Fluorescence Turn-on Research Articles

A fluorescence-electrochemiluminescence dual-mode sensor based on a "switch" system for highly selective and sensitive K-ras gene detection

Herein, an fluorescence (FL)−electrochemiluminescence (ECL) dual-mode biosensor is constructed based on the dual-signal “turn-on” strategy of functionalized metal-organic frameworks nanosheets (RuMOFNSs)-tetraferrocene for K-ras gene detection, and the mechanism of bursting through front-line orbital theory is explained for the first time. Amino-functionalized tetraferrocene-labeled probe DNA molecules are linked to RuMOFNSs by covalent amide bonds, acting as FL and ECL intensity switches. The target DNA, complementary to the probe DNA, triggers cyclic amplification of the target by nucleic acid exonuclease III (Exo III), repelling tetraferrocene reporter groups away from RuMOFNSs and inhibiting the electron transfer process and photoinduced electron transfer (PET) effect. These phenomena induce a double turn-on of FL and ECL signals with a high signal-to-noise ratio. The developed FL-ECL dual-mode sensing platform provides sensitive detection of the K-ras gene with detection limits of 0.01 fM (the detection range is 1 fM to 1 nM) and 0.003 fM (the detection range is 0.01 fM to 10 pM), respectively. In addition, the proposed dual-mode sensor can be easily extended to detect other disease-related biomarkers by changing the specific target and probe base sequences, depicting potential applications in bioanalysis and early disease diagnosis.

Fluorescence turn-on detection of human serum albumin based on the assembly of gold nanoclusters and bromocresol green.

As the most abundant protein in plasma, human serum albumin plays a vital role in physiological processes, such as maintaining blood osmotic pressure and carrying small-molecule ligands. Since the content of albumin in the human serum can reflect the status of liver and renal function, albumin quantitation is significant in clinical diagnosis. In this work, fluorescence turn-on detection of human serum albumin (HSA) had been performed based on the assembly of gold nanoclusters and bromocresol green. Gold nanoclusters (AuNCs) capped by reduced glutathione (GSH) were assembled with bromocresol green (BCG), and the assembly was used as a fluorescent probe for HSA. After BCG assembling, the fluorescence of gold nanoclusters was nearly quenched. In acidic solution, HSA can selectively bind to BCG on the assembly and recover the fluorescence of the solution. Based on this turn-on fluorescence, ratiometric HSA quantification was realized. Under optimal conditions, HSA detection by the probe possessed agood linear relationship in the range of 0.40-22.50mg·mL-1, and the detection limit was 0.27 ± 0.04mg·mL-1 (3σ, n = 3). Common coexisting components in serum and blood proteins did not interfere with the detection of HSA. This method has the advantages of easy manipulation and high sensitivity, and the fluorescent response is insensitive to reaction time.

Confined construction of CDs@MIL-101 as reusable and turn-on fluorescence sensor for highly sensitive water detection based on adsorption-solvent synergistic mechanism

Fabrication a reusable and highly sensitive fluorescence sensor in a controllable manner for water detection in solvents and humidity remains a top priority target yet a challenging task. In this work, carbon dots (CDs) are successfully implanted into confined metal–organic framework MIL-101(Cr) cages via “ship in the bottle” strategy and the confined CDs are firstly observed in MIL-101(Cr) cage showing high dispersion and high N content by advanced iDPC STEM technique. The highly selective water capture and sensitive changes of the fluorescence property with solution polarity and hydrogen-bonding donor acidity efficiently enhance the detection sensitivity and realize reusable turn-on detection based on the adsorption-solvent synergistic mechanism. Consequently, CDs@MIL-101 exhibits high sensitivity (detection limit: 0.036% and 0.028% v/v), wide response range (0.04–100% and 0.03–45%) and reusable stability for liquid water detection in acetone and tetrahydrofuran, respectively. Meanwhile, it can also detect gaseous water and the detection range is 23% to 85%. The analysis of real samples confirms the proposed method. Such construction strategy and detection mechanism will offer guidance for optimizing and expanding the detection performance of CDs, which is of great significance in the design and construction of novel multiphase sensors.

Encoded Fusion-Mediated MicroRNA Signature Profiling of Tumor-Derived Extracellular Vesicles for Pancreatic Cancer Diagnosis.

MicroRNAs (miRNAs) in tumor-derived extracellular vesicles (tEVs) are important cancer biomarkers for cancer screening and early diagnosis. Multiplex detection of miRNAs in tEVs facilitates accurate diagnosis but remains a challenge. Herein, we propose an encoded fusion strategy to profile the miRNA signature in tEVs for pancreatic cancer diagnosis. A panel of encoded-targeted-fusion beads was fabricated for the selective recognition and fusion of tEVs, with the turn-on fluorescence signals of molecule beacons for miRNA quantification and barcode signals for miRNA identification using readily accessible flow cytometers. Using this strategy, six types of pancreatic-cancer-associated miRNAs can be profiled in tEVs from 2 μL plasma samples (n = 36) in an isolation-free and lysis-free manner with only 2 h of processing, offering a high accuracy (98%) to discriminate pancreatic cancer, pancreatitis, and healthy donors. This encoded fusion strategy exhibits great potential for multiplex profiling of miRNA in tEVs, offering new avenues for cancer diagnosis and screening.

Benzothiazole-based novel fluorescence probe sensing 1, 3-diaminopropane

Amines are extensively present in biological systems and are abundantly used in research, industries and agriculture. Systematic detection and quantification of certain amines can help us in food quality control and diagnosis of many diseases. A Schiff base probe HL was designed and successfully synthesized. It was proposed as a sensor for the exclusive detection of 1, 3- diaminopropane through turn-on fluorescence response in a variety of solvents including water. Micromolar limits of detection was achieved in all these solvents. Mechanism of detection was proposed by investigating mass spectrometric and NMR results. These were corroborated with DFT/TD-DFT calculations. Spiking experiments performed in various real water samples revealed the potential of the sensor to be used in day-to-day applications. Paper strip experiments demonstrated the suitability of the probe for real-life applications.

Visualizing Drug Release from a Stimuli-Responsive Soft Material Based on Amine-Thiol Displacement.

In this research, we developed a photoluminescent platform using amine-coupled fluorophores, generated from a single conjugate acceptor containing bis-vinylogous thioesters. Based on the experimental and computational results, the fluorescence turn-on mechanism was proposed to be charge separated induced energy radiative transition for the amine-coupled fluorophore, while the sulfur-containing precursor was not fluorescent since the energy internal conversion occurred through vibrational 2RS- (R represents alkyl groups) as energy acceptor(s). Further utilizing the conjugate acceptor, we establish a new fluorogenic approach via a highly cross-linked soft material to selectively detect cysteine under neutral aqueous conditions. Turn-on fluorescence emission and macroscopic degradation occurred in the presence of cysteine as the stimuli, which can be visually tracked due to the generation of an optical indicator and the cleavage of linkers within the matrix. Furthermore, a novel drug delivery system was constructed, achieving controlled release of sulfhydryl drug (6-mercaptopurine) which was tracked by photoluminescence and high-performance liquid chromatography. The photoluminescent molecules developed herein are suitable for visualizing polymeric degradation, making them suitable for additional "smart" material applications.

Semicarbazide-based fluorescent probe for detection of Cu2+ and formaldehyde in different channels

Two fluorescent sensors with the receptor semicarbazide respectively at 7- (CAA) and 3-position (CAB) of coumarin were designed and synthesized. CAA exhibits fluorescence turn-on response to Cu2+ by triggering the intramolecular charge transfer (ICT) process via Cu2+-catalyzed hydrolysis, and can detect formaldehyde (FA) at different channel by inhibiting the photo-induced electron transfer (PET). However, CAB displays quite different responses: the photophysical properties hardly changed in the presence of FA; while a three-stage fluorescence response of fast quenching, steady increasing and slowly decreasing was found upon addition of Cu2+. The high selectivity enabled CAA a good candidate for quantification of Cu2+ and formaldehyde as well as bioimaging Cu2+ in living cells. Good linear relationships between the fluorescence intensity and analyte concentration were observed in the range of 0.1–30 μM for Cu2+ and 1.0–50 μM for FA, and their detection limits (LOD) were calculated to be 0.43 μM and 1.92 μM (3δ/k), respectively.

Fluorescent Turn-On Sensing of Zinc(II) and Alkaline Phosphatase Activity Using a Pyridoxal-5'-Phosphate Derived Schiff Base.

A novel Zn2+ ion and alkaline phosphatase (ALP) selective fluorescence turn-on sensor L was developed by reacting pyridoxal 5'-phosphate (PLP) with hydrazine. Sensor L shows significant flurescence enhancement at 476nm due to the formation of a L-Zn2+ complex in 1:1 binding stoichiometry with the association constant of 3.1⋅104M- 1. Using L, the concentration of Zn2+ can be detected down to 2.34 µM, and the practical utility of L was validated by quantifying Zn2+ in real water samples. Additionally, the receptor L was applied to mimic the dephosphorylation reaction catalysed by the enzyme ALP and the resulted fluorescence change was monitored to detect the ALP activity.

A "Turn-on" Fluorescent Probe Based on Phenothiazine for Selectively Recognizing ClO- and its Practical Applications.

Hypochlorous acid (HClO), a highly reactiveoxygen species, has important effects on human health. High selectivity and sensitivity remain challenges of fluorescent probes for detection of ClO- with a large Stokes shift. This work designed and synthesized a novel phenothiazine-based fluorescent probe TF which can detect ClO- by colorimetric and fluorescent dual signals. TF displayed turn-on fluorescence effect toward ClO- with high selectivity (≥ 28-folds) and sensitivity (LOD = 0.472μM), fast response time (< 1min) and large Stokes shift (150nm) in PBS (pH = 7.4, 40% DMSO). Meanwhile, TF can visualize ClO- on the mung bean sprouts model and apply as testing strips for portable and rapid detecting ClO- by the naked eyes. A phenothiazine-based fluorescent probe with large Stokes shift was synthesized and its responding rapidly ability to detect ClO- was studied.

Kiwi juice stabilized gold nanoclusters for fluorescence turn-on detection of copper ions

Gold nanoclusters (AuNCs)-based fluorescent probes with fluorescence turn-on property are necessary to address the challenges in fluorescence turn-off processes for sensing of copper ions (Cu2+) in present. Herein, we described a simple one-step method for fabrication of [email protected] with kiwi juice as both a reducing reagent and a capping ligand, and demonstrated its fluorescent probe's role. With addition of Cu2+, the fluorescence intensity of [email protected]2+ was enhanced obviously. The sensing principle was explored. The charge transfer between surface ligand and Cu2+, and the rigid structure of [email protected]2+ served important roles for monitoring of Cu2+. Then, a fluorescence turn-on protocol was successfully developed for highly selective detection of Cu2+ with a good linear relationship (R2 = 0.992) between the fluorescence intensity and the Cu2+ concentration, ranging from 5.0 µM to 35.0 µM. The limit of detection was 1.6 µM. Furthermore, after intraperitoneal injection, the Cu2+ metabolism process in rats was investigated with the proposed [email protected] as the fluorescent probes. It provides a feasible strategy for preparation of fruit juice protected AuNCs and opens an avenue to realize Cu2+ detection in real bio-samples with the AuNCs as the fluorescence turn-on probes.

Screening of aggregation-induced emission and multi-response acrylonitrile-bridging fluorescent molecules tailored for rapid turn-on detection of HClO as well as ratiometric visualizing of extreme basicity.

Realizing the rapid and sensitive tracing of multiple analysis indicators using single molecular probe through structural designing is urgently desired for exploring novel multi-response chemosensors. Herein, a series of acrylonitrile-bridging organic small molecules have been rationally designed. Among these donor-π-acceptor (D-π-A) compounds with efficient aggregation-induced emission (AIE) characteristics, a unique derivative, 2-(1H-benzo[d]imidazole-2-yl)-3-(4-(methylthio)phenyl) acrylonitrile, named MZS, has been screened out for multifunctional utilizing. First, probe MZS can respond to hypochlorous acid (HClO) through specific oxidation reaction, showing a marked fluorescence turn-on signal (I495). This special sensing reaction is ultra-fast with a rather low detection limit (LOD=13.6nM). Next, versatile MZS is also sensitive to the extreme pH fluctuation, displaying an intriguing ratiometric signal variation (I540/I450), facilitating the real-time and naked-eye visualizing, which is even stable and reversible. Furthermore, probe MZS has been used for the monitoring of HClO in real water and commercially available disinfectant spray samples with satisfactory results. We envision that probe MZS would be a flexible and powerful tool for monitoring of environmental toxicity and industrial operations under realistic scenarios.

Metal ion-induced assembly of dipeptide-attached perylenediimide for fluorometric "turn on" detection of biologically important small molecule.

A dipeptide-appended perylenediimide (PDI-CFF) fluorescent molecule was designed, synthesized, and characterized. Though the molecule does not dissolve in any individual solvent, it dissolves well in an organic/water mixed solvent system such as tetrahydrofuran/water. This new fluorescent molecule was self-assembled in a tetrahydrofuran/water mixture to form both nanofibrous network structures and a nano ring structure. It has shown nanofibril morphology by the interactions with ferric ions (PDI-CFF/Fe3+ system) with diminishing fluorescent property. Interestingly, L-ascorbic acid (LAA) interacts with the PDI-CFF/Fe3+ system, showing turn-on fluorescence. Another interesting feature is that the minimum detection limits for Fe3+ ions and LAA are at the submicromolar levels of 6.2 × 10-8 and 3 × 10-8 M, respectively. Moreover, the fluorescent (10μM) signals can be monitored by the naked eye under handheld UV lamp irradiation at 365 nm, and this is very convenient for the real application. In this study, the molecule offers the opportunity for processing these sequential fluorescence responses in order to fabricate a implication logic gate that includes NOT, AND, and OR simple logic gates using chemical stimuli (ferric ions and LAA) as inputs and fluorescence emission at 536 nm as output. The detailed mechanism of interactions of Fe3+ with PDI-CFF and LAA with the PDI-CFF/Fe3+ system is vividly studied by using Fourier transform infrared (FT-IR) analysis and fluorescence. Moreover, this new molecule was reusable for several times without significant loss of its activity. The construction of logic gates using biologically important molecules/ions holds future promise for the design and development of new bio-logic gates.

Analyte-Triggered Excited-State Intramolecular Proton Transfer- Delayed Fluorescence: A General Approach for Time-Resolved Turn-On Fluorescence Imaging.

The research of delayed fluorescence (DF) has been a hot topic in biological imaging. However, the development of analyte-triggered small molecule DF probes remains a considerable challenge. Herein a novel excited-state intramolecular proton transfer-delayed fluorescence (ESIPT-DF) approach to construct analyte-stimulated DF probes was reported. These new classes of ESIPT-DF luminophores were strategically designed and synthesized by incorporating 2-(2'-hydroxyphenyl)benzothiazole (HBT), a known ESIPT-based fluorophore, as acceptor with a series of classic donor moieties, which formed a correspondingly twisted donor-acceptor pair within each molecule. Thereinto, HBT-PXZ and HBT-PTZ exhibited significant ESIPT and DF characters with lifetimes of 5.37 and 3.65 μs in the solid state, respectively. Furthermore, a caged probe HBT-PXZ-Ga was developed by introducing a hydrophilic d-galactose group as the recognition unit specific for β-galactosidase (β-gal) and ESIPT-DF blocking agent and applied to investigate the influence of metal ions on β-gal activity on the surface of Streptococcus pneumoniae as a convenient tool. This ESIPT-DF "turn-on" approach is easily adaptable for the measurement of many different analytes using only a predictable modification on the caged group without modification of the core structure.

New "Destruction Seek to Survive" Strategy Based on a Serum Albumin Assembly with a Squaraine Molecule for the Detection of Peroxynitrite.

Peroxynitrite (ONOO-), a kind of active nitrogen species, plays an important role in biological systems. Overproduction of ONOO- is closely related to the pathogenesis of many diseases. Therefore, it is necessary to quantify intracellular ONOO- for differentiating health and disease states. Fluorescent probes with near-infrared (NIR) fluorescence can detect ONOO- with high sensitivity and selectivity. However, there is an inevitable problem that many NIR fluorophores are easily oxidized by ONOO- to give a false-negative result. To avoid this problem, herein, we ingeniously propose a "destruction to seek to survive" strategy to detect ONOO-. Two NIR squaraine (SQ) dyes were connected together to form a fluorescent probe (SQDC). This method utilizes the destructive effect of peroxynitrite on one of the SQ moieties of SQDC to eliminate the steric hindrance, enabling the other "survived" SQ segment to enter the hydrophobic cavity of bovine serum albumin (BSA) via the well-known host-guest interactions. The encapsulation of albumin protects the "survived" SQ from further attack of ONOO-. As a result, a NIR fluorescence turn-on response coming from the host-guest interaction between BSA and the "survived" SQ escaped from SQDC was found, which can be used for the detection of ONOO-. The assembly of SQDC mixed with BSA can be located in mitochondria to detect endogenous and exogenous ONOO- sensitively in living cells. As a proof-of-concept method, it is envisioned that this novel detection strategy with a simple assembly would become a powerful means for the detection of ONOO- when employing NIR fluorophores.

A novel near-infrared fluorescent probe with hemicyanine scaffold for sensitive mitochondrial pH detection and mitophagy study

Mitochondria, as an energy-producing powerhouse in live cells, is considered to be directly linked to cellular health. However, dysfunctional mitochondria and abnormal mitochondria pH would possibly activate mitophagy, cell apoptosis and intercellular acidification process. In this work, we synthesized a novel near infrared fluorescent probe (FNIR-pH) for measurement of mitochondrial pH based on the hemicyanine skeleton as a fluorophore. The FNIR-pH probe functioned as a mitochondrial pH substrate and exhibited quick and sensitive turn-on fluorescence responses to mitochondrial pH in basic solution due to the deprotonation of hydroxy group in the structure. From pH 3.0 to 10.0, the FNIR-pH exhibited almost 100-fold increase in fluorescence intensity at 766 nm wavelength. The FNIR-pH also displayed superior selectivity to various metal ions, excellent photostability, and low cytotoxicity, which facilitated further biological application. Owing to the proper pKa value of 7.2, the FNIR-pH paved the way for real-time monitoring of mitochondria pH changes in live cells and sensitive sensing of mitophagy. Moreover, the FNIR-pH probe was also implemented for fluorescent imaging of tumor-bearing mice to validate its potential application for in vivo imaging of bioanalytes and biomarkers.

Esterase Specific Fluorescent Probe: Mechanistic Understanding Using QM/MM Calculation and Cell States Discrimination.

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase's active site for hydrolyzing the substrate. In addition, the fluorescent turn-on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM-OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD-DFT calculation. Moreover, the substrate (PM-OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image-based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity.

Förster Resonance Energy Transfer between Multicolor Emissive N-Doped Carbon Quantum Dots and Gold Nanorods for the Detection of H2O2, Glucose, Glutathione, and Acetylcholinesterase

Multicolor emissive nitrogen-doped carbon quantum dots (CQDs) were synthesized by the hydrothermal method and characterized using spectroscopic and electron microscopic techniques. Förster resonance energy transfer (FRET) between CQDs and gold nanorods (AuNRs) has been investigated that occurs due to the distinctive overlap of transverse and longitudinal bands of AuNRs with the fluorescence (FL) spectra of CQDs. Significant FL quenching (turn-off) of CQDs by AuNRs and their recovery (turn-on) have been observed due to etching of AuNRs and end-to-end assemblies that lead to the detection of biomolecules like H2O2, glucose, glutathione (GSH), and acetylcholinesterase (AChE). The redox reaction of Au(0) with H2O2 results in the decomposition of AuNRs to give Au(I) ions, thereby inducing the fluorescence recovery of CQDs (turn-on). The interruption of the FRET phenomenon by the production of H2O2 from the reaction of glucose oxidase in the presence of glucose and a thiol-containing compound from the reaction of acetylthiocholine and the AChE enzyme causes the FL recovery of CQDs, respectively. Moreover, the assembly of AuNRs leads to the FRET disruption, and FL turn-on signals were found to be measures of GSH. In comparison to the UV–visible approach, the FL measurements through the FRET process are found to be more sensitive under the same reaction conditions. The practical applicability of the proposed sensing system has been verified using human plasma samples.

The switchable fluorescent detection of mercury, copper, and iodine in real water samples using bola-amphiphilic-tetraphenylethene

The main objectives of this study are the synthesis of fluorescent bola-amphiphilic-TPE and investigations of its wide range of photochemical properties. Initially, the TSC-TPE-TSC was synthesized for this purpose. Then, using UV–Vis and fluorescence spectroscopy, the AIE studies and interactions of TSC-TPE-TSC with a variety of metal ions were investigated in tap water, distilled water, and EtOH/H2O. The studies revealed that the TSC-TPE-TSC had turn-off sensor characteristics against Hg2+ and Cu2+ ions. Additionally, the interactions of TSC-TPE-TSC, TSC-TPE-TSC-Hg2+, and TSC-TPE-TSC-Cu2+ with anions were also studied in EtOH/H2O (v/v:1, pH 7).The TSC-TPE-TSC-Hg2+ displayed specific turn-on fluorescence characteristics against I¯ ions. Moreover, the computed LOD values for TSC-TPE-TSC/metals (Cu2+ or Hg2+) and TSC-TPE-TSC-Hg2+/I¯ were 2.42 (for Cu2+), 0.39 (for Hg2+), and 3.70 (for I¯), respectively. On the other hand, 1H NMR, FTIR, and DFT experiments were used to determine the binding's morphology. In all reversal studies, the alternate combination of HgCl2 and [Bu4N]I to TSC-TPE-TSC produced switchable off/on variation in all reversal assays. In the end, efficient methods were created in tap water, distilled water, and EtOH/H2O solvent systems for the detection of Hg2+, Cu2+, and I¯.

Red-emitting Styryl-BODIPY dye with donor-π-acceptor architecture: Selective interaction with serum albumin via disaggregation-induced emission

Serum albumin (SA) is an abundant protein in mammalian blood plasma and a biomarker for various diseases, such as cirrhosis or hepatitis in the case of the liver. The research of fluorescent probes for SA based on host-guest interactions provides an opportunity to study the nature of intermolecular interactions and thus reveals the structure information of SA at the molecular level. However, some commercially available fluorescent probes, such as boron dipyrromethenes (BODIPYs) have a general tendency to form aggregates under an aqueous environment, which is a drawback for their use due to the strong quenching of the emission. Additionally, short emission wavelength and small Stokes shift are the other common shortcomings. In this work, we explore this unfavourable property and provide an effective method for developing an “off-on” type probe for SA. A new probe named BOD-Red with red-emitting aggregation/disaggregation characters was then designed, consisting of donor-π-acceptor (D-π-A) architecture where boron-dipyrromethene (BODIPY) core as electron acceptor and N-phenylpiperazine as electron donor. The extended π-conjugation system provides not only the increase of emission wavelength due to the enhancement of intramolecular charge transfer strength, but also forms a higher rigid coplanar backbone, which can modulate the photophysical properties and aggregation process. Furthermore, the incorporated N-(2-hydroxyethyl) piperazine group behaves as a SA-binding receptor. BOD-Red formed aggregates in an aqueous solution and was non-emissive. In the presence of SA, it exhibited a pronounced enhancement of emission intensity centred at 630 nm owing to the disaggregation-induce emission. Notably, this probe in solution was non-responsive to any other tested proteins and small molecules. The mechanism responsible for the binding of BOD-Red with BSA was analyzed by a combined experimental and computational approach. The practical utility of BOD-Red as a fluorescent turn-on sensor was validated by quantifying BSA in real samples with reasonable recovery percentages.

A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine's Effect on Cellular Amino Acids via Inhibiting Cathepsin L.

Maintaining homeostasis of metabolites such as amino acids is critical for cell survival. Dysfunction of nutrient balance can result in human diseases such as diabetes. Much remains to be discovered about how cells transport, store, and utilize amino acids due to limited research tools. Here we developed a novel, pan-amino acid fluorescent turn-on sensor, NS560. It detects 18 of the 20 proteogenic amino acids and can be visualized in mammalian cells. Using NS560, we identified amino acids pools in lysosomes, late endosomes, and surrounding the rough endoplasmic reticulum. Interestingly, we observed amino acid accumulation in large cellular foci after treatment with chloroquine, but not with other autophagy inhibitors. Using a biotinylated photo-cross-linking chloroquine analog and chemical proteomics, we identified Cathepsin L (CTSL) as the chloroquine target leading to the amino acid accumulation phenotype. This study establishes NS560 as a useful tool to study amino acid regulation, identifies new mechanisms of action of chloroquine, and demonstrates the importance of CTSL regulation of lysosomes.