Detection Of Biothiols Research Articles

Piperazine-Coumarin based fluorescence probe with enhanced brightness and solubility for bio-thiol detection and esophageal carcinoma diagnosis

The development of novel fluorescent dyes for bio-thiol is of great importance in biological, clinical and pharmaceutical sciences. Given the importance of bio-thiol anticipating in numerous physiological processes, there is a great need to construct fluorescent biosensors with high quality to detect them. Fluorophores, especially those used in bio-system, usually require high-quality properties such as high brightness, good water solubility, bio-compatible and photostability. Herein, we reported a novel fluorescent probe based on piperazine-coumarin scaffold with enhanced brightness and solubility. To further demonstrate the potential clinical applications, we performed living cell fluorescence image and human esophageal carcinoma diagnosis. The result indicated that we were able to distinguish pathological tissue from normal tissue by applying this probe. Thus, we hope this design will be helpful to develop high-quality fluorophores for clinical diagnosis.

A Dual-emitting Two-dimensional Nickel-based Metal-organic Framework Nanosheets: Eu3+/Ag+ Functionalization Synthesis and Ratiometric Sensing in Aqueous Solution.

Using two-dimensional (2D) nickel-based metal organic framework (Ni-MOF) nanosheets as a matrix, Eu3+ and Ag+ were incorporated to synthesize Ag/Eu@Ni-MOF with double luminescence centers of Eu3+ ion (615 nm) and organic ligand (524 nm). And a ratiometric luminescence sensor is constructed based on Ag/Eu@Ni-MOF for sensitive detection of biothiols in aqueous solutions. The dual-emissive fluorescence properties can be tuned by changing the amounts of Ag+ ions doping. The results of temperature and pH effects on the fluorescence of Ag/Eu@Ni-MOF indicates that the Ag/Eu@Ni-MOF is a temperature-sensitive material and the fluorescence of Ag/Eu@Ni-MOF can keep stable over a wide pH range. Due to the binding of -SH in cysteine (Cys) and glutathione(GSH) with Ag+, the ligand luminescence was significantly inhibited by weakening the Ag + influence on the energy transfer process in the MOFs. Therefore, ratiometric fluorescent sensing of biomolecular thiols was realized based on the dual-emission Ag/Eu@Ni-MOF. More importantly, the fluorescence color change can be observed with naked eyes to realize visual detection. The ratiometric fluorescent sensor exhibits high performance for Cys and GSH detection with a wide linear range of 5-250 µM and a relatively low detection limit of 0.20 µM and 0.17 µM, respectively. Furthermore, the biothiols content in human serum was determined with satisfactory results. It proves the Ni-MOF nanosheets can be used as a stable matrix for construction luminescent MOFs for the first time, and validate the great potential of Ag/Eu@Ni-MOF as a ratiometric fluorescent probe for point-of-care testing (POCT) in disease diagnosis.

Polydopamine-mediated photothermal effect enables a new method for point-of-care testing of biothiols using a portable photothermal sensor

Abnormal levels of biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) are associated with many pathological diseases. The demand for bulky and costly apparatuses represents a big impediment for point-of-care testing of biothiols. Herein, we developed a novel photothermal sensor for biothiols based on the formation of polydopamine nanoparticles (PDA NPs) using a thermometer as readout. A zeolitic imidazolate framework (ZIF-67) was first fabricated through a facile one-pot strategy. Then dopamine (DA) was immediately polymerized into PDA NPs with excellent photothermal effects (PETs) in the presence of the ZIF-67. Biothiols could disintegrate ZIF-67 by substituting ligands, causing the reaction between biothiols and cobalt ions to form complexes, and release 2-methylimidazole (2MIM) that can slowly oxidize DA, thereby "weakening" the PETs signal, which enabled us to convert biological signals into temperature signals. Therefore, the quantitative detection of Cys, Hcy and GSH can be achieved by recording temperature changes, with detection limits as low as 0.158 μM, 0.181 μM and 0.207 μM, respectively. Furthermore, the photothermal sensor has been applied in detection of biothiols in fetal bovine serum with satisfactory recovery rates, correlating well with the results by standard Ellman method, signifying its promising potential in integrated biological detection system.

Gold–Nanoparticle Based Turn–on Fluorometric Sensor for Quantification of Sulfhydryl and Disulfide Forms of Biothiols: Measurement of Thiol/Disulfide Homeostasis

A simple and sensitive method was developed to determine biothiols in both sulfhydryl (–SH) and disulfide (S–S) forms using a fluorometric sensor composed of citrate–stabilized gold nanoparticles (AuNPs) prepared by electrostatic adsorption of water-soluble fluorophore Rhodamine–6G (Rh–6G) dye onto citrate–stabilized AuNPs resulting in the fluorescence resonance energy transfer in the turn-off mode. Due to the high surface affinities of biothiols to AuNPs, the fluorescence increment at 552 nm (turn-on mode) accompanying the release of Rh–6G indicated the thiol content. This assay also enables the differentiation of oxidized and reduced thiols and the measurement of thiol/disulfide homeostasis as a vital oxidative stress indicator using thiol-alkylating agent N-ethylmaleimide (NEM). After pretreatment with NEM, disulfides were measured in the presence of reduced thiols. The overall biothiol levels and thiol/disulfide ratios were measured using the proposed assay. This method was shown to be selective over various potential interfering compounds (e.g., polyphenols and plasma antioxidants) known to adversely affect redox-based assays. The proposed assay also enabled the naked-eye detection of biothiols by NP agglomeration and may be successfully applied to pharmaceutical samples containing thiols.

A novel dual-excitation and dual-emission fluorescent probe (CQDs-O-NBD) based on carbon quantum dots for detection and discrimination of Cys/Hcy and GSH/H2S in living cells

Cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) are essential active substance in human body. The identification and detection of biothiols and H2S are of great significance for the early diagnosis of certain diseases. In this paper, a new dual excitation and dual emission fluorescent probe, carbon quantum dot-O-7-nitrobenzo-2-oxa-1,3-diazole (CQDs-O-NBD), for the detection of biothiols and H2S was synthesized. The carbon quantum dots (CQDs-OH) (λex/em maxima = 377 nm/467 nm) were synthesized by a microwave-assisted method using hydroquinone as the carbon source. Through the reaction of the phenolic hydroxyl group of the carbon quantum dots and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), the CQDs-O-NBD was synthesized. Due to the static quenching (SQ) and the photo-induced electron transfer (PET) from CQDs to NBD, the fluorescence of the CQDs-O-NBD is very weak. After adding biothiols and H2S, the ether bond of the CQDs-O-NBD was broken through nucleophilic substitution reaction, which led to the release of the CQDs-OH (channel 1 is turned on) and generation of a new compound NBD-thiols (channel 2). The CQDs-O-NBD as a probe shows the high selectivity and sensitivity for Cys/Hcy/GSH/NaHS (LOD of 0.24 μM/0.21 μM/0.19 μM/0.18 μM) over other aminos and ions by channel 1. The new formed compounds from NBD and Cys/Hcy have strong fluorescence under 470 nm wavelength light excitation (channel 2 is turned on), while the formed compounds from NBD and GSH/NaHS have no fluorescence (no channel 2), so the CQDs-O-NBD can discriminate Cys/Hcy (LOD: 0.07 μM/0.06 μM) from GSH/NaHS. In addition, the CQDs-O-NBD shows low toxicity and can be applied to the detection of Cys in fetal bovine serum and imaging in living cells.

A long-wavelength fluorescent probe with a large Stokes shift for lysosome-targeted imaging of Cys and GSH

Biothiols including cysteine (Cys) and glutathione (GSH) are biological signaling molecules responsible for cell detoxification, cell metabolism and neutralization of reactive oxygen species. Here, we synthesized a long-wavelength fluorescent probe, DCIMA, for lysosome-targeted imaging of Cys and GSH in living cells. DCIMA is consisted of a dicyanoisophorone core modified with an acrylate group for biothiol detection through the Michael addition reaction and a morpholine group as the lysosome-targeting agent. The presence of the electron-donating morpholine group also enhances the intramolecular charge transfer mechanism of the probe, thereby enabling its long-wavelength fluorescence emission (670 nm) and large Stokes shift (180 nm). In concentration range of 0–30 μM, the probe was determined to react quickly with both Cys and GSH with low detection limits (<5 min, 35.2 nM for GSH and 34.8 nM for Cys) and achieve the sensitive fluorescence imaging of the biothiols located in the lysosomes of living cells.

Substitution-rearrangement-cyclization strategy to construct fluorescent probe for multicolor discriminative analysis biothiols in cells and zebrafish

Discriminative detection of biothiols (Cysteine, homocysteine and glutathione) is of great significance to clarificate their complex physiological processes, the occurrence and development of related diseases. However, similar structure and reactivity among such species pose huge challenges in developing fluorescent probes to distinguish among of them. In this work, a dual-site probe CTT reacted with the analytes to regulate molecular conjugation through substitution-rearrangement-cyclization strategy, utilizing a multi-channel signal combination mode to realize the distinguishing detection of the three biothiols. Cell and zebrafish imaging experiments sufficiently demonstrated that CTT could semiquantify biothiols, which will provide valuable chemical tool for elucidating the complex biological functions of biothiols.

Mitochondria-targeted fluorescent probe for rapid detection of thiols and its application in bioimaging

Biothiols are crucial reactive sulfur species in living cells, playing an essential role in maintaining the normal function of organisms. Effective detection of biothiols promotes the exploration of metabolic pathways and physiological functions of organisms. In this paper, a cyanine-based, mitochondria-targetable fluorescent probe, Cy-DNBS, was designed and synthesized. This probe uses a lipophilic cation unit as mitochondrial targeting site and DNBS group as the fluorescence quencher as well as the recognition site. The probe shows a distinct fluorescence emission at 604 nm upon rapid interaction with intracellular biothiols, owing to the removal of the 2,4-dinitrobenzene-sulfonyl (DNBS) group and the blockage of PET process. Moreover, the probe can not only image endogenous thiols, but also efficiently target mitochondria and evaluate thiol fluctuations under mitochondrial oxidative stress induced by H2O2. Collectively, the new low cytotoxic probe can serve as a promising tool for monitoring cellular thiols and further exploring elevant disease processes.

A Phenothiazine-HPQ Based Fluorescent Probe with a Large Stokes Shift for Sensing Biothiols in Living Systems.

Due to the redox properties closely related to numerous physiological and pathological processes, biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), have received considerable attention in biological science. On account of the important physiological roles of these biothiols, it is of profound significance to develop sensitive and selective detection of biothiols to understand their biological profiles. In this work, we reported an efficient fluorescent probe, PHPQ-SH, for detecting biothiols in vitro and vivo, based on the phenothiazine-HPQ skeleton, with DNBS (2,4-dinitrobenzenesulfonate) as the response unit. Probe PHPQ-SH exhibited brilliant sensing performances toward thiols, including a large Stokes shift (138 nm), excellent sensitivity (for GSH, LOD = 18.3 nM), remarkable fluorescence enhancement (163-fold), low cytotoxicity, rapid response (8 min), and extraordinary selectivity. Finally, the probe PHPQ-SH illustrated herein was capable of responding and visualizing biothiols in MCF-7 cells and zebrafish.

Colorimetric detection of individual biothiols by tailor made reactions with silver nanoprisms

We herein described a rapid, sensitive, and selective colorimetric sensing platform for biothiols in human serum, which relies on the dual functions of biothiols as anti-etching and aggregating agent for silver nanoprisms (AgNPRs). In principle, the target biothiols that bind to the surface of AgNPRs through Ag–S covalent interactions protect the AgNPRs from being etched by chloride ion (Cl−) in human serum, thus exhibiting the blue/purple color that is indicative of AgNPRs. On the other hand, the color of AgNPRs turned to yellow in the absence of biothiols or the presence of non-sulfur-containing amino acids, indicating the formation of small silver nanoparticles (AgNPs). Importantly, we found that individual biothiols (Hcy, Cys, and GSH) exert not only the anti-etching effect, but also the aggregating effect on AgNPRs, which can be modulated by simply tuning the pH conditions, and this consequently allows for the discriminative detection of each biothiol. Based on this simple and cost-effective strategy, we successfully determined the Hcy, Cys, and GSH in human serum with high sensitivity and selectivity within 10 min, demonstrating the diagnostic capability and potential in practical applications.

Advances of Carbon Quantum Dots for Fluorescence Turn-On Detection of Reductive Small Biomolecules

Small biomolecules in the human body involve in many important life activities, especially some small reductive biomolecules, which play important roles in maintaining human health and resisting diseases. Therefore, the rapid and accurate detection of small reductive biomolecules is of great significance. The fluorescence turn-on detection is a sensitive detection method with ease of operation. Carbon quantum dots (CQDs) are a type of novel zero-dimensional fluorescent carbon nanomaterial, and exhibit great advantages including good biocompatibility, low toxicity, ease of fabrication, low cost, good photostability and high fluorescence quantum yield, therefore, CQDs have great potential in the fluorescence turn-on detection of small biomolecules. In this paper, we summarized the development of CQDs in fluorescence turn-on detection of glucose, ascorbic acid, dopamine, biothiols and uric acid in recent three years. Finally, we discussed the opportunities and challenges of CQDs in the fluorescence turn-on detection of these small biomolecules. Achievements of CQDs in fluorescence turn-on detection of glucose, ascorbic acid, dopamine, biothiols and uric acid in recent three years are presented.

A red-emission fluorescence probe based on 1,4-addition reaction mechanism for the detection of biothiols in vitro and in vivo.

In this work, a new fluorescence probe (DC) with a donor-π-acceptor (D-π-A) structure was designed and synthesized for the detection of three kinds of biothiols (Cys, Hcy and GSH) in live cells and organisms. DC displayed an intense red-emission centered at 625nm. In the presence of biothiols, the nucleophilic addition reaction between the C=C double bond of DC and the sulfhydryl group (-SH) of biothiols occurred, resulting in obvious fluorescence quenching responses. DC exhibited high selectivity towards biothiols over other common bioactive species with low detection limits (0.26, 0.43, and 0.44µM for Cys, Hcy and GSH, respectively). In addition, DC displayed a rapid response to biothiols within 4min. The applications of DC in biothiols detection and imaging were then successfully demonstrated for the real-time monitoring of endogenous and exogenous biothiols in live cells and live animals.

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 3,5-dinitropyridin-2yl substituted naphthalimide-based fluorescent probe for the selective detection of biothiols and its application in cell-imaging.

A naphthalimide-based fluorescent probe was developed for the sensitive and selective detection of biothiols. The fluorescence of the probe was quenched by the electron-withdrawing 3,5-dinitropyridin-2-yl group via the photoinduced electron transfer process, and turned on by biothiol-triggered nucleophilic aromatic substitution. The sensing mechanism was confirmed by HPLC analysis and theoretical calculations. The probe shows a satisfactory response time of 30 min with low detection limits (Cys: 0.32 μM; Hcy: 0.88 μM; GSH: 0.46 μM). Furthermore, the probe was successfully utilized to detect endogenous and exogenous biothiols in HeLa cells.

Fabrication of a "Selenium Signature" Chemical Probe-Modified Paper Substrate for Simultaneous and Efficient Determination of Biothiols by Paper Spray Mass Spectrometry.

Significant efforts have been made to develop robust and reliable methods for simultaneous biothiols determination in different matrices, but there still exist the problems such as easy oxidation, tedious derivatization, and difficulty in discrimination, which brings unsatisfactory results in their accuracy and fast quantification in biological samples. To overcome these problems, a simultaneous biothiols detection method combining a "selenium signature" chemical probe and paper spray mass spectrometry (PS-MS) was proposed. In the strategy, the modified-paper substrate is used to enhance the analytical performance. Chemical probe Ebselen-NH2 that has a specific response to biothiols was designed and covalently fixed on the surface of an oxidized paper substrate. By the identification of derivatized product with distinctive selenium isotope distribution and employment of the optimized PS-MS method, qualitative and quantitative analysis of five biothiols including glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), N-acetylcysteine (Nac), and homocysteine (Hcy) were realized. Biothiols in plasma and cell lysates were measured with satisfactory results. The established method not only provides a novel protocol for simultaneous determination of biothiols, but also is helpful for understanding the biological and clinical roles played by these bioactive small molecules.

A new fluorescent ‘turn on’ probe for rapid detection of biothiols

ABSTRACT We designed and synthesised a novel molecular probe exhibiting high selectivity and sensitivity towards reactive sulphur species (RSS) over other amino acids and biologically relevant species, as well as scrutinised its spectroscopic behaviours under physiological conditions and in living milieu. We used an electrophilic cyanate group as a masking agent to block the excited state intramolecular proton transfer process of 2-(2-cyanato-3-methoxyphenyl)benzo[d]thiazole (HMBT-OCN), which readily hydrolyses to the highly fluorescent structure, 2-(2ʹ-Hydroxy-3ʹ-methoxyphenyl)benzothiazole (HMBT-OH), in the presence of reactive sulphur species.

Characterization of a novel styrylbenzimidazolium-based dye and its application in the detection of biothiols.

A novel styrylbenzimidazolium dye containing a maleimide group 5 was synthesized and characterized using proton nuclear magnetic resonance spectroscopy and mass spectrometry. The photophysical properties [ultraviolet-visible (UV-vis) light absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime] were investigated. Spectroscopic characterization of the novel styrylbenzimidazolium-based dye under various conditions is presented and its usefulness to detect biothiols proved. The addition of biothiols [l-cysteine (l-Cys), l-homocysteine (l-Hcy), l-glutathione (l-GSH)] to compound 5 in phosphate buffer (0.1 M, pH 7.4) containing 10% CH3 CN induced a 15-28-fold enhancement in fluorescence intensity at 410 nm. The limits of detection of compound 5 for l-Cys, l-Hcy, and l-GSH were estimated as 0.114, 0.118, and 0.059 μM, respectively. Evaluation of the cytotoxicity of 5 using the PrestoBlue assay for HeLa cells was also determined. The examined compound revealed a slight cytotoxicity against HeLa cells under experimental conditions.

A novel NIR fluorescence probe with cysteine-activated structure for specific detection of cysteine and its application in vitro and in vivo

Cysteine (Cys) as a vital antioxidant molecule and an effective biomarker for illness, plays an essential role in physiological functions and pathological processes. Extensive work has been done to explore the physiological functions of Cys and develop probes for detection of biothiols. However, the challenge to differentiate Cys from glutathione and homocystine remains. In this work, we constructed a novel near-infrared (NIR) probe, termed TMN-Cys, using TMN-NH2 and thionoesters. The probe could selectively detect Cys over homocysteine and glutathione in solution. It displayed a large Stokes shift (210 nm) upon treatment with Cys, and its detection limit was as low as 79 nM. Moreover, this probe showed low toxicity and was successfully employed in monitoring endogenous Cys in living cells and mice.

Nile-Red-Based Fluorescence Probe for Selective Detection of Biothiols, Computational Study, and Application in Cell Imaging.

A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm to 567 nm, which induces an obvious color change from blue to pink and exhibits a 35-fold fluorescence enhancement at 645 nm in red emission range. NRSH displays rapid (<1 min) response for H2S, which is faster than other biothiols (>5 min). The detection limits of probe NRSH towards biothiols are very low (22.05 nM for H2S, 34.04 nM for Cys, 107.28 nM for GSH and 113.65 nM for Hcy). Furthermore, NRSH is low cytotoxic and can be successfully applied as a bioimaging tool for real-time monitoring biothiols in HeLa cells. In addition, fluorescence mechanism of probe NRSH is further understood by theoretical calculations.

Enhancement of the Peroxidase-Like Activity of Iodine-Capped Gold Nanoparticles for the Colorimetric Detection of Biothiols.

A colorimetric assay was developed for the detection of biothiols, based on the peroxidase-like activity of iodine-capped gold nanoparticles (AuNPs). These AuNPs show a synergetic effect in the form of peroxidase-mimicking activity at the interface of AuNPs, while free AuNPs and iodine alone have weak catalytic properties. Thus, iodine-capped AuNPs possess good intrinsic enzymatic activity and trigger the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB), leading to a change in color from colorless to yellow. When added to solution, biothiols, such as cysteine, strongly bind to the interface of AuNPs via gold-thiol bonds, inhibiting the catalytic activity of AuNPs, resulting in a decrease in oxidized TMB. Using this strategy, cysteine could be linearly determined, at a wide range of concentrations (0.5 to 20 μM), with a detection limit of 0.5 μM using UV-Vis spectroscopy. This method was applied for the detection of cysteine in diluted human urine.