Synthesis, Crystal Structure and Biothiols Detection Capability of a New “off-on” Fluoresent Probe

Biothiols such as cysteine(Cys),homocysteine(Hcy)and glutathione(GSH)act as critical roles in maintaining biological redox homeostasis,which is crucial for a plenty of physiological and pathological processes.Therefore,the detection of biothiols is very important and beneficial for many applications.Herein,we have designed and developed a new crystal dimethyl 4-(2,4-dinitrophenylsulfonyloxy)pyridine-2,6-dicarboxylate(P)with 2,4-dinitrobenzene-1-sulfonyl(DNBS)pendant for light-up and detection of biothiols(Cys was selected as the analyte model).The fluorescence"off-on"switch is triggered by the cleavage of DNBS unit by the interaction with biothiols via nucleophilic aromatic substitution reaction.The turn-on fluorescent probe exhibited excellent selectivity and sensitivity toward biothiols.

Raman imaging analysis of intracellular biothiols independent of the aggregation of sensing substrates

The content of biothiols in cells is highly associated with the occurrence and development of several diseases.However,due to their active chemical properties,thiol-contained molecules are normally volatile during the detection process,rendering precise analysis of intracellular biothiols challenging.In this study,5,5’-dithiobis-(2-nitrobenzoic acid)(DTNB)is covalently modified on the surface of gold nanorods(AuNRs),constructing sensing substrates for in situ Raman imaging analysis of biothiols in cells.Au NRs are able to serve as ideal surface-enhanced Raman scattering substrates,and thus Raman signals of DTNB are greatly amplified by AuNRs.Meanwhile,the disulfide bond of DTNB can be broken by thiols,thereby releasing part of DTNB from the surface of AuNRs.As a result,three kinds of main biothiols are sensitively quantified with DTNB-modified AuNRs according to the variation of Raman signals,and DTNB-modified Au NRs exhibit far better analytical performance than a commercial probe.In addition,the sensing substrates can be readily delivered to cytoplasm with the transmembrane of Au NRs,and are capable of responding to biothiols in cells.Notably,the Raman approach is established by the breaking of chemical bonds rather than the aggregation of substrates,which is more inclined to analyze intracellular biothiols with a desirable spatial resolution.Therefore,fluctuation of biothiols in glioma cells is evidently observed via Raman imaging.Overall,this work provides an alternative strategy for designing Raman sensors to visualize active molecules in cells.

A pH-Regulated Colorimetric Sensor Array for Discrimination of Biothiols Based on Two Different-Sized β-Cyclodextrin-Functionalized AuNPs

The abnormal changes of biothiols are directly related to health condition of human body,and the effective identification and quantification of biothiols is of great significance for screening and diagnosis of disease.This study described the development of a p H-regulated colorimetric sensor array for discrimination of five kinds of biothiols including cysteine(Cys),glutathione(GSH),homocysteine(Hcy),cysteamine(CA)and N-acetylcysteine(NAC).The proposed sensor array was established using 5-nm-and 20-nm-sizedβ-cyclodextrin-functionalized gold nanoparticles(β-CD@Au NPs)as nonspecific receptor and signal transduction elements.Due to the different binding affinity between biothiols andβ-CD@Au NPs in various p H environments,the different aggregation behaviors of nanoparticles produced unique colorimetric response patterns,which were able to be distinguished by bare eyes and UV-vis spectrophotometer.Accordingly,principal component analysis(PCA)and hierarchical cluster analysis(HCA)were employed for pattern recognition and generated a clustering map for a clear differentiation of biothiols at the level ofμmol/L.Furthermore,it can be proved that the method was successfully applied to the analysis of biothiols in human urine samples.

Fluorescent probes for detection of biothiols based on “aromatic nucleophilic substitution-rearrangement” mechanism

Biothiols,including cysteine(Cys),homocysteine(Hey),and glutathione(GSH) play important roles in physiological processes,and the detection of thiol using fluorescent probes has attracted attention due to their high sensitivity and selectively and invasive on-time imaging.However,the similar structures and reactivity of these biothiols present great challenges for selective detection.This review focused on the the "aromatic nucleophilic substitution-rearrangement(SNAr-rearrangement) mechanism",which provided a powerful tool to design fluorescent probes for the discrimination between biothiols.We classify the fluorescent probes according to types of fluorophores,such as difluoroboron dipyrromethene(BODIPY),nitrobenzoxadiazole(NBD),cyanine,pyronin,naphthalimide,coumarin,and so on.We hope this review will inspire exploration of new fluorescent probes for biothiols and other relevant analytes.

A multifunctional upconversion nanoparticles probe for Cu^(2+)sensing and pattern recognition of biothiols

Lanthanide-doped upconversion nanoparticles(Ln-UCNPs)are a new type of nanomaterials with excellent fluorescence properties,which are well applied in fluorescent biosensing.Herein we developed a multifunctional probe based on the surface engineering of core-shell structure UCNPs with polyacrylic acid(PAA).The developed PAA/UCNPs probe could be highly selective to detect and respond to Cu^(2+) at different pH.Cu^(2+) could easily combine with the carboxylate anion of PAA to quench the fluorescence of UCNPs.Therefore,we creatively proposed a fluorescent array sensor(PAA/UCNPs-Cu^(2+)),in which the same material acted as the sensing element by coupled with pH regulation for pattern recognition of 5 thiols.It could also easily identify the chiral enantiomer of cystine(L-Cys-and D-Cys),and distinguish their mixed samples with different concentrations,and more importantly,it could be combined with urine samples to detect actual level of homocysteine(Hcys)to provide a new solution for judging whether the human body suffers from homocystinuria.

Dual-site fluorescent probe for highly selective and sensitive detection of sulfite and biothiols

A dual-site fluorescent probe with double bond and aldehyde as reactive sites, was designed for the selective detection of sulfite and biothiols. Sulfite reacts with conjugate bond selectively, while Cys responses with aldehyde and GSH occurs substitution reaction. Different interactions cause different absorption and fluorescence responses. Moreover, it could be further applied in imaging in living cells.

Bis-cyclometalated Ir(Ⅲ)Complex-Based Electrogenerated Chemiluminescence Sensor Array for Discriminating Three Biothiols

A simple and sensitive electrogenerated chemiluminescence(ECL)sensor array was developed for the discrimination of three biothiols,including homocysteine(Hcy),cysteine(Cys)and glutathione(GSH)using two bis-cyclometalated Ir(Ⅲ)complexes as ECL probes.Two aldehyde groups bearing bis-cyclometalated Ir(Ⅲ)complexes were selected as ECL probes,including[(bt)2Ir(phen-CHO)]PF_(6)(bt=2-phenylbenzothiazole,phen-CHO=1,10-phenanthroline-5-carboxaldehyde,probe 1)and[(ppy)_(2)Ir(phen-CHO)]PF_(6)(ppy=2-phenylpyridine,probe 2).A“signal on”ECL method was proposed for biothiols assay based on the increase in ECL intensity of two ECL probes by biothiols.Three biothiols can be detected with detection limits of 0.8,0.7 and 0.8μM by probe 1 and 0.3,0.4 and 0.5μM by probe 2 for Cys,Hcy and GSH,respectively.Two-element sensor array was assembled for the detection of three biothiols due to the different enhancement effects of biothiols on the ECL intensity of two ECL probes.The ECL sensor array combining with the principal component analysis was applied to discriminate three biothiols.This study demonstrates that the ECL sensor array using dual ECL reagents provides a promis-ing way for the discrimination of multiple biothiols with good sensitivity and simpleness.

Au nanoparticles based ultra-fast “Turn-On” fluorescent sensor for detection of biothiols and its application in living cell imaging

Au or other metal nanostructures have the ability to strongly quench the fluorescence of fluorophores.This feature has made AuNP-conjugates attractive for the construction of platforms for various bioanalytes to overcome the limitations of small molecule fluorophores(poor solubility,long reaction time).In this paper,an ultrafast"Turn-On"fluo rescent sensor for biothiols was constructed.The sensor is based on the fluorescent resonance energy transfer(FRET)effect between the fluorophore(PN)and AuNPs,which effectively quenches the fluorescence of the fluorophore.In the presence of thiols,PN is displaced and released from AuNP surfaces,and thus,the fluorescence is rapidly restored.The sensor features appreciable water solubility and ultrafast response time(a few seconds for Cys).In addition,it exhibits high selectivity and a detection limit as low as 12 nmol/L for Hcy.Moreover,the sensor presents good biocompatibility and has been successfully applied for imaging biothiols in living cells.

Discrimination of biothiols in different media with NBD-F as the probe

4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole（NBD-F） was employed as a colorimetric probe for differential detection of biothiols in different media. The spectral response and the selectivity of NBD-F toward thiols were significantly improved by surfactant micelles. Mercapto group exhibited high reactivity in all the solvents（including Tris–HCl buffer solution, CTAB and SDS micelles）. The 4-thioether derivatives of NBDF reacting with Cys and Hcy but not GSH could transfer to the corresponding 4-amino-substituents via intramolecular nucleophilic aromatic substitution, thus, GSH could be discriminated from Cys/Hcy. In CTAB micelles, the reaction product of NBD-F with Cys is non-fluorescent and it absorbs in longwavelength region. According to the spectral responses of NBD-F toward different low-molecularweight thiols, we could identify Cys, Hcy and GSH from each other.

Biothiol-specific fluorescent probes with aggregation-induced emission characteristics

Biothiols are important species in physiological processes such as regulating protein structures, redox homeostasis and cell signalling. Alternation in the biothiol levels is associated with various pathological processes, therefore non-invasive fluorescent probes with high specificity to biothiols are highly desirable research utilities. Meanwhile, fluorescent probes with aggregationinduced emission properties(AIEgens) possess unique photophysical properties that allow modulation of the sensing process through controlling the aggregation-disaggregation or the intramolecular rotational motions of the fluorophores. Herein we review the recent progress in the development of biothiol-specific AIEgens. In particular, the molecular design principles to target different types of biothiols and the corresponding sensing mechanisms are discussed, along with the potential of the future design and development of multi-functional bioprobes.

Recent development in fluorescent probes based on attacking of double bond and masking of functional group

The works on the procedure of fluorescent sensors for the detection of biological analytes are extremely momentous.Among diverse analytical approaches,fluorescence is the most eye-catching due to its high sensitivity,selectivity,rapidity,robustness,ease of measurement and non-destructive approaches.Herein,we show different fluorescent probes synthesized for estimation and detection of biological analytes(H_(2)S,SO_(3)^(2-)/HSO_(3)^(-),H_(2)O_(2),HOCl,HNO,ONOO^(-)).These probes were constructed by masking the functional groups (hydroxyl and amino) of fluorophore and formation of active C=C,C=N,C=O and N=N for specific analytes.In this review we concentrate on synthesis of the probe,their photophysical properties and applications to biological studies.

A multisite-binding fluorescent probe for simultaneous monitoring of mitochondrial homocysteine, cysteine and glutathione in live cells and zebrafish

Homocysteine(Hcy), cysteine(Cys) and glutathione(GSH) play crucial roles in redox homeostasis during mitochondria functions. Simultaneous differentiation and visualization of mitochondrial biothiols dynamics are significant for understanding cell metabolism and their related diseases. Herein, a multisitebinding fluorescent probe(MCP) was developed for simultaneous sensing of mitochondrial Cys, GSH and Hcy from three fluorescence channels for the first time. This novel probe exhibited rapid fluorescence turn-on, good water-solubility, high selectivity and large spectral separation for discriminating Cys, GSH and Hcy with 131-, 96-, 748-fold fluorescence increasement at 471, 520, 567 nm through different excitation wavelengths, respectively. Importantly, this probe was successfully applied to simultaneous monitoring of mitochondrial Cys, GSH, and Hcy in live cells and zebrafish from three fluorescence channels,promoting the understanding of the functions of Hcy, Cys and GSH.

Detection of biological thiols based on a colorimetric method

Biological thiols（biothiols）, an important kind of functional biomolecules, such as cysteine（Cys） and glutathione（GSH）, play vital roles in maintaining the stability of the intracellular environment. In past decades, studies have demonstrated that metabolic disorder of biothiols is related to many serious disease processes and will lead to extreme damage in human and numerous animals. We carried out a series of experiments to detect biothiols in biosamples, including bovine plasma and cell lysates of seven different cell lines based on a simple colorimetric method. In a typical test, the color of the test solution could gradually change from blue to colorless after the addition of biothiols. Based on the color change displayed, experimental results reveal that the percentage of biothiols in the embryonic fibroblast cell line is significantly higher than those in the other six cell lines, which provides the basis for the following biothiols-related study.

A red-emitting water-soluble fluorescent probe for biothiol detection with a large Stokes shift

In this work, we presented a fluorescent probe(MCQ-DNBS) for selective and sensitive detection of biothiols based on a methylated chromenoquinoline(MCQ) derivative. Probe MCQ-DNBS was constructed by masking the OH group in MCQ with a common sensing unit, 2,4-dinitrobezensulfonate group(DNBS) for biothiols. Due to the photo-induced electron transfer(PET) process between MCQ and DNBS, this probe was weekly fluorescent. Upon the addition of biothiols(Cys, Hcy and GSH), this probe emitted a strong red fluorescence(λ_(em max)=613 nm) with a large Stokes shift(115 nm). In addition,fluorescence imaging of biothiols in living cells was successfully realized using MCQ-DNBS as a detector.

Conjugated Polyelectrolyte-Ag＋ Fluorescent Switch for Biothiol Sensing

A new ＂off-on＂ switch for sensitive and selective fluorescence detection of biothiols[glutathione（GSH）, cysteine（Cys） and homocysteine（Hcy）] was developed based on an anionic conjugated polyelectrolyte（CPE）, pyridyl-functionalized poly（phenylene ethynylene）（Pl）. The fluorescence of P1 can be significantly quenched by Ag＋ due to complexation-mediated interpolymer aggregation. Furthermore, biothiols can efficiently recover the fluorescence intensity of P1 as a result of the stronger binding between thiol group and Ag＋, which dissociates PI from the P1/Ag＋ complex and disrupts interpolymer aggregation. Under optimum conditions, a good linear relation- ship in a range of 100--4200 nmol/L is obtained for GSH with a detection limit of 80 nmol/L（S/N=3）. As a result of specific interaction between the thiol group and Ag＋, the proposed method shows a high selectivity for biothiols. In addition, the CPE-based fluorescence ＂off-on＂ switch has been used to quantitatively detect total biothiols in cell lysates.

Glutathione-triggered non-template synthesized porous carbon nanospheres serve as low toxicity targeted delivery system for cancer multi-therapy

Nano material based drug delivery system have received great attention in clinical application due to their high therapeutic efficacy and lower side effects than classical method,multi-functional nanomaterial also have shown the excellent performance at cancer theranostic and durg tracking in vivo and in vitro.However,most of these works are influenced by the bio-toxicity of applied nanomaterials,which could influence the diagnostic results and treatment effect.Therefore,we have prepared a high biocompatibility porous carbon nanospheres(PCNs) based nano-system(PCN-siRNA-DOX-FA) for targeted drug delivery and the ranostic.The surface modifications have increased dispersion and stability of the PCNs,and folic acid(FA) had enhanced the active target ability for FA receptor positive cell lines.Moreover,through the siRNA structure and doxorubicin(DOX) loading,biological and chemical combined multi-therapy was achieved in cancerous cells.This constructed nano-system could positively improve the biotoxicity problem of nanomaterial and provide a potential platform for clinical cancer theranostic applications.

N-Acety-L-Cysteine-Stabilized Pt Nanozyme for Colorimetric Assay of Heparin

N-Acety-L-cysteine(NAC),enriched with multiple electron-rich groups involving-SH,-C=O,and-COOH,exhibits strong affinities to transition metal ions.Herein,NAC was adopted as the stabilizing agent to synthesize a series of peroxidase-like Pt nanoclusters(NCs)with different physicochemical properties.Both the cluster size and the charge state of NAC-Pt NCs are highly dependent upon the molar ratio of[K_(2)PtCl_(4)]/[NAC],contributing to different peroxidase mimicking activities.The most active Pt nanozyme(~1.7 nm),composed of 60%metallic Pt^(0) and 40%positively charged Pt^(2+)respectively,exhibits the K_(m) of 0.132 mM for 3,3′,5,5′-tetramethylbenzidine and 35 mM for H_(2)O_(2).Based on the heparin-activated enzymatic activ-ity of NAC-Pt at pH 6.0,an ultrasensitive test was established for quantitative determination of heparin,giving the linear response of 0.5-20μg/mL as well as the limit of detection of 2×10^(−3)μg/mL.This proposed method is also applicable in biological fluid for practical applications.Such a colorimetric method possesses several advantages involving high sensitiv-ity,short response,eco-friendly synthesis,low consumption of material and energy,free of expensive instrument,as well as simple operation.