Advances in fluorescence probes for detection of hydrogen polysulfides / 中国药科大学学报

@#Sulfane sulfur species in the reactive sulfur species family include hydrogen polysulfides (H2Sn, n ≥2), which play an essential role in physiological regulation and signal transduction. As a redox pair of H2S, H2Sn can be produced through oxidation or enzyme reaction and regulate protein interaction and enzyme activity.Research has revealed that H2Sn, with higher efficiency of protein S-sulfhydration than H2S, may be responsible for some physiological functions previously attributed to H2S.Therefore, real-time detection of H2Sn is crucial for studying its physiological activity and the relationship between H2S and H2Sn.Traditional detection methods, such as mass spectrometry, are not suitable for living organisms as they require tissue cell disruption.Instead, fluorescence probes are often used for in situ real-time detection due to their high sensitivity and specificity and low biological toxicity.This review summarizes the physiological regulatory activity of H2Sn, as well as the design strategy, response mechanism, fluorescence characteristics, and biological applications of H2Sn fluorescent probes based on the structure of the response group, with a prospect of the challenges and developments in this field.

Research progress on the detection methods and their application in ferroptosis / 药学学报

Ferroptosis is a novel type of cell death, which is distinguished from the traditional cell death pathways such as apoptosis, proptosis, necrosis and autophagy in terms of morphology, biochemistry and genetics. The main features of ferroptosis are the iron accumulation and lipid peroxidation. The regulation mechanism of ferroptosis involves glutathione metabolism, lipid peroxidation reactions and iron metabolism, which are closely related to the pathological process of tumor, aging, neurodegenerative diseases, ischemia reperfusion injury, cardiovascular and cerebrovascular diseases, kidney injury, hepatic fibrosis and so on. How to effectively study the role of ferroptosis regulation mechanism in the treatment of diseases becomes the hot spot and focus of the ferroptosis research. In recent years, with the in-depth study of ferroptosis, the identification, confirmation and the mechanism of ferroptosis have been developed significantly and have come forth continuously, in the meantime, techniques based on the morphology, biochemistry, molecular biology and genetics have been widely applied in the detection of ferroptosis. In order to deepen readers' understanding of ferroptosis and its detection methods, this paper will mainly review the current research progress on the detection methods and their application in ferroptosis, summarize and discuss their advantages and disadvantages in the detection of ferroptosis, this knowledge are crucial for better understanding and studying the biological function of ferroptosis.

The effect of different guanine base number on fluorescence intensity of DNA/ silver nanoclusters / 药学实践杂志

Objective To investigate the effect of different guanine base numbers on the fluorescence intensity of DNA/ silver nanoclusters through C-G base complementary pairing, in order to explore a new method for the construction of fluorescent probe switches. Methods Designed complementary sequences of aptamer parts of a series of silver nanoclusters by using the nucleic acid base complementary pairing principle, and investigated the effect of the number of G bases exposed in the aptamer sequence on the fluorescence signal. Results Base G could enhance the fluorescence signal intensity of silver nanoclusters, and the fluorescence signal strength was positively correlated with the number of G bases. The fitting linear equation was Y=1726.1X+8972.5, r=0.9789. Conclusion This study is a great reference for the regulation of fluorescence intensity of silver nanoclusters and the design of G quadruplet aptamer fluorescent probe switch.

Design, synthesis and biological application of affinity-based small molecular probe for Hsp90 endoplasmic reticulum paralogue of Grp94 / 中国药科大学学报

@#Glucose-regulated protein 94(Grp94), an endoplasmic reticulum resident Hsp90 paralog, has a limited set of client proteins. Selective inhibition of Grp94 has emerged as a new direction for the development of drugs targeting the Hsp90 chaperone system. Now Grp94-Probe, an affinity-based probe of Grp94, was designed and synthesized based on DDO-5813, a most potent Grp94-selective inhibitor we found previously. Using fluorescence polarization(FP)assay and double staining assay with ER-Red in cells, we confirmed the binding of Grp94-Probe with ER Grp94. The FR results showed that the probe exhibited high affinity for Grp94N(EC50=117. 9 nmol/L)without exhibiting obvious Hsp90α inhibition, Moreover, as a fluorescence probe molecule, Grp94-Probe could better distinguish the inhibitory activity of compounds for Grp94N. The results of fluorescence analysis in cells showed that Grp94-Probe could co-stain with ER-Red in the endoplasmic reticulum, and the fluorescence did not decay rapidly with time after 4 h of staining, which further indicated the binding of Grp94-Probe with Grp94 in cells. This Grp94 selective probe can be further used for biology evaluation of Grp94 inhibitor and exploration of Grp94 biological functions.

Establishment of a Method for the Identification of Vibrio Parahaemolyticus by Taqman-Probe Fluorescence Quantitative PCR Analysis / 现代检验医学杂志

Objective To establish a method for the identification of Vibrio parahaemolyticus based on Taqman-fluorescence probe quantitative PCR method targeting toxR gene.Methods Taking the standard strain of Vibrio parahaemolyticus (VPJS421) and other ommon pathogenic bacteria'standard strain as the research object,using the bio-software to design specific PCR primers and Taqman probe of Vibrio parahaemolyticus toxR gene and detected by fluorescence quantitative PCR instrument.Results ①The designed primers could amplify specific bands.②The amplification efficiency of the 0.5 μl probe in the amplification system was better than that of the 1.0μl probe.③The detection sensitivity of toxR gene of Vibrio parahaemolyticus by Taqman fluorescence quantitative PCR was 10-1 mg/L.④The detection method did not show positive amplification in detection of Enterococcus f aecalis,Staphylococcus aureus,Saprophytic staphylococcus,Enterobacter hormaechei,Pseudomonas aeruginosa,Escheri ch ia coli,Vibrio al ginol yticus,Vibrio vulnficus,Vibrio metschnikovii and Wbrio furnissii 10 other common pathogenic bacteria.The specificity was 100％.Conlusion The fluorescence quantitative PCR method for the identification of Vibrio parahaemolyticus was successfully established.The method was sensitivty and specificity,and it is suitable for rapid detection of Wbrio parahaemolyticus and has a good application value.

Principle and applications of fluorescent probes for intracellular redox detection / 上海交通大学学报（医学版）

Redox state indicated by reactive oxygen species (ROS) level is a cellular physiological feature.Redox status is exactly controlled by conserved system.Its alterations consequently modulate various biological activities and play important roles in cells and organism under physiological and pathological conditions.Hence detection of redox state is deemed as an important technique both in basic and clinical researches.The present article reviews the principle and current applications of fluorescent probes or sensors for redox detection.With comparison of properties of various probes,especially genetic coding fluorescence probes,much understanding will be gained.And the review helps us to understand how to apply the redox probes on the biological and clinical researches and eventually look forward to new applications in the cell and whole animal level.

Principle and applications of fluorescent probes for intracellular redox detection / 上海交通大学学报（医学版）

Redox state indicated by reactive oxygen species (ROS) level is a cellular physiological feature. Redox status is exactly controlled by conserved system. Its alterations consequently modulate various biological activities and play important roles in cells and organism under physiological and pathological conditions. Hence detection of redox state is deemed as an important technique both in basic and clinical researches. The present article reviews the principle and current applications of fluorescent probes or sensors for redox detection. With comparison of properties of various probes, especially genetic coding fluorescence probes, much understanding will be gained. And the review helps us to understand how to apply the redox probes on the biological and clinical researches and eventually look forward to new applications in the cell and whole animal level.

Highly Sensitive Detection of Spermine Based on Fluorescence Quenching of Gold Nanoclusters / 分析化学

A new type of fluorescent gold nanoclusters (MU-Au NCs) was prepared by hydrothermal synthesis method using ammonium benzoate murexide (MU) as reducing agent and protecting agent.The synthesis method was simple and rapid.Based on the fluorescence quenching ability of spermine, a turn off type fluorescence analysis method was established for rapid and ultra sensitive detection of spermine.The linear range for detection of spermine was 0.003-300 μmol/L and the detection limit was 1 nmol/L (S/N=3).The established analytical method of spermine provided theoretical basis and reference for construction of spermine biosensor and actual sample detection.

Application of PCR-fluorescent probe in identifying genotypes of HCV / 中华临床感染病杂志

Objective To evaluate the application value of polymerase chain reaction (PCR)-fluorescence probe method in identifying genotypes of hepatitis C virus (HCV).Methods One hundred and sixty six serum samples from patients with chronic HCV infection were collected nationwide from March to June 2016.HCV Core-E1 gene region was amplified and sequenced by nested reverse transcription-PCR (RT nested-PCR)and genetic subtypes were analyzed by phylogenetic tree,meanwhile HCV genotypes were also determined by PCR-fluorescent probe method.Kappa test was used to compare the consistency of two methods.Results Among 166 samples detected by RT nested-PCR,the genotype of 66 samples (39.8%) was 1 b,34 (20.5%)was 2a,16 (9.6%)was 3a,27 was 3b (16.2%),23 (13.9%)was 6a.Two samples with 3b genotype detected by RT nested-PCR were identified as 1 b by PCR-fluorescent probe.The consistency rate of two methods was 98.7% (164 /166),there was no significant difference between two methods (χ2 =0.0492,P >0.05).Conclusion PCR-fluorescence probe method can accurately identify HCV genotypes and can be used in clinic.

Facilitating the measurement of circulatory hydrogen sulfide with fluorescence probe-coated microplates / 北京大学学报(医学版)

Objective:The hydrogen sulfide (H2 S) role in pathogenesis of various diseases were wildly addressed in recent decade.The circulatory (plasma or serum) and biological fluid H2S measurement is still an enormous issues due to the technical limitation.This paper aimed to develop a novel measurement method based on fluorescence probe.Methods:Firstly,20 μL ethanol was used to dissolve 100 pmol fluorescence probe,then added in a 96-well plate.An equal volume of ethanol was also added to the blank well of the plate.The plate was placed in a dark room for about 1 h until the fluorescence probe was evenly coated in the 96-well microplate and dried.The plate was frozen at-20 ℃ for later use.Secondly,the plasma or serum sample was added with saturated ammonium sulfate buffer (pH 7.8) and then centrifuged to remove the proteins.The equal volume supernatant liquid was added to the probecoated well and the probe-uncoated well.The plate was incubated in a dark environment at 37 ℃ for 2 h.Finally,after incubation,the fluorescence density was acquired at λEx/λEm 340/445 nm in a microplate reader.The differences of the fluorescence density values between the probe-coated well and probeuncoated well were counted and H2S concentration of plasma/serum was calculated by standard curve with NaHiS.Results:The method had high sensitivity (from 0.3 to 100 μmol/L) and specificity for measuring H2S as compared with other biologically relevant reactive sulfur species and sulfur-containing amino acid.Serum H2S concentrations were assayed in 188 health volunteers using this method [(12.1 ±3.5) μmol/L,95％ CI:4.6-19.8 μmol/L],and the frequency distribution showed a normal tendency(one-sample Kolmogorov-Smirnov test,P ＞ 0.1).The serum H2S concentrations in 30 hypertension patients were decreased compared with 22 age-and gender-matched health individuals (paired-samples t test,t =9.937,P ＜ 0.001).There were no differences of H2S concentration in serum [(19.66 ±2.32) μmol/L] or plasma [(18.67 ±2.07) μmol/L],between the samples acquired from artery [(19.34 ±0.51) μmol/L] or vein [(18.99 ±0.50) μ mol/L] of male Wistar rats (repeated measurement of ANOVA,P =0.38).One week frozen samples did not affect the detection.The values of the repeated measurement did not differ (two-way ANOVA,P ＞ 0.05).Conclusion:The present method is easily performed with high sensitivity,specificity and repeatability for circulatory H2S.It is also quick and may apply for large samples.

Establishment of Muti-channel Taqman-Probe Fluorescence Quantitative PCR Identification MRSA Method / 现代检验医学杂志

Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR basing on mecA/nuc/fem B three gene combined detecting.Methods Taking the coagulase positive MRSA,which isolated from the clinical samples and confirmed by VITEK 2 compact microbial analyzer,as the research obj ect,designed mecA/nuc/fem B specific PCR primers and Taqman fluorescent probe by bio-software PrimerPremier 5 and Designer Beacon 7,FAM,HEX and ROX markers were used to label the fluorescent probe at 5’,and the end of 3’was labeled with BHQ1,detected by fluo-rescence quantitative PCR instrment.Results ①1 g/dl gel electrophoresis results showed that the primer’s specificity of mec A/nuc/fem B were good,and molecular weight of the amplification band consistent with the expected molecular weight and no non-specific amplification band.②Three genes were obtained specific amplification in a single tube single channel and single tube multiple channel detection in PCR,and the three gene amplification effect in a single tube single tube single chan-nel and multichannel PCR similar.Conclusion Successfully established a method of multi channel Taqman-probe fluores-cence quantitative PCR identification of MRSA,mec A/nuc/fem B combined detection can effectively differentiate coagulase negative and positive MRSA,improve the accuracy of identification.

The changes of mitochondrial membrane potential detected by confocal microscopy in spermatocyte induced by bisphenol A / 中国医学装备

Objective:To detect changes in mitochondrial membrane potential after sperm mother cell damage induced by bisphenol A by laser scanning confocal microscopy(LSCM) and underline its potential action mechanism.Methods: The cultured spermatogenic cells were divided into 3 groups, respectively. Then 0, 10μmol/L, 100μmol/L of Bisphenol A(BPA) were added into the culture, after 3 hours culture, fluorescence probe JC-1 was used to lable the three groups. The fluorescence intensity of JC-1 in mitochondrial was then detected by LSCM. LSCM software was used to analyze the fluorescence intensity. Change of the mitochondrial membrane potential was represented by the change of fluorescence colors (relative proportion of red and green fluorescence is commonly used to measure mitochondrial depolarization ratio). Results:The ratio between the red and green fluorescence in the control group, low dose group and high dose group had significant difference. Intracellular mitochondrial membrane potential of Bisphenol A treatment group was lower than that of the control group, while mitochondrial membrane potential of high dose group was lower than that of the low dose group. Conclusion: Bisphenol A could damage spermatocytes, and as the dose increased, the more serious the injury. The method could real-time monitor with high sensitivity the change of intracellular mitochondrial membrane potential.

Fluorescence agent and methylene blue in sentinel lymph node biopsy for patients with breast cancer / 中华内分泌外科杂志

Objective To analyze the value of sentinel lymph node biopsy (SLNB)in predicting axillary lymph node status by injection of fluorescence agent and methylene blue.Methods 156 breast cancer patients receiving surgery from Oct.2013 to Jun.2014 were studied and they were randomly divided into the experimental group(n =78) and the control group(n =78).The fluorescent agent combined with methylene blue and methylene blue were used respectively as tracers for SLNB.Axillary lymph nodes dissection was made during surgery and combined with pathology the status of sentinel lymph node(SLN) metastasis was distinguished between true negative,false negative,true positive,and false positive.Results A total of 164 SLNs were detected by the method of fluorescent agent combined with methylene blue with the detection rate of 97.44％.An average of 2.10 SLNs were detected for each patient.The accuracy rate was 97.44％,the sensitivity was 97.44％,the false negative rate and the false positive rate was 0％ and 0％.A total of 139 SLNs were detected by the method of methylene blue with the detection rate of 89.74％.An average of 1.78 SLNs were detected for each patient.The accuracy rate was 89.74％,the sensitivity was 89.74％,the false negative rate and the false positive rate was 10.26％ and 3.85％.There was statistical difference between the two groups in the average detection number and the false negative rate (P ＜ 0.05)while no statistical difference was found in the detection rate,accuracy,or sensitivity between the two groups (P ＞ 0.05).Conclusion Fluorescent agent combined with methylene blue as tracer for lymph nodes has the advantages of higher detection rate and less trauma,which is worth of clinical application.

Establishment of a real-time quantitative RT-PCR assay for rapid detection of hepatitis E virus in serum / 国际检验医学杂志

Objective To establish a method for the rapid detection of hepatitis E virus (HEV)from serum samples based on fluorescence quantitative PCR.Methods (1 )One-hundred HEV sequences including our country popular three major genotypes were obtained from the GeneBank with the Vector NTI software.The proper sequence was selected to design and synthesize the primers of the fluorescence quantitation and the Taqman probe.(2)The amplification region PCR fragment was transcribed in vitro to synthesize cRNA standard,at the same time the trace serum virus lysate was introduced into a universal real-time TaqMan PCR assay.(3)10 clinical serum samples were collected from the patients with clinical hepatitis E and detected by using the established method for further verifying this method.Results This detection technique could effectively detect the serum samples in the pa-tients with genotype I and genotype IV hepatitis E positive,while the serum detection in the patients with other virus infectious dis-eases had the negative results,which verified that this RT-PCR detection technique had higher specificity and good reliability.The detection results from 10 clinical serum samples further verified that this method was rapid,convenient and sensitive with good re-peatability.Conclusion A fluorescence quantitative RT-PCR detection technique suitable for detecting main genotypes of HEV in China population is established,which can meet the demand of early and rapid diagnosis for HEV.

Clinical application of real-time fluorescence quantitive PCR for detecting Streptococcus pneumoniae / 中国生化药物杂志

Objective To establish an assay for the detection of Streptococcus pneumoniae by real-time fluorescence quantititive polymerase chain reaction (PCR).Methods Special primers and probe for the autolysin A (lytA)gene were designed.The sensitivity and specificity of primers and probe were studied,and cut-off of cycle threshold was assayed.158 clinical specimens were confirmed by real-time fluorescence quantitative PCR and bacterial culture method.Results Primer and probe design for LytA gene could sensitively detect serotype Streptococcus pneumoniae strains of common pathogenic,and the sensitivity was 100 copies.Among 35 strains of Streptococcus pneumoniae,34 cases were detected to be positive for Streptococcus pneumoniae by real-time fluorescence quantitative PCR,while 1 case was detected to be negative;among 15 strains of non-Streptococcus pneumoniae, all were detected to be negative.Among the 158 clinical sputum specimens,34 cases with Streptococcus pneumoniae were detected by real-time fluorescence quantitative PCR,while only 10 cases with Streptococcus pneumoniae were detected by the culture method.White blood cells count and time in hospital of cases with Streptococcus pneumoniae were higher than those of cases without Streptococcus pneumoniae (P <0.05 ). Conclusion Real-time fluorescence quantitative PCR is a sensitive and specific assay for the detection of Streptococcus pneumoniae.It can be used for the diagnosis of Streptococcus pneumoniae.

Highly Fluorescent Carbon Nanoparticles for Sensitive Detection of IronⅢ / 分析化学

A simple preparative strategy was proposed for the preparation of water-soluble, bright blue-emitting carbon nanoparticles ( CNPs ) at different temperatures by hydrothermal treatment using bean curd residue as carbon source for the first time. When the temperature reaches 240 ℃, quantum yield ( QY) can be up to 15. 24%. Most importantly, such fluorescent CNPs can be served as an effective sensing platform for label-free sensitive and selective detection of Fe3+ions with a detection of limit as low as 50 nmol/L, which is nearly 2 orders of magnitude lower than the maximum level of Fe3+ions in the drinking water permitted by the European Community. These outstanding properties give promising potential for reliable detection of Fe3+ in intracellular fluid and water samples, even bioimaging and optical imaging without being hazard in the future.

Determination of Moroxydine Residue in Tomatoes Using CdTe Quantum Dots as Fluorescence Probes / 分析化学

A novel method for the detection of moroxydine ( ABOB) residue in tomatoes was developed based on the enhancement of the fluorescence intensity of CdTe quantum dots( QDs) in the presence of ABOB. The factors influencing the performance of the QDs fluorescent probes were investigated, and the optimum conditions were determined:the concentration of mercaptoacidic acid ( TGA) capped-CdTe quantum dot was 1×10-4 mol/L, and the reaction time was 20 min at pH=5. 6. Under the optimum conditions, the relative fluorescence intensity increases linearly proportional to the ABOB concentration in the range of 1. 0×10-12-5. 0×10-10 mol/L with a limit of detection of 5. 2×10-13 mol/L, R=0. 9981, the recovery was 97%-106%, without obvious interference on the determinations of moroxydine from the common coexisting ions, antibiotics, and vitamins. The proposed method has been successfully applied in the detection of trace moroxydine hydrochloride residue in tomatoes.

Optical Molecular Imaging for Diagnosing Intestinal Diseases

Real-time visualization of the molecular signature of cells can be achieved with advanced targeted imaging techniques using molecular probes and fluorescence endoscopy. This molecular optical imaging in gastrointestinal endoscopy is promising for improving the detection of neoplastic lesions, their characterization for patient stratification, and the assessment of their response to molecular targeted therapy and radiotherapy. In inflammatory bowel disease, this method can be used to detect dysplasia in the presence of background inflammation and to visualize inflammatory molecular targets for assessing disease severity and prognosis. Several preclinical and clinical trials have applied this method in endoscopy; however, this field has just started to evolve. Hence, many problems have yet to be solved to enable the clinical application of this novel method.

Study on DNA protection of polysaccharide and flavonoids in Artemisia annua L. from different areas with fluorescence probe / 中国药学杂志

OBJECTIVE: To investigate the DNA protection of polysaccharide and flavonoids in 14 kinds of Chinese herbs of Artemisia annua L. from different growing places such as Hunan, Chongqing, Beijing etc. METHODS: The fluorescence integration intensity of DNA, EB and extracts containing polysaccharide and flavonoids from 14 kinds of Artemisia annua L. were determined with EB as a sensitive fluorescence probe. Constant D was used to directly denote the degree of interaction between the drug molecules with DNA. According to the size of D, the protective action of polysaccharide and flavonoids on DNA was discussed. RESULTS: The order of interaction degree of polysaccharide in extracts with DNA was as follows; Hunan Huaihua > Beijing > Shanxi Yushe > Hunan (Xinzhou cultivation) > Shanxi Xinzhou cultivation in room > Chongqing (Xinzhou cultivation) > Hebei Fuping > Shanxi Dingxiang Nanzhuang > Chongqing > Shanxi Xinzhou Luye > Shanxi Yuxian > Shanxi Grey artemisia (Yangqu) ; The order of interaction of flavonoids in extracts with DNA through determination was as follows; Shanxi Xinzhou Luye > Hunan(Xinzhou cultivation) > Shanxi Xinzhou cultivation in room > Chongqing(Xinzhou cultivation) > Wild Artemisia(Shanxi Xinzhou) > Grey artemisia (Shanxi Yangqu) > Shanxi Dingxiang Nanzhuang > Chongqing > Hebei Fuping > Beijing > Shanxi Yushe > Iron rod Artemisia(Shanxi Xinzhou) > Shanxi Yuxian > Hunan Huaihua. The interaction of artemisia polysaccharide and flavonoids with DNA showed a definite dose-effect relationship. CONCLUSION: The extracts of Artemisia annua L. could interact with DNA, but the degree of interaction was different. The bigger the D was, the stronger the interaction was. Copyright 2012 by the Chinese Pharmaceutical Association.

Clinical application of fluorescent probe PCR for detection of mycoplasma pneumoniae / 中国小儿急救医学

Objective To explore the clinical application of FP-PCR method to detect MP-DNA.Methods Five hundred and sixty-three children suspected of MP infection were enrolled in experimental group. FP-PCR was adopted to detect MP-DNA. MP-DNA was re-detected later in 60 children. At the same time,MP-Ab (MP antibody) was detected by means of particle agglutination. MP-Ab was re-detected one or two weeks later. Also 20 healthy children were selected as the control group. Results The positive rate of MP-DNA and MP-Ab were 34. 99% and 35.52% respectively,which showed no significant difference (x2 =0. 31, P > 0. 05). The coincidence of the two methods was 97. 69%. But the positive rate of MP-DNA was significantly higher than that of MP-Ab in the early stage(30. 48% vs 10. 16%) (x2 = 74. 46, P < 0. 05).The sensitivity and specificity of FP-PCR were 96. 00% and 98.62% respectively. The result of reviewed MP-DNA was consistent with the clinical diagnosis. Conclusion FP-PCR method is very sensitive, convenient and stable. It is fit for the clinical application ,especially the diagnosis of early MP infection. It helps to identify those who had been infected with MP before.