ANALYST
·
2018
·Vol. 143
·期 4
·914-919
·
IF 3.864
Yaoqi Wang,Jiaxue Gao,Xianying Meng,Zhenxin Wang
Highly accurate analysis of single-nucleotide polymorphisms (SNPs) plays an important role in both disease diagnostics and personalized medicine development. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of papillary thyroid carcinoma (PTC) related mutation points including BRAFV600E (t1m), NRAS codon 61 (t2m), TERT promoter g.1295228 (t31m) and TERT promoter g.1295250 (t32m) with high sensitivity and selectivity by the attachment of polyvalent ssDNA modified 13 nm gold nanoparticles (ssDNAs@GNPs) followed by silver deposition for signal enhancement. The microarray-based RLS assay provides a detection limit (S/N = 3) at the sub-nanomolar level for the target ssDNAs and determines allele frequencies as low as 0.2% for t1m, 0.2% for t2m, 0.5% for t31m, and 0.5% for t32m in the cocktail of target ssDNAs, respectively. The practicability of the DNA microarray-based RLS assay is demonstrated by profiling of t2m in 50 clinical thyroid tissue samples of PTC patients, and satisfactory results are obtained.
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Biosensors-Basel
·
2021
·Vol. 11
·期 11
·451
·
IF 5.519
Ping Ouyang,Chenxin Fang,Jialun Han,Jingjing Zhang,Yuxing Yang,Yang Qing,Yubing Chen,Wenyan Shang,Jie Du
The qualitative and quantitative determination of marker protein is of great significance in the life sciences and in medicine. Here, we developed an electrochemical DNA biosensor for protein detection based on DNA self-assembly and the terminal protecting effects of small-molecule-linked DNA. This strategy is demonstrated using the small molecule biotin and its receptor protein streptavidin (SA). We immobilized DNA with a designed structure and sequence on the surface of the gold electrode, and we named it M1-Biotin DNA. M1-Biotin DNA selectively combines with SA to generate M1-Biotin-SA DNA and protects M1-Biotin DNA from digestion by EXO III; therefore, M1-Biotin DNA remains intact on the electrode surface. M1-Biotin-SA DNA was modified with methylene blue (MB); the MB reporter molecule is located near the surface of the gold electrode, which generates a substantial electrochemical signal during the detection of SA. Through this strategy, we can exploit the presence or absence of an electrochemical signal to provide qualitative target protein determination as well as the strength of the electrochemical signal to quantitatively analyze the target protein concentration. This strategy has been proven to be used for the quantitative analysis of the interaction between biotin and streptavidin (SA). Under optimal conditions, the detection limit of the proposed biosensor is as low as 18.8 pM, and the linear range is from 0.5 nM to 5 μM, showing high sensitivity. The detection ability of this DNA biosensor in complex serum samples has also been studied. At the same time, we detected the folate receptor (FR) to confirm that this strategy can be used to detect other proteins. Therefore, this electrochemical DNA biosensor provides a sensitive, low-cost, and fast target protein detection platform, which may provide a reliable and powerful tool for early disease diagnosis.Keywords:terminal protection;electrochemical DNA biosensor;streptavidin;Exonuclease III;small-molecule-linked DNA
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SENSORS AND ACTUATORS B-CHEMICAL
·
2017
·
IF 5.401
Xiao Fang Zhang,Na Li,Yu Ling,Li Tang,Nian Bing Li,Hong Qun Luo
As one of the DNA damage repair enzymes, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. By linked bridge hybridizing-induced split G-quadruplex (SQ), we demonstrate here the construction of a simple and sensitive DNA machine for the detection of UDG activity. The satisfactory split G-quadruplex sequences (SQS) were successfully selected to be the ingredients of SQ formation. In this work, UDG recognized and removed the uracil bases from the stem of hairpin DNA (HP). And then, HP with a low melting temperature hybridized with designed SQS, forming a three-way DNA structure with an SQ. With the addition of Thioflavin T, a dramatical enhancement of fluorescence intensity was presented due to the G-quadruplex/Thioflavin T complex formation. The detection limit was as low as 7.8 × 10 −3 U/mL. And we also successfully investigated the performance of UDG activity in the HeLa cell lysate. This optical DNA machine with the merits of being simple, rapid, and economical was flexibly suitable for not only active UDG assay but also diverse target detection by adjusting the recognition region of the HP.
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ANALYST
·
2015
·Vol. 140
·期 19
·6660-6670
·
IF 4.107
Qing Zheng,Hao Wu,Zongxu Shen,Wenyu Gao,Yu Yu,Yuehui Ma,Weijun Guang,Quangui Guo,Rui Yan,Junzhong Wang,Kejian Ding
A label-free electrochemical DNA sensor was fabricated by deposition of polyaniline and pristine graphene nanosheet (P/Gratios) composites in different mass ratios, DNA probe and bovine serum albumin (BSA) layer by layer on the surface of a glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS) was employed to monitor every step of fabrication of P/Gratio-based DNA sensors and to evaluate the detection results in terms of the hybridization of complementary DNA, mutant DNA and non-complementary DNA. The results illustrate that the P/Gratio-based DNA sensor could highly efficiently detect complementary DNA from 0.01 pm to 1 μm and discriminate single-nucleotide polymorphisms (SNPs). In the process of detection, double-stranded DNA (dsDNA), resulting from hybridization of a DNA probe, escaping from or remaining on the sensor surface, was monitored by changing the ratio of polyaniline (PANI) to graphene, which was decided by the competition between the electrostatic interaction and Brownian motion.
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BIOSENSORS & BIOELECTRONICS
·
2014
·
IF 6.451
Xiaoying Jing,Xianqing Cao,Li Wang,Tian Lan,Yiyan Li,Guoming Xie
A sensitive and selective electrochemical method was developed for the detection of DNA methylation , determination of DNA methyltransferase (MTase) activity and screening of MTase inhibitor. Methylene blue (MB) was employed as electrochemical indicator and DNA-modified gold nanoparticles (AuNPs) were used as signal amplification unit because the DNA strands in this composite have strong adsorption ability for MB. First, the thiolated single-stranded DNA S1 was self-assembled on gold electrode, hybridization between the lower portion of DNA S1 and its complementary DNA S2 formed an identical double-stranded tetranucleotide target sequence for both DNA adenine methylation (Dam) MTase and methylation-resistant endonuclease Mbo I, then the upper portion of DNA S1 was hybridized with its complementary DNA S3 modified on AuNPs to bring the DNA S3-AuNPs amplification units onto the electrode. The DNA S1/S2/S3-AuNPs bioconjugate has lots of DNA strands, and they can adsorb abundant MB. Mbo I endounuclease could not cleave the identical target sequence after it was methylated by Dam MTase. On the contrary, the sequence without methylation could be cleaved, which would decrease the amount of adsorbed MB. The presence of redox-active MB was detected electrochemically by differential pulse voltammetry (DPV). Thus, the activity of Dam MTase and methylation status were sensitively converted to the DNA S3-AuNPs amplified DPV signals. The DPV signal demonstrated a linear relationship with logarithm of Dam concentration ranging from 0.075 to 30 U/mL, achieving a detection limit of 0.02 U/mL ( S / N =3). Also, screening of Dam MTase inhibitor 5-fluorouracil was successfully investigated using this fabricated sensor.
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ELECTROCHIMICA ACTA
·
2013
·
IF 3.777
Zhenning Xu,Mo Wang,Huanshun Yin,Shiyun Ai,Liang Wang,Junling Pang
A sensitive electrochemical method for detection of DNA methylation and assay of DNA methyltransferase (Dam) was investigated based on methylation-sensitive restriction endonuclease, DNA-Au bio bar code and enzymatic signal amplification. After the DNA probe was hybridized with complementary DNA sequence, and the obtained double-strand DNA (dsDNA) was further methylated by Dam MTase, it could be successively digested by Dpn I at the methylated sites. As a result, the bio bar code and streptavidin-horseradish peroxidase (S-HRP) could not be conjugated on the electrode surface through the specific interaction between biotin and S-HRP, where biotin was labeled at the DNA-Au bio bar code. Thus, only a low electrochemical reduction signal was obtained. However, the hybridized probe could hybrid with DNA-Au bio bar code, and then S-HRP could be conjugated on the electrode surface in the absence of Dam MTase and Dpn I, so a larger reduction current of enzymatic product was obtained. The change of the reduction current of enzymatic product was used to detect DNA methylation and Dam MTase activity. The detection limit of Dam MTase was 0.02 unit/mL (S/N = 3). Moreover, the inhibitions of mitomycin C, paclitaxel, chlorogenic acid and epicatechin were also investigated. Therefore, this method provided a sensitive platform for monitoring DNA methylation and DNA MTase activity.
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ACS Sensors
·
2024
·
IF 8.2
Xiaolu Xing,Yuhao Zhao,Haiyan Zhao,Ying Zhang,Ziqiu Chen,Bowen Liu,Bin Ren
Surface-enhanced Raman scattering (SERS) represents a promising avenue for DNA detection as it offers intrinsic chemical insights with high sensitivity compared to conventional methods. However, label-free and quantitative detection of unmodified DNA by SERS remains a major challenge in DNA analysis. To overcome this challenge, we propose a positively charged plasmonic nanosurface for DNA capture and quantitative analysis. Highly sensitive and uniform SERS enhancement was realized by a three-dimensional plasmonic array supporting well-designed hybrid plasmonic modes. Subsequently, the plasmonic array was modified with an electrostatically functionalized PDDA (poly(diene-dimethylammonium-chloride)) self-assembled monolayer in a single step. The effectiveness of the resulting PDDA-SERS substrate was demonstrated by the label-free and quantitative detection of base content and base mutation in hybridized DNA. The PDDA-SERS substrate provides a robust platform for SERS analysis not only of DNA but also of other electronegative analytes.
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JOURNAL OF FLUORESCENCE
·
2015
·Vol. 25
·期 5
·1267-1277
·
IF 1.927
Huang Weidong,Wang Yue,Wang Zhimin
Single-molecule detection (SMD) and single-molecule fluorescence resonance energy transfer (smFRET) were conducted using Cy3- and Cy5-labeled single-strand DNAs (ssDNAs) either immobilized on substrates or encapsulated in microdroplets. High-quality fluorescent images were obtained using a total internal reflection fluorescence microscope (TIRFM). In the substrate system, deposition of a low concentration of fluorescence molecules on substrates through electrostatic adsorption showed that most of the fluorescence spots were single molecules, and the mean value of signal to noise ratio (S/N) reached 6.9 ± 0.34. smFRET analysis was conducted through immobilization of donor- and acceptor-labeled oligonucleotides on substrates. In the droplet system, fluorophor-labeled oligonucleotides were injected into T-type microfluidics. Single and double fluorophor-labeled DNA molecules encapsulated in droplets were detected, the FRET efficiency and inter-dye distance of a single donor-acceptor pair were measured accurately. smFRET was conducted detailedly in the tortuous channel for the first time.
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MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS
·
2015
·
IF 2.415
Haibo Mu,Qianjin Liu,Hong Niu,Dongdong Wang,Jiangjiang Tang,Jinyou Duan
Toxic DNA–protein crosslinks (DPCs) can result from exposure to radiation or chemotherapeutic agents. DPCs can also accumulate during aging or stress. However, the cellular mechanisms underlying clearance of DPCs remain largely unknown. Here, we have identified an important role of autophagy in the processing of DPCs induced by three representative agents: formaldehyde, a chemical used widely in industry; UV light; and camptothecin, a cytotoxic anticancer drug. Autophagy inhibitors, 3-methyladenine (3-MA) or chloroquine (CQ), promoted the accumulation of DPCs in damaged cells and injured organs. siRNA-mediated silencing of Atg5 or Atg7, two essential components for the formation of the autophagosome, gave similar results. In contrast, the autophagy inducer rapamycin (RAP) attenuated DPCs in vitro and in vivo . Our findings reveal the importance of autophagy in controlling the level of DPCs, and may open up a new avenue for understanding the formation and clearance of this detrimental DNA adduct.
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CURRENT CANCER DRUG TARGETS
·
2022
·Vol. 22
·期 9
·771-783
·
IF 2.907
Cui Zhi,Sun Shiqun,Li Jia,Li Jianing,Sha Tong,He Jie,Zuo Linjing
Background: Inhibitor of growth 4 (ING4) level was reported to be decreased in head and neck squamous cell carcinoma (HNSC) tissue, however, it is unknown whether and how ING4 participates in regulating the development of oral squamous cell carcinoma (OSCC). Objective: This study
aimed to investigate the role and mechanism of ING4 in OSCC. Methods: ING4 was forced to up- or down-regulated in two OSCC cell lines, and its effects on the malignant behavior of OSCC cells were investigated in vitro. The ubiquitination level of NF-kB p65 in ING4 upregulated cells was
measured by co-immunoprecipitation. Moreover, the effects of ING4 on the methylation level of ALDH1A2 were evaluated by methylation-specific polymerase chain reaction (MSP) assay. The role of ING4 in OSCC growth in vivo was observed in nude mice. Results: Our results showed that the expression
of ING4 in OSCC cell lines was lower than that in normal oral keratinocyte cells. In vitro, ING4 overexpression inhibited the proliferation, migration, and invasion of OSCC cell lines and ING4 silencing exhibited opposite results. We also demonstrated that ING4 overexpression promoted the
ubiquitination and degradation of P65 and reduced DNA methyltransferase 1 (DNMT1) expression and Aldehyde dehydrogenase 1A2 (ALDH1A2) methylation. Moreover, overexpression of p65 rescued the suppression of malignant behavior, induced by ING4 overexpression. In addition, ING4 negatively regulated
the growth of OSCC xenograft tumors in vivo. Conclusion: Our data evidenced that ING4 played a tumor-repressing role in OSCC in vivo and in vitro via NF-ΚB/DNMT1/ALDH1A2 axis.
研究领域:
肿瘤学
·
细胞生物学
·
分子生物学
·
癌症研究
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Biosensors-Basel
·
2025
·Vol. 15
·期 10
·700
·
IF 5.6
Yu Qin,Jiali Xie,Shujun Zhen
Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5–9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.
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ANALYTICAL CHEMISTRY
·
2019
·
IF 6.35
Ding Wang,Yaqin Chai,Yali Yuan,Ruo Yuan
In this work, a rigid DNA tetrahedron (TDN) scaffold was synthesized to precisely control the interenzyme distance by randomly anchoring two pairs of horseradish peroxidase (HRP)/glucose oxidase (GOx) at the vertices of TDN, which could not only avoid the drawbacks of poor controllability and biocompatibility from traditional scleroid skeletons, but also overcome the defect of imprecise regulation for interenzyme distance caused by DNA origami. Impressively, by varying the side length of TDN scaffold, the interenzyme distance was precisely regulated, thus, an optimal TDN scaffold with highest catalytic efficiency was acquired and subsequently applied for constructing an ultrasensitive biosensor for DNA detection with a low detection limit of 3 fM. This strategy paved an avenue for developing new reliable scaffold to precisely regulate the catalytic efficiency of enzyme cascade reaction with ultimate applications in bioanalysis and clinical diagnosis.
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