Specific Fluorescent Probes Research Articles

Long-term tracking of endoplasmic reticulum autophagy by a fluorescent probe with a long alkyl chain

Autophagy is a process through which cytoplasmic materials are degraded in lysosome. It is critical in the maintenance of cellular homeostasis. Thus, visualization and tracking of the autophagy is very important for understanding complex intracellular events. To monitor autophagy, various specific fluorescent probes to organelles are often used, such as mitochondria probes for mitophagy, lipid droplets probes for lipophagy. However, an endoplasmic reticulum (ER) probe for ER-phagy is still lack and challenging. Herein, we presented an ER fluorescent probe (HTMP-16) with a long alkyl chain. By strong hydrophobic interaction between the long alkyl chain and the lipid bilayers in ER, HTMP-16 can exclusively locate in the ER for a long time. At the same time, HTMP-16 exhibits excellent self-delivery to living cell, which is a prerequisite of a tracking probe. Furthermore, utilizing HTMP-16, in-situ and real-time long-term tracking of ER-phagy has been realized in living cells. Besides, considering its high sensitivity to viscosity, HTMP-16 can visualize the viscous changes of ER induced by temperature changes or tunicamycin.

A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells.

Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO- macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

Kinetics of lipid radical formation in lipoproteins from β-thalassemia: Implication of cholesteryl esters and α-tocopherol

Vascular complications in β-thalassemia are associated with oxidative modification of lipoproteins under high oxidative stress. The lipid components of lipoproteins are oxidized via lipid peroxidation and produce lipid radicals (L•) as the key initial intermediates. Modification of lipid components, therefore, might result in alterations in the rate and products of lipid peroxidation. In this study, the kinetics of L• formation during the 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH)- and hemin-induced oxidation of low-density and high-density lipoproteins (LDL and HDL) from β-thalassemia patients and healthy volunteers were investigated using a specific and sensitive fluorescence probe for L•. Kinetic parameters, including initial lag time, propagation rate and total L• production, were calculated by monitoring a fluorescence-active NBD-Pen-L• adduct. Oxidation of thalassemia lipoproteins exhibited a significantly shorter lag time but a slower propagation rate of L• formation when compared with healthy lipoproteins. LDL showed higher resistance to oxidation during the initiation phase but higher L• formation than HDL. Our results indicated that the levels of α-tocopherol determined the initial lag time, whereas the levels of core lipids and cholesteryl esters, especially cholesteryl linoleate (CL), determined the propagation rate and total L• production. The difference in potency of AAPH and hemin supported that hemin preferentially targeted core lipids. Moreover, analysis of 13-hydroxyoctadecadienoic acid cholesteryl ester (13-HODE-CE)/CE ratio indicated that thalassemia lipoproteins have higher susceptibility to oxidation than healthy lipoproteins. In conclusion, our findings suggested that CL and α-tocopherol were implicated in the susceptibility of lipoproteins to lipid peroxidation in physiological and pathological conditions of β-thalassemia.

Surface imprinted polymer on dual emitting MOF functionalized with blue copper nanoclusters and yellow carbon dots as a highly specific ratiometric fluorescence probe for ascorbic acid

The rational incorporation of metal organic frameworks (MOFs) into molecular imprinted polymers (MIPs) shows excellent potential for improving various merits from both approaches and elimination of the drawbacks of the counterparts. In this work, surface imprinted polymer on zeolitic imidazole framework-8 (ZIF-8) are presented as a highly selective probe for detection of ascorbic acid (AA). An MIP layer was surface imprinted on nano-sized ZIF-8 with AA as a template, and blue copper nanocluster (B-CuNCs) and yellow carbon dots (Y-CDs) as fluorescent signal transducers. The MIP capped ZIF-8 (MIP@ZIF-8) offered selective cavities for AA detection. The integrated system of MIP@ZIF-8 consisted of two emitters, CuNCs (λem 420 nm) used as a reference signal inserted in the pores of the ZIF-8, and yellow carbon dot (CDs, λem 560 nm) used as a probe and inserted in the polymer layer. The yellow emission of MIP@ZIF-8 nanocomposite was selectively quenched after addition of ascorbic acid, while the blue emission remained unaffected and used as an internal reference signal. The MIP@ZIF-8 ratiometry probe showed a dynamic linear response to AA in the range 4.0 µM – 22.0 µM with a detection limit 1.56 µM. The ratiometry-based fluorescence probe was successfully used for detection AA in drug samples and human serum with satisfactory recoveries of 92.5–106.11 % and 81.69 – 93.89 % respectively. Additional to high selectivity and sensitivity obtained from this MIP-MOF design, the presented probe, showed highly stability in room temperatures under well-lit room, which qualifies the probe for long-term use.

Ultrasensitive near-infrared fluorescence probe activated by nitroreductase for in vivo hypoxia detection

The fluorescence probe has attracted much attention in the detection of hypoxia-related pathological conditions with unique advantage. However, in vivo imaging the early stages of diseases formation and development remains a great challenge, mainly due to the lack of highly sensitive and specific fluorescence probe. Herein, a high-efficiency near-infrared fluorescence probe YQ-NO2 was synthesized to detect hypoxia via responding to nitroreductase (NTR) in living subjects. In vitro confocal-mediated competitive binding inhibition and flow cytometry analyses indicated YQ-NO2 was of high specificity on hypoxic cells. In vivo near-infrared imaging showed that the probe had the laudable characteristics of a low background inference, fast response and prolonged imaging time in the tumor-bearing mice. Furthermore, it had the property of high affinity and potent permeability, allowing for continuously monitoring cell hypoxia to evaluate longitudinal and dynamic change in the tumor area. Meanwhile, ischemic liver and enteritis sites could be accurately and timely imaged in mice. In addition, it was observed that the fluorescence intensity of the probe in tumor was significantly higher than that in inflammation. The probe has failed to exhibit any bio-toxicity in cells and animals treated with high doses. Ultimately, this near-infrared fluorescence probe will offer an imaging tool for characterizing the hypoxia microenvironment at the tiny lesions in vivo.

Design and Development of Benzothiazole-Based Fluorescent Probes for Selective Detection of Aβ Aggregates in Alzheimer's Disease.

The formation and accumulation of amyloid beta (Aβ) peptide are considered the crucial events that are responsible for the progression of Alzheimer's disease (AD). Herein, we have designed and synthesized a series of fluorescent probes by using electron acceptor-donor end groups interacting with a π-conjugating system for the detection of Aβ aggregates. The chemical structure of these probes denoted as RMs, having a conjugated π-system (C═C), showed a maximum emission in PBS (>600 nm), which is the best range for a fluorescent imaging probe. Among all these probes, RM-28 showed an excellent fluorescence property with an emission maximum of >598 nm upon binding to Aβ aggregates. RM-28 also showed high sensitivity (7.5-fold) and high affinities toward Aβ aggregates (Kd = 175.69 ± 4.8 nM; Ka = 0.5 × 107 M-1). It can cross the blood-brain barrier of mice efficiently. The affinity of RM-28 toward Aβ aggregates was observed in 3xTg-AD brain sections of the hippocampus and cortex region using a fluorescent imaging technique, as well as an in vitro fluorescence-based binding assay with Aβ aggregates. Moreover, RM-28 is highly specific to Aβ aggregates and does not bind with intracellular proteins like bovine serum albumin (BSA) and α-synuclein (α-Syn) aggregates. The results indicate that the probe RM-28 emerges as an efficient and veritable highly specific fluorescent probe for the detection of Aβ aggregates in both in vitro and in vivo model systems.

Development of a novel highly specific fluorescent probe for detecting HOCl in living cells and zebrafish, as well as real water samples

Hypochlorous acid (HOCl), a member of reactive oxygen species (ROS), plays an important role in living systems. Highly sensitive probes for detection of hypochlorous acid are in great demand. A novel chromene derivative fluorescent probe was constructed for determining hypochlorous acid. When HOCl was absent, the probe itself exhibited strong fluorescence intensity, while the fluorescence was quenched with the presence of HOCl. The fluorescent probe exhibited high selectivity and sensitivity for HOCl in a linear response range of 0 -15 μM with a low detection limit of 0.12 μM. The probe has been successfully applied for the confocal imaging of HOCl in the RAW 264.7 cells and zebrafish with low cell toxicity. It also was used to detect HOCl in tap water and lake water.

A specific turn-on fluorescence probe for determination of nitazoxanide based on feasible oxidation reaction with hypochlorite: Applying cobalt ferrite nanoparticles for pre-concentration and extraction of its metabolite from real urine samples

Nitazoxanide is an antimicrobial compound that was originally developed as an antiprotozoal drug. Recently nitazoxanide has been identified as broad-spectrum antiviral agent and redirected for the remediation of some respiratory tract viral infections. In this study, the spectrofluorimetric technique has been applied to determine Nitazoxanide (NTX) in tablets or its metabolite, tizoxanide (TZD), in human urine samples. The developed methodology is based on oxidizing NTX (non-fluorescence) into a highly fluorescent product by sodium hypochlorite. The fluorescence emission intensity was measured at 436.5 nm after fluorescence excitation at 362.5 nm. After optimizing all conditions, the analytical procedures and bio-analytical steps were evaluated and validated using ICH and FDA criteria, respectively. The method linearity, LOQ, and LOD values of NTX were 1.0–5.0 µg/mL, 0.434, and 0.143 µg/mL, respectively. The other novelty side of the presented work is the application of cobalt ferrite (CoFe2O4) nanoparticles (NPs) as a magnetic solid-phase for the pre-concentration and extraction process. The synthesized magnetic nanoparticles were characterized by scanning electron microscope and zeta sizer techniques. Finally, the utilized magnetic nanoparticles exhibited good recovery results for pre-concentration and extraction of NTX or its metabolite from spiked and real human urine samples, respectively.

A highly sensitive and selective enzyme activated fluorescent probe for in vivo profiling of carboxylesterase 2

Carboxylesterase 2 (CES2) is a serine-type hydrolase that plays important roles in xenobiotic detoxification and lipid metabolism. Its abnormal expression is highly associated with diseases such as diabetes and carcinoma. To date, intense attention has been attracted to CES2 targeted drug discovery and disease diagnosis. Thus, to further explore the physiological function of CES2 is of great importance. However, until now, most medical research on CES2 function and activity assays is still dependent on conventional methods, which could hardly specify CES2 activity. Therefore, there is an urgent need to develop efficient tools for selective measurement and sensing of endogenous CES2 in complicated biological system. In this study, we report the design and construction of an enzyme-activated fluorescent probe for CES2 activity sensing. The acquired probe DXMB was characterized as a highly specific and sensitive fluorescent probe for CES2 and possessed superior binding affinity, overall catalytic efficiency, and reaction velocity when compared with the reported CES2 probes. By application of DXMB into living system, it was capable of sensing endogenous CES2 in living cells, dynamic monitoring CES2 in zebrafish development, and visualizing tissue distribution of CES2 in nude mice. Most importantly, abnormally elevated CES2 activity in the intestine of diabetic model mice was first revealed, while significantly decreased CES2 activity in the liver was validated by DXMB. These results indicated that DXMB could serve as a vital tool for further CES2-related biological and medical research.

MtDNA specific fluorescent probe uncovering mitochondrial nucleoids dynamics during programmed cell death under super-resolution nanoscopy

Mitochondrial nucleoids or mitochondrial DNA (mtDNA) encodes for a variety of enzymes and proteins that are essential for oxidative phosphorylation, mitochondrial fussion/fission and apoptotic processes. However, visulization of mtDNA dynamics in response to external stumili has not yet been achieved. Herein, we developed a fluorescent probe, named BDP, that is capable of specifically bind to mtDNA in vitro and in living cells, without interfering mitochondrial functions. Its large Stokes-Shift and red-emission tail render its suitability for stimulated emission depletion (STED) visulization of mtDNA dynamics in living cells. We sucessfully demonstrated for the first time how apoptotic induced anti-cancer drug could impact on mitochondrial nucleoids, and the morphology evolution of mtDNA from segmentation to dispersion was recorded, in a single mitochondria at nanoscale.

Development of a multi-task formaldehyde specific fluorescent probe for bioimaging in living systems and decoration materials analysis

Formaldehyde (FA) is one of the ubiquitous chemical pollutants in the indoor environment. Excessive inhalation of FA could cause asthma, stupor and even cancer. In addition, FA is the main member of reactive carbonyl species (RCS), it is mainly produced in nucleus and involved in a variety of life activities in organisms. The disorders of FA metabolism could affect the methylation and demethylation of nucleic acids, leading to cognitive impairment and even could cause neurodegenerative diseases, heart disorders and cancers, etc. it is urgent to develop novel tools to monitor FA in the environment and endogenous FA in the nucleus. Herein, we have designed a robust fluorescent probe (Nu-DH) targeted the nucleus to recognize FA. Nu-DH has high selectivity, large turn-on fluorescence (up to 120-fold) signal and sensitive detection for recognizing FA. Furthermore, in molecular docking calculation and titration experiments, Nu-DH has good affinity with DNA and RNA. Most importantly, the unique probe Nu-DH targets nucleus well and has been effectively implemented in zebrafish or in decoration materials for FA imagery. We found that many decoration materials will release a certain amount of FA, among which the FA release amount of white latex is the largest. We expect Nu-DH to become an effective tool for the diagnosis and monitoring of related diseases due to the influence of FA in vivo and in environment.

Study on cryopreservation of Guanzhong dairy goat semen with bovine semen seminal plasma

There are phospholipase and yolk agglutinase in the seminal plasma of dairy goat. The existence of these enzymes will hydrolyze lecithin in yolk into lysophosphatides and cause the change of pH value of diluent, resulting in toxic and side effects on sperm. In order to explore the effect of bovine seminal plasma replacing different doses of dairy goat seminal plasma (0%, 25%, 50%, 100%) on semen freezing quality of Guanzhong dairy goat, we used computer-aided sperm analysis (CASA) system, specific fluorescent probe detection technology, hypo osmotic swelling test (HOST), oxidation index determination and in vivo fertilization verification test to study the semen cryopreservation of Guanzhong dairy goat. The results showed that if we used bovine seminal plasma instead of 100% dairy goat seminal plasma, the sperm motility, plasma membrane functionality and acrosome integrity rate were the highest after thawing, reaching 67.04%, 38.12% and 68.83% respectively, which was significantly higher than that of other groups (P < 0.05); In terms of oxidation indexes, the values of SOD and GSH-PX in sperm of dairy goats were 199.78 U/mL and 133.55 U/L, respectively, which were significantly higher than those in other groups (P < 0.05), and the values of ROS and MDA were 397.58 U/mL and 5.16 nmol/L, respectively, which were significantly lower than those in other groups (P < 0.05); The results of artificial insemination also showed that using bovine seminal plasma instead of 100% of dairy goat seminal plasma could significantly improve the fertilization ability of sperm. The quality of frozen Guanzhong dairy goat sperm can be improved by using bovine seminal plasma instead of dairy goat seminal plasma and using bovine diluent.

Boronic acid-containing polymeric nanomaterials via polymerization induced self-assembly as fructose sensor

Owing to the reversible binding to diols, investigation of a simple and scalable strategy for the building of boronic acid-containing materials for fructose sensor is highly desirable but still challenging. Here, polymerization induced self-assembly is introduced to synthesis of core-shell spherical nanomaterials with boronic acid-rich shell in one-pot. Poly(4-vinylphenylboronic acid) 39 - b -polystyrene 242 (PVPBA 39 - b -PS 242 ) nanospheres with the diameter of 135 nm and PVPBA 230 - b -PS 1275 nanospheres with the diameter of 527 nm are prepared by PISA of styrene with PVPBA as chain transfer agent in methanol/H 2 O. The sensing applications of these two nanospheres are investigated. The fluorescence detection of 8-hydroxyquinoline by PVPBA 39 - b -PS 242 nanospheres is realized with high sensitivity at the ppm level, which is more sensitive than PVPBA 39 chain. PVPBA- b -PS nanospheres/alizarin red S complex and PVPBA- b -PS nanospheres/quercetin complex are built as a specific and sensitive fluorescence probe for fructose. The detection limit (LOD) of PVPBA- b -PS/ARS complex achieves 42.6 nM, while the LOD of PVPBA-b-PS/QC achieves 1.83 nM. • Nanospheres with boronic acid-rich shell are synthesized by PISA and designed as sensors for detection of fructose. • Via the evaluation by 8-hydroxyquinoline, these core-shell nanoshperes show higher sensitivity than PVPBA chains. • The detection limit of PVPBA-b-PS/ARS complex achieves 42.6 nM for fructose, while that of PVPBA-b-PS/QC achieves 1.83 nM.

Visualization of endogenous formaldehyde in the nucleus via a robust activatable fluorescent probe

As one of the most basic substances in the live system, the improper regulation of nucleic acid behavior may cause cell dysfunction and induce serious diseases. As the main storage location of nucleic acids, the nucleus is also an important site for the production and transfer of endogenous formaldehyde (FA) in live cells. However, the disorder of FA metabolism could affect the methylation and demethylation of nucleic acids, leading to cognitive impairment and even could cause Alzheimer’s disease, sarcosinemia and dwarfism, etc. Therefore, the design of FA fluorescent probes that can precisely target the nucleus is of great significance for the detection of nuclear FA. Herein, the first nucleus-targeting FA specific fluorescent probe (Nu-FA) was rationally designed and demonstrated. Encouraged by the advantageous affinity to DNA and RNA in molecular docking calculations and titration experiments, and the excellent specific response to FA in spectroscopy experiments, Nu-FA has been successfully employed for the first time to visualize endogenous FA in the nucleus and monitored the removal of endogenous FA from the nucleus by exogenous drug. This effective probe would provide an unprecedented approach to enable exploring FA in the nucleus and greatly promote basic research on related diseases caused by FA.

MtDNA-Specific Ultrasensitive Near-Infrared Fluorescent Probe Enables the Differentiation of Healthy and Apoptotic Cells.

Mitochondrial DNA (mtDNA) as a class of important genetic material is easily damaged, which can result in a series of metabolic diseases, hereditary disease, and so on. mtDNA is an ultrasensitive indicator for the health of living cells due to the extremely short physiological response time of mtDNA toward damage (ca. 5.0 min). Therefore, the development of specific ultrasensitive fluorescent probes that can in real-time monitor mtDNA in vivo are of great value. With this research, we developed a near-infrared twisted intramolecular charge transfer (TICT) fluorescent probe YON. YON is a thread-like molecule with an A-π-D-π-A structure, based on the dicyanoisophorone fluorophore. The molecular design of YON enabled the specific binding with dsDNA (binding constant (K) = 8.5 × 105 M-1) within 1.3 min. And the appropriate water-oil amphiphilicity makes YON significantly accumulate in the mitochondria, enabling the specific binding to mtDNA. The fluorescence intensity at 640 nm of YON enhanced linearly with increasing concentrations of mtDNA. Dicyanoisophorone as the strong electron-withdrawing group that was introduced into both ends of the molecule resulted in YON being a classic quadrupole, so it could ultrasensitively detect trace mtDNA. The minimum detection limit was 71 ng/mL. Moreover, the large Stokes shift (λex = 435 nm, λem = 640 nm) makes YON suitable for "interference-free" imaging of mtDNA. Therefore, YON was used to monitor trace changes of mtDNA in living cells; more importantly, it could be used to evaluate the health of cells by monitoring microchanges of mtDNA, enabling the ultrasensitive evaluation of apoptosis.

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase.

The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the α1 or α2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method was validated by Sanger sequencing using mice of known genotype, and by comparing results with a previous two-step method that used PCR amplification followed by gel electrophoresis. In addition, we clarified inconsistencies in published sequences, updated numbering to current reference sequences, and confirmed the continued presence of the mutations in the colony. It is expected that a wider availability of these models and a more efficient genotyping protocol will advance studies of the Na,K-ATPase and its cardiotonic steroid receptor.

Newly found K+-Thioflavin T competitive binding to DNA G-quadruplexes and the development of a label-free fluorescent biosensor with extra low detection limit for K+ determination in urine samples

The determination of potassium ion K+ in body fluids is important in health monitoring and diagnoses. One of the interesting and simple methods for K+ detection is the use of label-free biosensors based on DNA G-quadruplexes (GQs) coupled with a specific fluorescent probe, such as Thioflavin T (ThT), which lights up when bound with K+-stabilized GQs. However, these biosensors are not generally sensitive. In this work, we found a solution: at a low concentration, K+ competes with ThT in binding to a bimolecular GQ or a tetramolecular GQ, resulting in a decrease in ThT fluorescence emission with increasing K+. Therefore, we developed a label-free turn-off fluorescent K+ sensor. The sensor provides a very low detection limit of 21.87 ± 0.59 nM. Other possible interfering components in urine did not exert any effect even at quantities that were 10-fold greater than their upper limit of normal concentrations found in urine samples. With its only requirement of diluting samples, the developed low-cost label-free probe and simple sensor was successfully applied to the direct detection of K+ in normal urine samples with high accuracy (recoveries ranged from 90% to 100%).

A Novel Levulinate‐Based Highly Specific Colorimetric Fluorescent Probe for Bisulfite Detection in Live Cells and Zebrafish

AbstractBisulfite (HSO3−) is associated with many physiological processes in living systems. High concentration of HSO3− may lead to many diseases. Herein, a simple levulinate‐based colorimetric fluorescent probe for HSO3− detection was designed and synthesized. The probe showed high selectivity for HSO3− detection with color changes from light pink to deep pink. Under the optimal test conditions, there is a good linear relationship between fluorescence intensity and concentrations of HSO3− in the range of 0–50 μM with detection limit of 1.04 nM. In addition, the probe was successfully applied to monitor HSO3− in living cells and zebrafish, which proved that the probe can be used to detect HSO3− in living systems.

A smart small molecule as specific fluorescent probe for sensitive recognition of mitochondrial DNA G-Quadruplexes

G-quadruplex (G4) has become an essential target for the therapy of human genetic diseases (cancer included) with important biological functions for regulation and monitoring. In contrast, little information about G-quadruplexes in mitochondria is known due to the limited development of fluorescent ligands for mitochondrial DNA (mtDNA) G4 detection, urging the design of fluorogenic detection ligands. Herein, we designed and synthesized a robust aggregation-induced emission ligand SPN for recognition of mtDNA G4s, which integrates a G4-active photosensitizer (BZT-triphenylamine) molecule and a mitochondrial targeting functional group (lipophilic triphenylphosphonium cation). Using SPN as the mtDNA G4s sensor, we surprisingly found that SPN could achieve sensitive recognition, sensing, and stabilization of the G4s with the detection limit as low as 0.89 nM. SPN can be highly colocalized with mitochondria, allowing monitoring of mtDNA G4s effectively in vitro. Remarkably, the fluorescence intensity in cancer cells is significantly stronger than that in normal cells, suggesting the potential applications of SPN in cancer diagnosis. This study provides a promising example of designing a fluorescent probe targeting and sensing mtDNA G4s and offers a feasible chemical tool for investigating mtDNA G4s.