ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Shuyu Zhu,Shiying Zhou,Liyuan Deng,Jiangbo Dong,Tao Gu,Xinyu He,Liang Jin,Changjun Hou,Danqun Huo
Background Alkaline phosphatase (ALP) can catalyze the hydrolysis of phosphate esters under alkaline conditions, mediate the dephosphorylation of substances such as proteins and nucleic acids. ALP can participate in physiological activities such as bone mineralization, cell division, and immune regulation. Abnormal ALP activity is associated with various diseases, making it an important clinical biomarker. In recent years, highly sensitive detection technologies based on isothermal amplification have developed rapidly. The Rolling Circle Amplification (RCA) technology uses T4 DNA ligase to connect the 5'-phosphate group and 3'-hydroxyl terminus of the padlock probe into a ring, achieving efficient amplification through continuous rolling circle replication. Results Leveraging this characteristic, we developed a highly sensitive sensing method (RCA-LG) based on RCA-generated G43 long nanowires for ALP detection. T4 DNA ligase circularizes the RCT into a template. Subsequently, Phi29 DNA polymerase amplifies the primer along the circular template, producing G43 long nanowires that bind with Thioflavin T (ThT) to generate fluorescent signals. When ALP is present, it dephosphorylates the 5' end of RCT, preventing circular template formation and thereby inhibiting the amplification reaction. The RCA-LG method can detect ALP concentrations as low as 1.0×10 -6 U/mL within 2.5 h. We propose a novel one-step RCA approach that integrates ligation and amplification reactions into a single step, simplifying operations while enhancing amplification efficiency. Furthermore, we physically separated the ALP dephosphorylation and RCA processes to avoid potential contamination from repeated sample handling. Significance and Novelty This strategy takes advantage of the characteristics of RCA without the need for redundant phosphorylation modifications. Moreover, we have proposed an improved one-step RCA method combined with physical partitioning, enabling highly sensitive and specific detection of ALP and allowing the detection of this target in complex samples. Furthermore, the RCA-LG strategy can also play an important role in the screening of enzyme inhibitors.
研究领域:
生物化学
·
分子诊断
·
医学检测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL SCIENCES
·
2018
·Vol. 34
·期 2
·149-153
·
IF 1.355
Xiao Fang ZHANG,Hong Mei XU,Lei HAN,Nian Bing LI,Hong Qun LUO
The detection of disease-related DNA is of great significance for early and accurate diagnosis and therapy. In this work, we successfully achieved the sensitive detection of target DNA based on a thioflavin T (ThT)-induced G-quadruplex fluorescent biosensor. ThT, a water-soluble fluorescent dye, can induce G-rich sequences to form G-quadruplexes and obtain an obviously enhanced fluorescence. In this work, it was employed to construct a biosensor for the detection of HIV. When the target HIV existed, the hairpin DNA probes would be opened in succession and release the completely exposed G-rich sequence to combine with ThT. The simple and rapid biosensor performed satisfactory selectivity; it also exhibited sensitivity with a detection limit of 2.4 nM. With good performance in human serum, we believe that this optical biosensor has the potential to be applied to the practical detection of target DNA.
研究领域:
生物化学
·
分子生物学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2016
·Vol. 8
·期 48
·8461-8465
·
IF 1.915
Hongyan Wang,Jine Wang,Lijun Xu,Yajie Zhang,Yang Chen,Hongxia Chen,Renjun Pei
Aptamers are single-stranded oligonucleotides which are selected and are capable of binding certain targets tightly and specifically. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) has been used to select aptamers for various targets such as proteins, metal ions, small molecules and so on. For the first time, we have used a modified SELEX technology to obtain DNA aptamers for a fluorogenic dye, thioflavin T (ThT). DNA aptamers with low micromolar dissociation constants were achieved after only 8 rounds of selection and the final minimized 33-mer aptamer showed 191-fold fluorescence enhancement in a saturated concentration. The truncated DNA aptamer showed excellent selectivity and specificity for ThT.
研究领域:
分子生物学
·
生物化学
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF FLUORESCENCE
·
2026
·
IF 3.4
Hu Xuan,Zhang Jingyi,Shen Fei,Yang Qian,Xing Changrui,Yuan Jian,Li Guanglei
Endotoxin (lipopolysaccharide, LPS), a major toxic component of Gram-negative bacteria. It shows high stability and resistance to conventional sterilization. Therefore, rapid and effective detection of LPS is crucial to mitigate its threats to human health. This study developed a sensitive, simple, and rapid LPS detection method. It uses a label-free fluorescent aptamer sensor based on an LPS-specific aptamer and the commercial fluorescent probe Thioflavin T (ThT). A novel LPS-detecting aptamer (G4-LA47) was synthesized by attaching guanine-rich DNA sequences to both ends of the LPS-specific aptamer LA27. The G4-LA47 aptamer retained high affinity for LPS, while its terminal guanine-rich sequences formed a G-quadruplex structure. Upon binding with ThT, the G-quadruplex/ThT complex exhibited strong fluorescence. The presence of LPS unfolds of the G-quadruplex, releases free ThT and reduces the fluorescence intensity. LPS was quantitative detected by monitoring fluorescence intensity changes. The linear range was 10–80 ng/mL. The LOD and LOQ were 3.26 ng/mL and 10.86 ng/mL, respectively. The sensor was successfully applied to juice and drinking water samples, with recoveries ranging from 93.34% to 113.29%. This label-free fluorescent aptamer sensor demonstrates high sensitivity, specificity, and simplicity, showing promising potential for practical applications.
✓ 已验证
DOI
PubMed
高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2020
·
IF 3.232
Yuanyuan Dai,Ying Zhang,Wanliang Liao,Weiping Wang,Liang Wu
A label-free fluorescence aptasensor was developed for the rapid detection of tetracycline (TET) based on G-quadruplex structure of TET aptamers and G-quadruplex specific dye Thioflavin T (ThT). The fluorescence of free ThT is essentially weak in aqueous solution, whereas it selectively identifies the G-quadruplex of aptamers to form the G-quadruplex/ThT conjugates, resulting in an enormous increase of the fluorescence intensity . However, the fluorescence intensity of G-quadruplex/ThT conjugates was drastically suppressed due to the release of free ThT from G-quadruplex/ThT conjugates after the addition of TET via specific binding with TET aptamers. The key factors affecting sensitivity and selectivity including the reaction medium, binding time of ThT to TET aptamers, incubation time between TET aptamers and TET, concentration of ThT and TET aptamers were investigated in detail. The optimal conditions were as follows: ultrapure water as reaction medium, binding time of 5 min, incubation time of 1 min, 9.0 μmol/L ThT and 0.03 μmol/L aptamers. A good linear relationship (correlation coefficient of 0.9973) was obtained between the fluorescence quenching efficiency ( F 0 − F ) / F 0 and the logarithm of TET concentration in the range of 0.01–1.0 μmol/L. The limit of detection was 0.001 μmol/L (S/N = 3). The proposed assay was applied for the detection of TET in the spiked honey and milk samples with recoveries ranging from 93.5% to 106.9%. The developed label-free fluorescence aptasensor showed advantages of high specificity, low cost and short time-consuming, illustrating potential application for on-site detection of TET in foodstuffs.
研究领域:
分析化学
·
食品科学
·
生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
LANGMUIR
·
2017
·
IF 3.833
Zhe Qin,Ying Sun,Baohuan Jia,Dan Wang,Yan Ma,Gang Ma
Thioflavin T (ThT) is widely used as a fluorescent probe for amyloid fibril detection. Yet the exact kinetic mechanism of ThT binding onto amyloid fibril remains elusive. Previously reported kinetic studies using ThT-fluorescence-detected kinetic design suggested two completely different ThT-binding mechanisms. In one study, a multistep sequential binding mechanism onto a single ThT-binding site was suggested. In another study, a one-step parallel binding mechanism onto multiple ThT-binding sites was suggested. The discrepancy is likely due to the incapability of ThT-fluorescence-detected kinetic design to differentiate the two above-mentioned mechanisms. Considering the weakness of the ThT-fluorescence-detected approach, we investigated the ThT-binding mechanism onto the amyloid fibril of hen egg white lysozyme (HEWL) using a new approach, ThT-absorbance-detected kinetic design. Our new results suggest that ThT binds to HEWL fibril through the one-step parallel binding mechanism. We hope our work can offer some new insights into the interactions between dye molecules and amyloid fibrils.
研究领域:
生物化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2016
·
IF 4.035
Yan-Yun Li,Xiao-Qin Jiang,Ling-Fei Lu,Min Zhang,Guoyue Shi
In this work, we presented a simple, label-free and rapid-responsive fluorescence assay for iodide (I − ) detection based on “molecular beacon (MB)”-hosted thioflavin T (ThT), achieving a limit of detection as low as 158 nM. The proposed method exhibited very good selectivity to I − ions over other anions interference due to the strong binding force between I − ions with Hg 2+ . Upon the addition of I − ions, it would capture Hg 2+ from a T–Hg 2+ –T complex belonging to the MB-like DNA hairpin structure, which eventually quenched the initial fluorescence as output. In addition, it was successfully applied for operation of an integrated DNA logic gate system and to the determination of I − in real samples such as human urine.
研究领域:
分析化学
·
生物化学
·
分子生物学
·
生物医学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2022
·Vol. 414
·期 20
·6149-6156
·
IF 4.478
Qin Shanshan,Chen Xuliang,Xu Zhichen,Li Tao,Zhao Shuhong,Hu Rui,Zhu Jiang,Li Ying,Yang Yunhuang,Liu Maili
Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT–based method. We believe this ht-G3/ThT–based label-free strategy could be widely applied for RNA detection.
研究领域:
生物化学
·
分子生物学
·
分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Yi Liang,Ke Huang,Jin-Wen Liu,Yumin Wang
Thioflavin T as split G-quadruplex fluorescent dye was researched. • A novel sensing method based on CoOOH nanoflakes and HCR was proposed. • Label-free and sensitive detection of miRNA-21 was successfully realized. To date, various fluorescent probes for G-quadruplex have been reported. However, the development of fluorescent dyes for split G-quadruplex formed on double-stranded DNA structure still remained largely unexplored. Herein, we have demonstrated that thioflavin T (ThT) was able to intercalate into split G-quadruplex sequence and the formed split G-quadruplex-ThT structure exhibited remarkable fluorescence enhancement. By integrating cobalt oxyhydroxide (CoOOH) nanoflakes and hybridization chain reaction (HCR), a sensitive and label-free biosensing strategy for miRNA detection was proposed. In our design, miRNA-21 was chosen as the model target and it was capable of triggering HCR with two split G-quadruplex sequence-contained hairpin probes, resulting in the generation of long chain-like HCR product. Unlike hairpin probes, the HCR product cannot be adsorbed onto CoOOH nanoflakes because of lower affinity. Consequently, in the presence of ThT, numerous split G-quadruplex-ThT structures on HCR product were formed and the target miRNA was converted into amplified and activatable fluorescence signal. Experimental results demonstrated that this novel sensing strategy can be applied for sensitive and selective detection of miRNA. Moreover, the analysis of miRNA in complex biological media was successfully realized, offering a robust, label-free and cost-effective platform for miRNA-related disease diagnosis and biomedical research. A novel fluorescence biosensing strategy based on cobalt oxyhydroxide (CoOOH) nanoflakes and hybridization chain reaction (HCR) was developed for sensitive and label-free detection of miRNA-21. Download: Download high-res image (161KB) Download: Download full-size image
✓ 已验证
DOI
高影响因子
JOURNAL OF CHEMICAL RESEARCH
·
2023
·
IF 1.4
Zhe Chen,Yunxia Wang
A label- and enzyme-free fluorescence assay based on thioflavin T–induced G-quadruplexes is developed to sensitively and specifically detect telomerase activity. Thioflavin T has a dual role as an efficient inducer and fluorescent probe, and the incorporation of thioflavin T into the thioflavin T–induced G-quadruplexes results in an intense fluorescence enhancement. In the presence of thioflavin T and K+, G-quadruplexes are formed by elongation of the telomerase substrate primer that is catalyzed by telomerase extracted from cancer cells. Thus, the telomerase activity in cancer cell extracts can be evaluated by measuring the thioflavin T fluorescence. More importantly, thioflavin T can specifically recognize and bind to G-quadruplexes, whereas it cannot recognize single- and double-stranded DNAs, which leads to the thioflavin T–based fluorescence assay exhibiting a reduced background and improved signal-to-noise ratio. As a result, the proposed assay has the linear range from 5 to 200 HeLa cells and the detection limit is 34 HeLa cells, which holds great potential for use in the detection of telomerase activity and the diagnosis of cancer.
研究领域:
生物化学
·
分子生物学
·
癌症研究
✓ 已验证
产品关联
DOI
高影响因子
Food Additives and Contaminants Part A-Chemistry Analysis Control Exposure & Risk Assessment
·
2025
·
IF 2.2
Xinyue Yin,Qi Pu,Chumeng Wang,Yuhong Xiang,Nengsheng Ye
Kanamycin (KANA) plays a key role in the treatment of bacterial infections and has been widely used in animal husbandry. However, its overuse causes antibiotic residues in animal-derived foods. Determination methods for KANA are urgently needed for food safety. Most of the developed fluorescent aptamer sensors for detecting KANA use parental aptamer (kana-Apt) as recognition unit. However, excessive bases tend to form secondary structures and lead to high background or nonspecific signals. In this study, two fluorescent sensors based on one (kana1-Apt) and two (kana1/kana2-Apt) split fragments were developed for KANA detection. The LODs of the kana1-Apt/ThT system and kana1/kana2-Apt/ThT systems were 4.88 nM and 4.53 nM, respectively. In addition, satisfactory recoveries of the kana1-Apt/ThT system and kana1/kana2-Apt/ThT system were obtained in the detection of KANA in milk, which were 97.6%-104.5% and 98.4%-105.9%, respectively. Moreover, the results indicated that the kana1-Apt fragment plays a critical role in recognition. In conclusion, the results of the present study provide a novel strategy for molecular detection based on split aptamers.
研究领域:
食品安全
·
生物传感
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
FOOD CONTROL
·
2024
·
IF 5.6
Lidong Pang,Qunchao Su,Jingqi Cheng,Biqi Liu,Chaoxin Man,Ling Guo,Xinyan Yang,Qianyu Zhao,Yujun Jiang
The development of a simple, rapid, sensitive, and accurate method for detecting Escherichia coli O157:H7 in complex matrices is critical for ensuring food safety. In this study, a fluorescent biosensor employing aptamer (Apt) -functionalized magnetic beads and cascade strand displacement amplification (SDA) was devised for the detection of E. coli O157:H7 in milk samples. E. coli O157:H7 is captured by the aptamer, causing the complementary DNA (cDNA) to not bind to the aptamer and to be released into the supernatant. Following magnetic separation, the cDNA present in the supernatant initiates the opening of the hairpin (HP) structures. This enables primers to attach to the opened HP, triggering the initial SDA process, which generates a substantial quantity of double-stranded DNA (dsDNA) and recycles cDNA to open more HP structures. The dsDNA activates a second SDA, producing single-stranded DNA (ssDNA) for template binding and instigating a third SDA process. Triple SDA leads to the production of numerous G-quadruplex sequences and the formation of a G-quadruplex structure in the presence of K + . The inclusion of ThT into the G-quadruplex significantly enhances fluorescence. Under optimal conditions, this approach can detect E. coli O157:H7 at levels as low as 10 1 CFU/mL in pure cultures within 2.5 h, with a broad linear detection range of 10 1 –10 6 CFU/mL. This method exhibits remarkable specificity towards the target bacteria over non-target bacteria. Furthermore, the technique has been effectively utilized for the detection of E. coli O157:H7 in milk samples. Notably, the method involves only two main steps, identification and amplification, significantly reducing contamination risks and minimizing operational steps.
研究领域:
食品安全
·
生物传感
·
分子生物学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）