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.

基于香豆酮并色酮生物硫醇荧光探针的合成及应用研究

为监测具有高度生物学活性的谷胱甘肽(GSH),半胱氨酸(Cys)和高半胱氨酸(Hcy)等生物硫醇的生物功能,基于丙烯酸酯对生物硫醇的识别,通过三步反应设计合成香豆酮并色酮的荧光探针JA检测生物硫醇。探针JA具有反应快(10 min响应Cys)、抗干扰性强(Stokes位移150 nm)、毒性低和生物相容性好等特点,能对活细胞内外源性的生物硫醇进行荧光成像。探针JA为生物硫醇的灵敏检测提供了一种简单有效的方法。

以卡托普利为模板银纳米团簇的制备及生物硫醇的检测研究

生物硫醇在生物体内的代谢过程中起着重要作用,如体内半胱氨酸(Cys)和谷胱甘肽(GSH)含量异常会引发一系列疾病。为了建立一种快速、定量检测生物硫醇的荧光分析方法,本文以卡托普利(Captopril,Capt)为模板,采用湿化学还原法成功合成了发红色荧光的银纳米团簇(Capt-Ag NCs),对Capt-Ag NCs的形貌、结构、元素组成和光学性能等进行了表征。基于生物硫醇小分子对Capt-Ag NCs的荧光猝灭作用建立了定量检测Cys和GSH的新方法,在最佳实验条件下,Cys和GSH的线性范围分别为1~100μmol/L和2~180μmol/L,检出限分别为0.054μmol/L和0.23μmol/L。该方法可用于人血浆中Cys的测定,回收率为95.3%~103.3%。实验结果表明,CaptAg NCs作为一种新型信号关闭荧光探针,可用于Cys和GSH等生物硫醇的检测。

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 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.

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.

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.

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.

结直肠癌细胞WS480中硫化氢和生物硫醇的检测与成像研究

目的 旨在设计、合成一种新型荧光探针,用于结直肠癌细胞中生物硫醇和硫化氢的检测与成像,期望为结直肠癌的早期诊断提供新工具和新方法。方法 设计的探针WZL-1通过4步反应从商业易得的廉价原料合成而得,并对其光学性能、细胞毒性以及活细胞荧光成像进行了详细评价。在评价中,考虑了探针的荧光特性、对生物硫醇和硫化氢的敏感性,以及其在活细胞成像中的适用性。结果 在生理条件下探针WZL-1对生物硫醇半胱氨酸、同型半胱氨酸、谷胱甘肽和硫化氢在630 nm处的发射强度均显著增强。探针表现出卓越的灵敏度,在仅需30 s的极短时间内,WZL-1能够迅速准确地检测目标分子,且荧光强度至少可以维持50 min。WZL-1荧光探针的线性范围广泛,其对GSH的检测线低至554 nm,探针在0~30μmol/L浓度范围内细胞存活率超过90%。结论 荧光探针WZL-1能与结直肠癌细胞WS480内的生物硫醇和硫化氢发生特异性反应,并释放出红色荧光信号,而其他物质分子则不与其反应,其可用于细胞中各种生物硫醇和硫化氢的实时监测与成像。

一种检测生物硫醇和硫化物的NBD荧光探针

文中合成了一种新型的荧光探针LD-TPA-NBD,系统研究了探针响应生物硫醇和硫化物的性质。结果表明,探针LD-TPA-NBD在380 nm和475 nm的激发波长下,可以特异性的识别不同的活性硫物质,对Cys、GSH、Hcy、HS-和HSO3-的检测限分别为47.7 nM、72.2 nM、76.9 nM、0.34μM和0.207μM(λ_(ex)=380 nm)。此外,通过研究探针LD-TPA-NBD的荧光动力学,发现探针的荧光信号在3~7 min内即可达到平衡。探针LD-TPA-NBD可为用于检测活性硫物质的荧光探针的构建提供参考。

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.

N,Br-CDs作为荧光传感器连续检测银离子和生物硫醇

银离子(Ag^(+))是水体污染中最主要的有害重金属之一,由于其生物积累性和潜在毒性,Ag^(+)能够与酶/蛋白质结合并导致其失活.此外,生物硫醇,如谷胱甘肽(GSH)、半胱氨酸(Cys)在细胞生长、代谢和维持适当的氧化还原状态中发挥重要作用.因此,有效检测生物液体中的Ag^(+)和生物硫醇在生物化学和临床化学中具有重要意义.本工作以4-溴邻苯二胺和多巴胺为前体,通过水热法合成了氮溴共掺杂CDs(N,Br-CDs).N,Br-CDs具有强烈的红光,发射波长为640 nm,相对量子产率高达24.6%.再通过Ag^(+)猝灭其荧光,生物硫醇恢复其荧光,我们构建了一种荧光开关传感器用来连续检测Ag^(+)和生物硫醇,此方法已适用于人尿中生物硫醇的检测.本工作设计的荧光开关传感器用来连续检测Ag^(+)和生物硫醇有以下亮点:首先设计出红色荧光进行检测,这是目前极少被报道的;其次是相较于其他传感器,该传感器具有灵敏度极高、操作简单、线性范围较宽等优势.

3-氨基香豆素衍生的荧光探针

香豆素是常用的荧光生色团,在发展新型高效的荧光探针中有重要应用。3-氨基香豆素是一种α,β-脱氢氨基酸内酯,为其发展成为有多种生物活性与其他功能分子提供了独特的结构基础。迄今为止,以3-氨基香豆素为原料发展了一系列荧光探针分子,快速检测金属离子和生物分子。综述了以3-氨基香豆素为原料发展的一系列新的荧光探针,这些探针主要用于选择性地高效探测Hg^(2+)、Ag+、Cu^(2+)、Cu+、Pd^(2+)、Zn^(2+)和Cr^(2+)等多种金属离子、硫离子、半胱氨酸和β-巯基乙醇等生物硫醇、H_(2)O_(2)和活性蛋白质。

一种检测生物硫醇荧光探针的合成与应用

基于硫醇诱导的迈克尔加成反应阻断探针的光诱导电子转移过程(PET)合成了一种基于氟化硼络合二吡咯甲川(Bodipy)类染料的荧光探针,该探针具有高灵敏度和选择性,可在生理条件下检测硫醇。利用核磁和高分辨质谱对探针结构进行了表征。当向探针溶液加入硫醇(0~1 000μmol/L)时,可在探针溶液的绿色光谱区域引起一个显著的荧光增强响应(增强至150倍)。同时,探针可以检测相对较低浓度的硫醇,对于含有硫醇的氨基酸(半胱氨酸、谷胱甘肽和高半胱氨酸)的检出限分别为4.5×10^(-7),1.2×10^(-7),2.1×10^(-7)mol/L。此外,相对于其他氨基酸,探针对硫醇具有较高的选择性和灵敏度。该方法成功实现了细胞内硫醇的荧光成像,证明该荧光探针在生物体系中具有潜在的应用能力。

柱前衍生高效液相色谱-荧光检测法测定水稻中7种巯基化合物

建立了水稻中半胱氨酸（Cys）、谷胱甘肽（GSH）和植物螯合肽（phytochelatin，PC：PC2、PC3、PC4、PC5、PC6）7种巯基化合物的柱前衍生高效液相色谱-荧光检测分析方法。样品经0.1％三氟乙酸（ TFA）（含6.3 mmol／L 二乙烯三胺五乙酸（DTPA））超声提取，然后以单溴二胺（mBrB）为衍生剂在 pH 8.0的4-羟乙基哌嗪丙磺酸（HEP-PS）缓冲溶液中衍生化。采用的色谱分离柱为 Agilent Eclipse plus Cl8柱，流动相为0.1％TFA（pH 2.5）和100％乙腈（ ACN），梯度洗脱，流速为0.8 mL／min。荧光检测的激发波长和发射波长分别为380 nm 和470 nm。结果表明，7种巯基化合物在0.7-100.0 mg／L范围内，峰面积与质量浓度之间的线性关系良好（ r2≥0.9991）；检出限为0.03-0.20 mg／L；加标回收率为89.26％-99.42％，相对标准偏差为2.05％-5.87％。该方法准确、灵敏度高、重现性好，为水稻中巯基化合物的研究提供了检测手段。