, which is difficult to be detected. At present, many of the potential roles of D-amino acids, such as the conversion of D-amino acids to each other, have not yet been fully revealed. Hence, we synthesized fluorescent probe (R)-5 by condensation of 1,1'-Bi-2-naphthol (BINOL) and 2-(Aminomethyl)pyridine with Schiff base, which can recognize both D-arginine and D-glutamic acid at low concentrations. Meanwhile, (R)-5 can be applied to paper-based sensors for the detection of arginine and glutamate in living cells and for food amino acid detection.

Construction of a mitochondria-targeted probe to monitor cysteine levels in cancer cells and zebrafish.

Cysteine (Cys) plays an indispensable role as an antioxidant in the maintenance of bioredox homeostasis. We have constructed an efficient fluorescent probe Mito-Cys based on the binding of indole and naphthol. The acrylic ester group serves as a recognition switch for specific detection of Cys, which undergoes Michael addition and intramolecular cyclization reactions, thereby ensuring the chemical kinetics priority of Cys compared to other biothiols. The probe has good water solubility, large Stokes shift (137 nm), with a detection limit of 21.81 nM. In addition, cell imaging experiments have shown that the probe has excellent mitochondrial targeting ability (R = 0.902). The probe can distinguish between Cys, homocysteine (Hcy) and glutathione (GSH), and can detect Cys specifically and quickly (100 s) to ensure accurate quantitative analysis of Cys changes in cells. More importantly, the probe confirms that ferroptosis inducing factors trigger thiol starvation in mitochondria, which helps to gain a deeper understanding of the physiological and pathological functions related to Cys and ferroptosis.

Circular dichroism spectrum of (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol in albumin: Alterations caused by complexation-Experimental and in silico investigation.

This study describes the interaction of human serum albumin (HSA) with the binol derivative (R)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (R-BrB), which has its optical activity based on the prohibitive energetic barrier for conversion into the enantiomer (S)-(+)-3,3'-dibromo-1,1'-bi-2-naphthol (S-BrB). The objective was to assess the ability of HSA to differentiate axial enantiomers based on their binding efficiency and their impact on the CD spectra. We discovered that both enantiomers were effective ligands, and the CD signal disappeared when equimolar amounts of R-BrB and S-BrB were simultaneously added, indicating no preference for either enantiomer. The complexation resulted in a significant signal increase at 250 nm and a bathochromic effect at 370 nm. Molecular docking simulations were performed, and the lower energy pose of R-BrB was selected for DFT calculations. The theoretical CD spectra of free and complexed R-BrB were obtained and showed alterations corroborating the experimental results. By comparing the difference spectrum (HSA:R-BrB minus HSA) with the spectrum of free RBrB in water or ethyl alcohol, we concluded that the CD signal intensification was due to the increased solubilization of R-BrB upon binding to HSA.

Enantiopure Corral[4]BINOLs as Ultrastrong Receptors for Recognition and Differential Sensing of Steroids.

The precise recognition and sensing of steroids, a type of vital biomolecules, hold immense practical value across various domains. In this study, we introduced corral[4]BINOLs (C[4]BINOLs), a pair of enantiomeric conjugated deep-cavity hosts, as novel synthetic receptors for binding steroids. Due to the strong hydrophobic effect of their deep nonpolar, chiral cavities, the two enantiomers of C[4]BINOLs demonstrated exceptionally high recognition affinities (up to 1012 M-1) for 16 important steroidal compounds as well as good enantioselectiviy (up to 15.5) in aqueous solutions, establishing them as the most potent known steroid receptors. Harnessing their ultrahigh affinity, remarkable enantioselectivity, and fluorescence emission properties, the two C[4]BINOL enantiomers were employed to compose a fluorescent sensor array which achieved discrimination and sensing of 16 structurally similar steroids at low concentrations.

Heterologous production of (-)-geosmin in Saccharomyces cerevisiae.

(-)-Geosmin has high demand in perfumes and cosmetic products for its earthy congenial aroma. The current production of (-)-geosmin is either by distillation of sun-baked soil or by inefficient chemical synthesis because of the presence of multiple chiral centers. Fermentation processes are not viable as the titers of the Streptomyces sp. based processes are low. This work presents an alternative route by the heterologous synthesis of (-)-geosmin in Saccharomyces cerevisiae. The enzyme involved is the bifunctional geosmin synthase that catalyzes the conversion of farnesyl diphosphate to germacradienol and germacradienol to geosmin. This study evaluated the activity of many orthologs of geosmin synthase when expressed heterologously in S. cerevisiae. When the well-characterized CAB41566 from Streptomyces coelicolor origin was tested, germacradienol and germacrene D were detected but no geosmin. Bioinformatic analysis based on high/low identities to N-terminal and C-terminal domains of CAB41566 was carried out to identify different orthologs of geosmin synthase proteins from different bacterial and fungal origins. ADO68918 of Stigmatella aurantiaca origin showed the best activity among the tested orthologs, not only in terms of geosmin production but also an order of magnitude higher total abundance of the products of geosmin synthase as compared to CAB41566. This study successfully demonstrated the production of (-)-geosmin in S. cerevisiae and offers an alternative, sustainable and environment-friendly approach to producing (-)-geosmin.

Polyketide Trimming Shapes Dihydroxynaphthalene-Melanin and Anthraquinone Pigments.

Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.

Improved Quantification of Geosmin and 2-Methylisoborneol in Farmed Fish Using Stable Isotope Dilution Gas Chromatography-Mass Spectrometry.

A requisite to improving the taste and odor attributes of farmed fish is the availability of accurate and practical analytical methods to quantify 2-methylisoborneol (MIB) and geosmin (GSM). Solid-phase microextraction (SPME) enables reliable measurement of nanogram per liter quantities of MIB and GSM in water. In contrast, direct headspace (HS)-SPME of biological matrices with variable proximate compositions can increase bias and uncertainty in off-flavor determinations. Analytical recovery plays a crucial role in the accurate determination of MIB and GSM in fish, and this study investigates strategies to maximize and account for this recovery factor. MIB and GSM values in off-flavor catfish and trout were measured using direct HS-SPME and distillation as sample preparation techniques. Trout samples prepared by distillation yielded 10-fold higher GSM recoveries than those from direct HS-SPME (31% versus 3%). A stable isotope dilution method (SIDM) was implemented by routinely spiking samples with known quantities of deuterium-labeled MIB and GSM, allowing for the correction of sample-to-sample recovery deviations. SIDM-determined GSM values generated recoveries of 106 and 95% for direct HS-SPME and distilled trout, respectively. Aspects of the strategies and techniques presented can be incorporated into existing analytical methods to improve the accuracy and sample throughput. Particularly, routine inclusion of SIDM in the evaluation of MIB and GSM can facilitate identification of reliable practices to control off-flavors in aquaculture.

Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells.

Selective labeling of the protein of interest (POI) in genetically unmodified live cells is crucial for understanding protein functions and kinetics in their natural habitat. In particular, spatiotemporally controlled installation of the labels on a POI under light control without affecting their original activity is in high demand but is a tremendous challenge. Here, we describe a novel ligand-directed photoclick strategy for spatiotemporally controlled labeling of endogenous proteins in live cells. It was realized with a designer labeling reagent skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released and underwent a proximity coupling reaction with nucleophilic amino acid residues on the POI in live cells. With fluorescein as a marker, this photoclick strategy enables time-resolved labeling of carbonic anhydrase subtypes localized either on the cell membrane or in the cytoplasm and a discriminable visualization of their metabolic kinetics. Given the versatility underlined by facilely tethering other functional entities (e.g., biotin, a peptide short chain) via acylation or (in cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry opens up enormous opportunities for discovering dynamic functions and mechanistic interrogation of endogenous proteins in live cells.