Novel dual-site fluorescent probe for monitoring cysteine and sulfite in living cells

Fluorescent probes have been considered to be efficient tools for the visualization of physiological and pathological processes. Herein, a dual-site fluorescence probe denoted as LC-1 was developed for the detection of cysteine (Cys) and its metabolite SO₃²⁻. The probe was shown to be highly sensitive to Cys and SO₃²⁻ with a turn-on mode fluorescence signal through two emission channels under excitations at wavelengths of 320 nm and 440 nm. Notably, the LC-1 probe was also observed to be satisfactorily sensitive to Cys and SO₃²⁻ in the presence of other amino acids and reactive oxygen species (ROS). Meanwhile, LC-1 was shown to have low cytotoxicity and was successfully applied for imaging the metabolism of Cys in living cells.

A dual-site fluorescence probe LC-1 was developed for the detection of Cys and SO₃²⁻.     

Glucose measurements with sensors and ultrasound

Accurate monitoring of the blood glucose level in diabetics is essential in preventing complications. Generally, conventional over-the-counter glucose meters require frequent painful finger punctures to obtain samples, which makes a noninvasive method preferable. The purpose of this study was to demonstrate that glucose levels can be measured transdermally with the combination of the low-profile cymbal array and an electrochemical glucose sensor consisting of amperometric electrodes and a novel glucose oxidase hydrogel. Interstitial fluid glucose concentrations can be determined with the electrochemical glucose sensor after the skin is made permeable to glucose by ultrasound (US) (20 kHz) with the thin (< 7 mm) and light (< 22 g) cymbal array. Using this array to deliver insulin into hyperglycemic rats, our previous experiments demonstrated that blood glucose levels would decrease 233.3 mg/dl with 5 min of US exposure. With the sensor and array, our goal was to determine the glucose levels of hyperglycemic rats noninvasively and evaluate the possible bioeffects. A total of 12 anesthetized rats were placed into two groups (US exposure group and control group) and the array (I(SPTP) = 100 mW/cm(2)) with a saline reservoir operating for 20 min was affixed to the abdomen. The array was removed and an electrochemical glucose sensor was placed on the exposed area to determine the glucose concentrations through the skin. Comparison was made using a commercial glucose meter with the blood collected from a jugular vein. The average blood glucose level determined by the sensor was 356.0 +/- 116.6 mg/dl, and the glucose level measured by the commercial glucose meter was 424.8 +/- 59.1 mg/dl. These results supported the use of this novel system consisting of the electrochemical glucose sensor and the cymbal array for glucose monitoring.     

A fast-responsive fluorescent turn-on probe for nitroreductase imaging in living cells

Nitroreductase (NTR) may be more active under the environment of hypoxic conditions, which are the distinctive features of the multiphase solid tumor. It is of great significance to effectively detect and monitor NTR in the living cells for the diagnosis of hypoxia in a tumor. Here, we synthesized a novel turn-on fluorescent probe NTR-NO₂ based on a fused four-ring quinoxaline skeleton for NTR detection. The highly efficient probe can be easily synthesized. The probe NTR-NO₂ showed satisfactory sensitivity and selectivity to NTR. Upon incubation with NTR, NTR-NO₂ could successively undergo a nitro reduction reaction and then generate NTR-NH₂ along with significant fluorescence enhancement (30 folds). Moreover, the fluorescent dye NTR-NH₂ exhibits a large Stokes shift (Δλ = 111 nm) due to the intramolecular charge transfer (ICT) process. As a result, NTR-NO₂ displayed a wide linear range (0–4.5 μg mL⁻¹) and low detection limit (LOD = 58 ng mL⁻¹) after responding to NTR. In addition, this probe was adopted for the detection of endogenous NTR within hypoxic HeLa cells.

Probe NTR-NO₂ was effectively reduced in the presence of NTR generating a highly fluorescent product.     

A sensitive BODIPY-based fluorescent probe for detecting endogenous hydroxyl radicals in living cells

The hydroxyl radical (˙OH) has been suggested to play very vital roles in many physiological and pathological processes. However, selective detection of ˙OH is highly challenging owing to its extremely high reactivity and short lifetime. Herein, we designed and synthesized a sensitive “turn on” fluorescent probe for detecting endogenous ˙OH based on a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) platform. The probe had shown good properties including high sensitively, ideal selectively and low cytotoxicity, and was successfully employed to image endogenous hydroxyl radical in living cells.

A novel “turn-on” NIR fluorescent probe was designed and used for monitoring endogenous hydroxyl radical in living cells, which also showed higher selectivity toward hydroxyl radical over other reactive oxygen/nitrogen species.     

A coumarin Schiff's base two-photon fluorescent probe for hypochlorite in living cells and zebrafish

Selective and sensitive fluorescent probes for ClO⁻ are desirable due to the importance of ClO⁻ in biological processes. Here, a coumarin Schiff''s base, compound 1, has been developed and successfully used as a one- and two-photon fluorescent probe for ClO⁻ with high selectivity. This probe can recognize ClO⁻ with obvious color change from yellow-green to colorless and green to blue fluorescence emission, which can be observed by the naked eye. The properties of low cytotoxicity and good cell permeability allow it to be used for ClO⁻ detection in living cells and zebrafish by both one- and two-photon microscopy imaging. All these results indicate that the compound is a sensitive probe with potential for analysis of ClO⁻ in biological samples. The mechanism by which probe 1 recognizes ClO⁻ is possibly nucleophilic addition followed by hydrolysis.

A coumarin Schiff''s base compound can selectively recognize ClO⁻ and can be successfully applied to the detection of ClO⁻ in living cells and zebrafish by one- and two-photon fluorescence modes.     

A novel ratiometric fluorescent probe for rapid detection of hydrogen peroxide in living cells

In this work, we present a new ratiometric fluorescent probe JNY-1 for rapid and convenient detection of H₂O₂. The probe could selectively and sensitively respond to H₂O₂ within 10 min. In addition, this probe was successfully applied for monitoring and imaging of H₂O₂ in liver cancer HepG2 cells under physiological conditions.

A new ratiometric fluorescent probe JNY-1 for sensitive detection of H₂O₂ is presented with selectivity over other reactive oxygen species, reactive nitrogen species, and biologically relevant species. Imaging of H₂O₂ in liver cancer HepG2 cells was achieved.     

Glucose monitoring in type 2 diabetes

A major goal in the treatment of type 2 diabetes is to maintain blood glucose values in the normal or near normal range. A system of monitoring glycemic control, which includes self-monitoring of blood glucose (SMBG) by the patient, and frequent glycated protein assays, is an important tool for achieving this goal. Optimal use of SMBG by the patient is best accomplished through comprehensive glucose monitoring education, which includes appropriate monitor selection and use and strategies to use monitoring results to improve glycemic control.     

Detection of single fluorescent proteins inside eukaryotic cells using two-photon fluorescence

Imaging single fluorescent proteins in a live cell is a challenging task because of the strong cellular autofluorescence. Autofluorescence can be minimized by reducing fluorescence excitation volume. Total internal reflection fluorescence (TIRF) microscopy has been routinely used to reduce excitation volume and detect single protein molecules in or close to cell membrane. However, the limited penetration depth of evanescent field excludes imaging of single fluorescent proteins that reside deep inside a eukaryotic cell. Here we report detection of single fluorescent proteins inside eukaryotic cells by two-photon fluorescence (TPF) microscopy. TPF has an excitation volume less than 0.1 femtoliter (fL). Cell autofluorescence under TPF is low and thus enables us to detect single enhanced green fluorescent proteins (EGFP) and single monomeric teal fluorescent proteins (mTFP1.0) that reside several microns deep inside the cell. Discrete stepwise photobleaching of TPF was observed for both proteins inside the cell. Quantitative analysis of single-molecule fluorescence trajectories show that mTFP1.0 is about twofold brighter than EGFP, while its fluorescence on-time before bleaching is about 10 fold shorter. These findings demonstrate the sensitivity of TPF for imaging of eukaryotic cells at single-molecule level and will be useful for measurement of protein stoichiometry inside the cell.     

Glucose monitoring in the acutely ill patient with diabetes mellitus

The Diabetes Control and Complications Trial (DCCT) clearly demonstrated that improved blood-glucose control results in the decreased occurrence and progression of microvascular complications. The progression of acute medical conditions, commonly found in hospitalized diabetic patients, are also related to glycemic control. Glycemic control in the hospitalized setting is measured by point-of-care blood-glucose monitors. These monitors provide immediate feedback so that subcutaneous or infused insulin can be adjusted in a more timely and physiologic manner. The practitioner must become familiar with some of the limitations of these systems to ensure the accuracy of blood-glucose results.     

Rational design of a selective and sensitive “turn-on” fluorescent probe for monitoring and imaging hydrogen peroxide in living cells

As one type of reactive oxygen species (ROS), hydrogen peroxide (H₂O₂) plays a key role in regulating a variety of cellular functions. Herein, a fluorescent probe N-Py-BO was well designed and synthesized and its ability for detecting H₂O₂ by fluorescence intensity was evaluated. In the design, the arylboronate ester group was acted as a reaction site for H₂O₂. Upon reaction with H₂O₂ under physiological conditions, the boronate moiety in the probe was oxidized, followed by detachment from the probe and as a result, a “turn-on” fluorescence response for H₂O₂ was acquired. Due to the D–A structure formation between N,N′-dimethylaminobenzene and the –CN group and the linkage by thiophene and C C bonds to increase the conjugate length, this probe showed a remarkable red shift of emission wavelength (650 nm) as well as a large Stokes shift (214 nm). An excellent linear relation with concentrations of H₂O₂ ranging from 2.0 to 200 μM and a good selectivity over other biological species were obtained. Importantly, taking advantage of the low toxicity and good biocompatibility, the developed probe was successfully applied to monitoring and imaging H₂O₂ and its level fluctuation in living cells, which provided a powerful tool for evaluation of cellular oxidative stress and understanding the pathophysiological process of H₂O₂-related diseases.

A fluorescent probe N-Py-BO was well designed and synthesized and its ability for detecting H₂O₂ by fluorescence intensity was evaluated.     

Two-photon fluorescent probes for detecting the viscosity of lipid droplets and its application in living cells

Lipid droplets (LDs) are storage organelles at the centre of lipid and energy homeostasis, which act as vital hubs of cellular metabolism and the key to maintaining lipid and energy homeostasis. We synthesized a new two-photon fluorescent probe (CIV) that could detect the viscosity of lipid droplets. The probe is constructed via the typical ICT system of D–π–A using carbazole as the donor and imidazole as the acceptor. With the increase in viscosity from PBS to 99% glycerol, the fluorescence intensity of CIV increased by 13-fold, showing sensitivity and specificity towards viscosity. In addition, CIV showed low toxicity and excellent biocompatibility in cytotoxicity tests, and was successfully used for living cell LD imaging. Taken together, the results widen the way for the development of novel fluorescent probe-based the visualization LDs and detection in solutions, physiology and pathology.

A novel two-photon fluorescence probe (CIV) can detect the viscosity and locate lipid droplets in living cells.     

A real-time ratiometric fluorescent probe for imaging of SO2 derivatives in mitochondria of living cells

A real-time ratiometric fluorescent probe (IN-CZ) for highly selective detection of sulfite was designed and synthesized, which is based on modulating the intramolecular charge transfer (ICT) of the hemicyanine dye platform. The mechanism of using the probe is mainly through the Michael addition that occurs between IN-CZ and sulfite with a detection limit of 2.99 × 10⁻⁵ M. IN-CZ displays a fast response (within 1 minute) and is highly selective for SO₃²⁻/HSO₃⁻ over ROS, biologically relevant ions, biological mercaptans and other reactive species. More importantly, IN-CZ was suitable for ratiometric fluorescence imaging in living cells, by real-time monitoring of SO₃²⁻/HSO₃⁻ changes in mitochondria targeted in living cells.

A real-time ratiometric fluorescent probe (IN-CZ) for highly selective detection of sulfite was designed and synthesized, which is based on modulating the intramolecular charge transfer of the hemicyanine dye platform.     

Structure modulation on fluorescent probes for biothiols and the reversible imaging of glutathione in living cells

The detection of small molecular biothiols (cysteine, homocysteine and glutathione) is of great importance, as they involve in a series of physiological and pathological processes and are associated with many diseases. To realize the real-time monitoring of a specific biothiol, a rapid and reversible probe is required. Therefore, three probes, namely, o-MNPy, m-MNPy and p-MNPy, with pyridine substituted α, β-unsaturated ketone as the recognition site, were reported here, and the reactivity of the recognition site was finely tuned by the connection mode of the pyridine unit. To single out the optimal one, the response performances of three probes toward each biothiol were systemically studied, taking the differences of the intracellular contents of three biothiols into account during the evaluation. Biothiols reacted with the probes through Michael addition, and results showed that the slight structural variations could affect the performances of the probes obviously. p-MNPy with the pyridine unit connected to the recognition site through the para-position of the nitrogen atom, revealed the best sensing ability among the three probes. It demonstrated rapid response, good selectivity and sensitivity, excellent pH adaptability to Cys and GSH, and displayed reversible detection toward GSH. Finally, p-MNPy was successfully applied to track the GSH fluctuations under the oxidative stress stimulated by H₂O₂ in living cells.

A reversible fluorescent probe for GSH was obtained through structure modulation, by which the intracellular GSH fluctuation was imaged.     

用标记技术实时观察中性粒细胞吞噬病原体

目的　试图建立一种快速、直接判断中性粒细胞的吞噬、杀菌功能的标准方法 ,并对该方法进行分析与评价。方法　将带有绿色荧光蛋白 (GFP)基因的PRSETB质粒转化为大肠埃希菌BL2 1 ,利用BL2 1 高效表达GFP后发出强烈绿色荧光的特点 ,用电荷耦合器件 (CCD)耦合荧光显微镜连续记录中性粒细胞对大肠埃希菌粘附、内吞、消化和残体外排的全过程。结果　粘附过程 :BL2 1 在中性粒细胞膜表面停顿约 1 5s(1 5 0± 0 15s) ,为抗原识别过程 ;内吞过程 :中性粒细胞膜内陷、伸出伪足将大肠埃希菌包裹 ,并向细胞中心移动 ,持续时间可达 132s(132± 14s) ;消化过程 :当大肠埃希菌进入中性粒细胞胞体后 ,其杆状体逐渐变短 ,GFP发出的荧光强度逐渐变弱 ,持续时间约为 35 0s(35 0± 32s) ;残体外排过程 :可观察到溶酶体残体由中性粒细胞中心向胞膜运动 ,最后排出 ,持续时间约为 140s(140± 2 7s)。结论　该方法利用光子学显像和基因标记相结合的技术可以实时、连续观察中性粒细胞对大肠埃希菌的吞噬杀菌过程 ,对临床治疗有重要意义。     

An effective biocompatible fluorescent probe for bisulfite detection in aqueous solution,living cells,and mice

Sulfur dioxide, an air pollutant, is easily hydrated to sulfites and bisulfites and extremely harmful to human health. On the other hand, endogenous sulfur dioxide is the fourth gasotransmitter. In view of the above, it is worth developing an effective method for the detection of these compounds. In this paper, a novel colorimetric fluorescent probe (Hcy-Mo), based on hemi-cyanine, for bisulfites is reported. Hcy-Mo shows excellent selectivity for bisulfites over various other species including cysteine, glutathione, CN⁻, and HS⁻, and undergoes 1,4-addition reactions at the C-4 atom of the ethylene group. The reaction can be completed in 30 s in a PBS buffer solution and displays high sensitivity (limit of detection is 80 nM) for bisulfites. Test paper experiments show that the probe can be used for bisulfite detection in aqueous solutions. In addition, Hcy-Mo exhibits excellent cell permeability and low cytotoxicity for the successful detection of bisulfites in living MDA-MB-231 cells and in living mice, implying that this probe would be of great benefit to biological researchers for investigating the detailed biological and pharmacological functions of bisulfites in biological systems.

Sulfur dioxide, an air pollutant, is easily hydrated to sulfites and bisulfites and extremely harmful to human health.     

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

Ferritin self-assembly has been widely exploited for the synthesis of a variety of nanoparticles for drug-delivery and diagnostic applications. However, despite the crucial role of ferritin self-assembly mechanism for probes encapsulation, little is known about the principles behind the oligomerization mechanism. In the present work, the novel “humanized” chimeric Archaeal ferritin HumAfFt, displaying the transferrin receptor-1 (TfR1) recognition motif typical of human H homopolymer and the unique salt-triggered oligomerization properties of Archaeoglobus fulgidus ferritin (AfFt), was site-selectively labeled with N-(1-pyrenyl)maleimide on a topologically selected cysteine residue inside the protein cavity, next to the dimer interface. Pyrene characteristic fluorescence features were exploited to investigate the transition from a dimeric to a cage-like 24-meric state and to visualize the protein in vitro by two photon fluorescence microscopy. Indeed, pyrene fluorescence changes upon ferritin self-assembly allowed to establish, for the first time, the kinetic and thermodynamic details of the archaeal ferritins oligomerization mechanism. In particular, the magnesium induced oligomerization proved to be faster than the monovalent cation-triggered process, highly cooperative, complete at low MgCl₂ concentrations, and reversed by treatment with EDTA. Moreover, pyrene intense excimer fluorescence was successfully visualized in vitro by two photon fluorescence microscopy as pyrene-labeled HumAfFt was actively uptaken into HeLa cells by human transferrin receptor TfR1 recognition, thus representing a unique nano-device building block for two photon fluorescence cell imaging.

Pyrene fluorescence changes upon ferritin self-assembly allowed to establish the kinetic and thermodynamic details of the archaeal ferritins oligomerization mechanism and was successfully visualized in vitro by two photon fluorescence microscopy.     

A methylene blue-based near-infrared fluorescent probe for rapid detection of hypochlorite in tap water and living cells

A methylene blue-based near-infrared fluorescent probe was designed for the selective determination of hypochlorite (ClO⁻), over other reactive oxygen species or interfering agents. Acetylated methylene blue was synthesized by introducing the acetyl group into the methylene blue framework, which can specifically recognize exogenous and endogenous ClO⁻. The acetylated methylene blue fluorescent probe was characterized by ¹H NMR, ¹³C NMR and HRMS. The response process and possible mechanism were studied using products of the probe. The emission response of the probe to ClO⁻ presented good linear relationship in the 0–60 μM concentration range, with the detection limit of 0.1 μM (measured at 660 nm and 690 nm). The absorption and emission wavelengths of acetylated methylene blue are both in the near-infrared region; in addition, the probe itself and the degradation products were well-dissolved in water and have almost no toxicity. The probe was used for intracellular ClO⁻ imaging and showed a large fluorescence enhancement (about 200-fold increase).

We developed a MB-based probe to detect OCl⁻, whose product is almost non-toxic. The fluorescence enhancement times are large.     

A novel near-infrared fluorescent probe for highly selective recognition of hydrogen sulfide and imaging in living cells

A novel near-infrared fluorescent probe (L) based on a 1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano[2,3-g]quinoxalin-7-one scaffold has been synthesized and characterized. Probe L displays highly selective and sensitive recognition to H₂S over various anions and biological thiols with a large Stokes shift (125 nm) in THF/H₂O (6/4, v/v, Tris–HCl 10 mM, pH = 7.4). This probe exhibits turn-on fluorescence for H₂S through HS⁻ induced thiolysis of dinitrophenyl ether. Confocal laser scanning micrographs of MCF-7 cells incubated with L confirm that L is cell-permeable and can successfully detect H₂S in living cells.

A novel “off–on” fluorescent probe (L) for H₂S detection with NIR emission and imaging H₂S in living cells.     

Phenothiazine and semi-cyanine based colorimetric and fluorescent probes for detection of sulfites in solutions and in living cells

Four hemicyanine probes for selectively detecting sulfites (HSO₃⁻/SO₃²⁻) have been constructed by the condensation reaction of 7-substituted (CN, Br, H and OH) phenothiazine aldehyde with 1-ethyl-2,3,3-trimethylindolium iodide. All four probes show a fast and sensitive response to HSO₃⁻/SO₃²⁻via a Michael addition, with a detection limit lower than 40 nM based on monitoring their UV/vis absorption changes. Although all four probes display an increase in fluorescence when responding to HSO₃⁻/SO₃²⁻, the increment is larger for the probe with an electron-withdrawing group than the probe with an electron-donating group, except for Br. Thus, among four probes the 7-cyano probe (PI-CN) possesses the largest fluorescent response to HSO₃⁻/SO₃²⁻, and the lowest detection limit (7.5 nM). More expediently and easily, a film and a test paper with PI-CN have been prepared to detect HSO₃⁻/SO₃²⁻ in a sample aqueous solution selectively. Finally, the detection of HSO₃⁻/SO₃²⁻ by PI-CN in biological environments has been demonstrated by cell imaging.

Four 7-substituted phenothiazine hemicyanines display a substituent effect on the fluorescence response toward sulfites. The CN-substituted probe exhibits the best sensing behavior.     

全胰切除患者应用胰岛素治疗的血糖监测及护理

总结5例全胰切除患者应用胰岛素治疗的血糖监测及护理。对患者各阶段应用胰岛素的特点进行分析,把握血糖监测时机,做好相应处理,同时做好康复指导,可有效缓解症状及并发症的发生。