A highly sensitive electrochemical aptasensor was developed for the detection of kanamycin,employing a DNA signal amplification strategy that combines RecJf exonuclease-assisted target recycling with a hybridization c...A highly sensitive electrochemical aptasensor was developed for the detection of kanamycin,employing a DNA signal amplification strategy that combines RecJf exonuclease-assisted target recycling with a hybridization chain reaction(HCR).The sensing platform is constructed by covalently immobilizing double-stranded DNA(dsDNA)comprised of a kanamycin-specific aptamer and its thiol-modified complementary strand(SH-CDNA)onto a gold electrode.In the presence of kanamycin and RecJf exonuclease,the aptamer selectively binds to kanamycin,dissociating from the dsDNA complex.The RecJf exonuclease then cleaves the aptamer,releasing kanamycin and initiating a cycle of repetitive binding and release.The residual SH-CDNA on the electrode triggers an HCR between two types of ferrocene-labeled hairpin DNA,forming an elongated stable dsDNA nanostructure.This results in an amplified electrochemical signal proportional to the logarithm of kanamycin concentration over a range of 0.01–10 nmol/L,with a remarkable detection limit of 1.8 pmol/L.The aptasensor's performance was validated by analyzing spiked kanamycin in pharmaceutical eye drops and milk samples,yielding recovery rates between 95.6%and 104.8%and relative standard deviations from 1.4%to 4.2%.With its exceptional selectivity and sensitivity,this aptasensor offers a compelling alternative to traditional HPLC for rapid on-site detection of kanamycin.Capitalizing on the specificity of aptamers,the sensor design presented herein serves as a valuable blueprint for engineering detectors of other molecules,with significant implications for analytical chemistry and food safety monitoring.展开更多
DNA nanomaterials hold great promise in biomedical fields due to its excellent sequence programmability,molecular recognition ability and biocompatibility.Hybridization chain reaction(HCR)is a simple and efficient iso...DNA nanomaterials hold great promise in biomedical fields due to its excellent sequence programmability,molecular recognition ability and biocompatibility.Hybridization chain reaction(HCR)is a simple and efficient isothermal enzyme-free amplification strategy of DNA,generating nicked double helices with repeated units.Through the design of HCR hairpins,multiple nanomaterials with desired functions are assembled by DNA,exhibiting great potential in biomedical applications.Herein,the recent progress of HCR-based DNA nanomaterials for biosensing,bioimaging and therapeutics are summarized.Representative works are exemplified to demonstrate how HCR-based DNA nanomaterials are designed and constructed.The challenges and prospects of the development of HCR-based DNA nanomaterials are discussed.We envision that rationally designing HCR-based DNA nanomaterials will facilitate the development of biomedical applications.展开更多
In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5...In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5′-CAC CGC T-3′and 5′-ATT TGC CTT TTG GGG ACG GATA-3′)at two terminals creates a red-emitting AgNC nucleation sequence(rNS,5′-CAC CGC TAT TTG CCT TTT GGG GAC GGATA-3′).We found that the presence of a toehold fragment(5′-TGCCC-3′)in HP1 could silence the rNS.Upon the addition of a target nucleic acid,HCR of HP1 and hairpin probe 2(HP2)could be initiated,resulting in the formation of long chain of DNA duplexes with multibranched rNS.As the toehold fragment in HP1participated in generating duplexes,a strong emission of rNS-templated AgNCs was observed at 670 nm.More significantly,a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting AgNCs nucleation sequence.On the one hand,the blocker could increase the signal-to-noise-ratio of the constructed biosensor,and on the other hand,the blocker also helped to prepare ratiometric HCR-AgNCs assay with self-calibrating ability to strengthen its reproducibility.Compared with the traditional HCR-AgNCs sensors,the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability,which is important for the fabrication of biosensors in various fields for practical biosensing applications.展开更多
Accurate signal amplification in living cells is highly important in biomedical research and medical diagnostics.Benefiting from its enzyme-free,efficient isothermal signal amplification ability,hybridization chain re...Accurate signal amplification in living cells is highly important in biomedical research and medical diagnostics.Benefiting from its enzyme-free,efficient isothermal signal amplification ability,hybridization chain reaction(HCR)plays an important role in intracellular signal amplification;however,HCR fails the accurate signal amplification in the situation when the properties of some biological targets and analogues are too similar.Particularly,their signal amplification accuracy for mature mi RNAs is unsatisfactory due to the signal interference of precursor micro RNAs(abbreviated as pre-mi RNAs),which also contain the sequence of mature mi RNAs.Herein,we develop the first example of size-selective hybridization chain reaction probe for accurate signal amplification,which achieved accurate and sensitive biosensing of mature mi RNAs in living cancer cells.Our probe,termed as q Tcage,consists of a DNA nanocage for size-selective responsive to mature mi RNAs,as well as a quadrivalent tetrahedral DNA structure for HCR signal amplification.Benefiting from the size-selectivity of DNA nanocage,shorter mature mi RNAs(19–23 nt)rather than longer pre-mi RNAs(60–70 nt)could enter the cavity to release triggers strand,which activates HCR reaction for fluorescence signal recovery.The probe efficiently reduces signal interference of pre-mi RNAs and improves the imaging sensitivity for intracellular mature mi RNAs,which was successfully applied for mature mi RNAs imaging during drug treatment.Overall,this strategy provides the hybridization chain reaction with the feature of size-selective ability,which holds promise for further accurate signal amplification in biological processes study and clinical diagnostics.展开更多
Although immobilization-free and label-free electrochemical DNA(E-DNA)biosensors have engaged tremendous interest due to their superior properties,such as easy operation,time-saving and cost-saving,most of them are fa...Although immobilization-free and label-free electrochemical DNA(E-DNA)biosensors have engaged tremendous interest due to their superior properties,such as easy operation,time-saving and cost-saving,most of them are fabricated in homogeneous modes and usually produce high background current.In the present work,we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction(HCR)for one-pot nucleic acid detection with zero background.The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins,resulting in the generation of HCR products with multibranched arms,which can be captured onto the electrode viaπ-πstacking interactions between multibranched arms and reduced graphene oxide(rGO).Prior to the incubation process with an electrode,two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes.Thus,an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized.This strategy also presents additional merits of simplicity and cheap cost,since probe immobilization,signal tag labeling,and multiple incubation processes are avoided.Therefore,the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.展开更多
The fabrication of sensitive sensors with high selectivity is highly desirable for the detection of some important biomarkers,such as nucleic acids,proteins,small molecules and ions.DNA hybridization chain reaction(HC...The fabrication of sensitive sensors with high selectivity is highly desirable for the detection of some important biomarkers,such as nucleic acids,proteins,small molecules and ions.DNA hybridization chain reaction(HCR) and DNA supersandwich self-assembly(SSA) are two prevalent enzyme-free signal amplification strategies to improve sensitivity of the sensors.In this review,we firstly describe the characteristics about DNA HCR and DNA SSA,and then summarize the advances in the one-dimensional DNA nanostructures assisted by HCR and SSA.This review has been divided into three parts according to the two signal amplification methods and highlights recent progress in these two strategies to improve the detection sensitivity of proteins,nucleic acids,small molecules and ions.展开更多
Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases.Herein,a triple signal amplification strategy is developed for trace mic...Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases.Herein,a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR),hybridization chain reaction (HCR) and enzyme catalytic amplification.Four DNA hairpins(H1,H2,H3,H4) are employed to form an ultralong double-strand DNA (dsDNA) structure,which is initiated by target miRNA-21.As H3 and H4 are labeled with horseradish peroxidase (HRP),numerous HRPs are loaded on the long dsDNA,producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H_(2)O_(2)) and 3,3,5,5-tetramethylbenzidine (TMB) reaction strategy.Compared with single signal amplification,the triple signal amplification strategy shows higher electrochemical response,wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity,reproducibility and stability.Taking advantage of the triple signal amplification strategy,the proposed electrochemical biosensor can detect miRNA-21 in 10 He La cell lysates,suggesting that it is a promising method for fruitful assay in clinical diagnosis.展开更多
Assembling and ordering nanomaterials into desirable patterns are considerably significant,since the properties of nanomaterials depend not only on the size and shape,but also on the spatial arrangement among the coll...Assembling and ordering nanomaterials into desirable patterns are considerably significant,since the properties of nanomaterials depend not only on the size and shape,but also on the spatial arrangement among the collective building blocks.In this work,the DNA self-assembly technology of hybridization chain reaction(HCR) provided a convenient method to yield long double-strand DNA(dsDNA) to install gold nanoparticles(AuNPs) into one dimensional assembly along the skeleton of dsDNA.Interestingly,the tunable length of AuNPs assemblies along dsDNA chain could be achieved by adjusting the reaction time of HCR,which is based on the formation of covalent bond between Au and the-SH group of DNA.Compared with weak light scattering of single AuNP,these AuNPs assemblies could be clearly imaged under the dark field microscopy,indicating that the light scattering was greatly improved after assembling.展开更多
Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification...Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification strategy to meet this purpose.This strategy relies on three DNA probes,Apt-trigger,H1-AТP aptamer duplex and hairpin H2.The Apt-trigger probe consists of two com sequence for specific recognition of the target cells.and a trigger sequence for the HCR assembly.Theроnents:an aptamer duplex structure of H1-ATP aptamer causes the tochold in hairpin H1 to be hidden,preventing the strand-ent displacement reaction between haipin H1 and Apt-trigger.Upon activation with ATP the ATP aptamer will blnd to ATP to dissoci iate from hairpin H1,thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface.Benefiting from aptamer recogni-tion and ATP-activated HCR amplification,this strategy can not only perform sensitive quantitative anal-ysis with a detection limit of 25 cells in 200 ul.of binding buffer,but also show desirable specificity and accuracy for identifylng target cells from control cells and mixed cell samples,Imporantly,this method retains stable and good perfor mance for target cell detection in 10%fetal bovine ser rum,den onstrating great potential for clinical diagnosis in complex biological matrices.Furthermore,this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.展开更多
DNAzyme has emerged as a promising gene silencer for therapeutic applications.However,DNAzymebased gene therapy against tumors is still challenged by undesired cytotoxicity in normal cells.Herein,we explored the poten...DNAzyme has emerged as a promising gene silencer for therapeutic applications.However,DNAzymebased gene therapy against tumors is still challenged by undesired cytotoxicity in normal cells.Herein,we explored the potential application of hybridization chain reaction(HCR)for endogenous miRNA-activated DNAzyme-based gene silencing,which performing amplified signal and cellular onsite therapy in one system.The DNA probes,called HCR-tDz circuits,are designed by combining HCR and therapeutic DNAzyme(tDz).The miRNA-21 is selected as the diagnostic target for its distinctive overexpression in cancerous cells,and the HCR is employed to amplify the miR-21 recognition event as well as the tDz activation.The massively repeated DNAzyme activated by miR-21-triggered HCR is utilized to catalytically cleave early growth response-1 mRNA and implement a cellular gene-silencing strategy.Results show that HCR-tDz nanostructure enables amplified miR-21 imaging in living cells.Furthermore,this nanosystem can effectively inhibit the proliferation of cancer cells with high specificity,thus resulting in sensitive diagnostic signaling and accurate therapeutic effects.展开更多
Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impe...Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.展开更多
The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH...The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.展开更多
Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling...Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan（GV3）, Dazhui（GV14）, Zusanli（ST36） and Ciliao（BL32） and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ（ROCKⅡ） of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.展开更多
An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(...An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(Exo Ⅲ-ATC)and hybridization chain reaction(HCR)synergistic triple signal amplification.The MrGO-AuNPs is a superior carrier for capture DNA(cDNA)and acts as magnetic media for automatic separation and adsorption.This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode.Additionally,the three DNA hairpins(cDNA,methylene blue(MB)labeled HP1 and HP2)further contribute to biosensor specificity and selectivity.When cDNA captures Hgt,it activates Exo Ⅲ-ATC due to the formation of a sticky end in the DNA stem via thymine-Hig-thymidine(T-Hg^(2+)-T),this leads to the hydrolysis of self-folded DNA by Exo Ⅲ-ATC to form"key"DNA(kDNA).The kDNA subsequently initiates HCR,resulting in massive super-sandwich structures(kDNA-[HP1/HP2])carrying signaling molecules on MrGO-AuNPs,and this overall structure serves as a signal probe(SP).Leveraging magnetic adsorption,the SP was automatically adsorbed onto the magneto-glass carbon electrode(MGCE),generating an amplified signal.This biosensor's detection limit(LOD)was 3.14 pmol/L,far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA.The biosensor also showed excellent selectivity when challenged by interfering ions,and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring.展开更多
Natural enzymes,such as horseradish peroxidase(HRP),are a class of important biocatalysts with the high specificity,but their catalytic efficiency is usually unsatisfactory.Thus,the higher catalytic efficiency induced...Natural enzymes,such as horseradish peroxidase(HRP),are a class of important biocatalysts with the high specificity,but their catalytic efficiency is usually unsatisfactory.Thus,the higher catalytic efficiency induced by the confinement effect is promising in optical sensing systems.In this work,a dark-field light scattering sensing platform was fabricated by the confinement effect of HRP from hybridization chain reaction(HCR)and then released to solution by the toehold-mediated strand displacement reaction(TSDR).Then,HRP catalyzed the 3,3,5,5-tetramethylbenzidine(TMB)to TMB^(2+)with the assistance of hydrogen peroxide,which etched the gold nanorods(Au NRs)with the weakened light scattering.The single-particle assay was established based on the decreased light scattering intensity of AuNRs under dark-field microscope.The proposed assay revealed excellent analytical performance within a linear range from 25 pmol/L to 600 pmol/L,and a low limit of detection of 3.12 pmol/L.Additionally,it also manifested satisfactory recovery of mi RNA-21 in human serum samples.The high sensitivity,excellent specificity,and universal applicability make this sensing platform promising for disease diagnosis.展开更多
Circulating microRNAs(miRNAs)play a pivotal role in the occurrence and development of acute myocardial infarction(AMI),and precise detection of them holds significant clinical implications.The development of luminol-b...Circulating microRNAs(miRNAs)play a pivotal role in the occurrence and development of acute myocardial infarction(AMI),and precise detection of them holds significant clinical implications.The development of luminol-based luminophores in the field of electrochemiluminescence(ECL)for miRNA detection has been significant,while their effectiveness is hindered by the instability of co-reactant hydrogen peroxide(H_(2)O_(2)).In this work,an iron single-atom catalyst(Fe-PNC)was employed for catalyzing the luminol-O_(2) ECL system to achieve ultra-sensitive detection of myocardial miRNA.Target miRNA triggers a hybridization chain reaction(HCR),resulting in the generation of a DNA product featuring multiple sticky ends that facilitate the attachment of Fe-PNC probes to the electrode surface.The Fe-PNC catalyst exhibits high promise and efficiency for the oxygen reduction reaction(ORR)in electrochemical energy conversion systems.The resulting ECL biosensor allowed ultrasensitive detection of myocardial miRNA with a low detection limit of 0.42 fM and a wide linear range from 1 fM to 1.0 nM.Additionally,it demonstrates exceptional performance when evaluated using serum samples collected from patients with AMI.This work expands the application of single-atom catalysis in ECL sensing and introduces novel perspectives for utilizing ECL in disease diagnosis.展开更多
Exosomal miRNAs,as potential biomarkers in liquid biopsy for cancer early diagnosis,have aroused widespread concern.Herein,an electrochemical biosensor based on DNA“nano-bridge”was designed and applied to detect exo...Exosomal miRNAs,as potential biomarkers in liquid biopsy for cancer early diagnosis,have aroused widespread concern.Herein,an electrochemical biosensor based on DNA“nano-bridge”was designed and applied to detect exosomal microRNA-21(miR-21)derived from breast cancer cells.In brief,the target miR-21 can specifically open the hairpin probe 1(HP1)labeled on the gold electrode(GE)surface through strand displacement reaction.Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction(HCR)between two kinetically trapped hairpin probes:HP2 immobilized on the GE surface and biotin labeled HP3 in solution.Cascade HCR leads to the formation of DNA“nano-bridge”tethered to the GE surface with a great deal of“piers”.Upon addition of avidin-modified horseradish peroxidase(HRP),numerous HRP were bound to the formed“nano-bridge”through biotin-avidin interaction to arouse tremendous current signal.In theory,only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted.Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L,as well as little cross-hybridization even at the single-base-mismatched level.Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line.To our knowledge,this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection.In a word,the high sensitivity,selectivity,low cost make the proposed method hold great potential application for early point-of-care(POC)diagnostics of cancer.展开更多
To dissolve the bottleneck problem of life and biomedical science in detection of biomolecules with low abundance and acquisition of ultraweak biological signals,highly sensitive analytical methods coupling with the s...To dissolve the bottleneck problem of life and biomedical science in detection of biomolecules with low abundance and acquisition of ultraweak biological signals,highly sensitive analytical methods coupling with the specificity of biological recognition events have been quickly developed by the exploring of signal amplification strategies.These strategies have extensively been introduced into the development of highly sensitive immunosensing methods by combining with highly specific immunological recognition.They can be divided into two groups.One group of strategies attempts to transfer the immunological recognition event into large number of reporter probes or signal probes for signal readout by employing nano/micro-materials as vehicles for multi-labeling and/or molecular biological amplification for increasing the abundance of the signal molecules.The other uses nanomaterials or enzyme mimics as catalytic tools/tags to obtain enhanced detection signal.This review focuses on the significant advances in design of signal amplification strategies for highly sensitive immunosensing.展开更多
基金financially supported by Central Government Guided Local Science and Technology Development Fund Project(guikeZY22096017)Natural Science Foundation of Guangxi Province(2024GXNSFDA010036)National Natural Science Foundation of China(22164014,U23A2089)。
文摘A highly sensitive electrochemical aptasensor was developed for the detection of kanamycin,employing a DNA signal amplification strategy that combines RecJf exonuclease-assisted target recycling with a hybridization chain reaction(HCR).The sensing platform is constructed by covalently immobilizing double-stranded DNA(dsDNA)comprised of a kanamycin-specific aptamer and its thiol-modified complementary strand(SH-CDNA)onto a gold electrode.In the presence of kanamycin and RecJf exonuclease,the aptamer selectively binds to kanamycin,dissociating from the dsDNA complex.The RecJf exonuclease then cleaves the aptamer,releasing kanamycin and initiating a cycle of repetitive binding and release.The residual SH-CDNA on the electrode triggers an HCR between two types of ferrocene-labeled hairpin DNA,forming an elongated stable dsDNA nanostructure.This results in an amplified electrochemical signal proportional to the logarithm of kanamycin concentration over a range of 0.01–10 nmol/L,with a remarkable detection limit of 1.8 pmol/L.The aptasensor's performance was validated by analyzing spiked kanamycin in pharmaceutical eye drops and milk samples,yielding recovery rates between 95.6%and 104.8%and relative standard deviations from 1.4%to 4.2%.With its exceptional selectivity and sensitivity,this aptasensor offers a compelling alternative to traditional HPLC for rapid on-site detection of kanamycin.Capitalizing on the specificity of aptamers,the sensor design presented herein serves as a valuable blueprint for engineering detectors of other molecules,with significant implications for analytical chemistry and food safety monitoring.
基金supported in part by National Natural Science Foundation of China(Nos.22225505,22174097).
文摘DNA nanomaterials hold great promise in biomedical fields due to its excellent sequence programmability,molecular recognition ability and biocompatibility.Hybridization chain reaction(HCR)is a simple and efficient isothermal enzyme-free amplification strategy of DNA,generating nicked double helices with repeated units.Through the design of HCR hairpins,multiple nanomaterials with desired functions are assembled by DNA,exhibiting great potential in biomedical applications.Herein,the recent progress of HCR-based DNA nanomaterials for biosensing,bioimaging and therapeutics are summarized.Representative works are exemplified to demonstrate how HCR-based DNA nanomaterials are designed and constructed.The challenges and prospects of the development of HCR-based DNA nanomaterials are discussed.We envision that rationally designing HCR-based DNA nanomaterials will facilitate the development of biomedical applications.
基金supported by the National Natural Science Foundation of China(22304062)the Zhejiang Provincial Natural Science Foundation of China(LTGY24B050002)+2 种基金the Program for Science and Technology of Jiaxing(2023AY40028)the Baiqing Foundation of Jiaxing University(CD70621010)Springer Nature or its licensor(e.g.a society or other partner)holds exclusive rights to this article under a publishing agreement with the author(s)or other rightsholder(s)author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law。
文摘In this work,we proposed a ratiometric silver nanoclusters(AgNCs)fluorescent assay by designing a bifunctional-blockeraided hybridization chain reaction(HCR).Hairpin probe 1(HP1)containing two special DNA fragments(5′-CAC CGC T-3′and 5′-ATT TGC CTT TTG GGG ACG GATA-3′)at two terminals creates a red-emitting AgNC nucleation sequence(rNS,5′-CAC CGC TAT TTG CCT TTT GGG GAC GGATA-3′).We found that the presence of a toehold fragment(5′-TGCCC-3′)in HP1 could silence the rNS.Upon the addition of a target nucleic acid,HCR of HP1 and hairpin probe 2(HP2)could be initiated,resulting in the formation of long chain of DNA duplexes with multibranched rNS.As the toehold fragment in HP1participated in generating duplexes,a strong emission of rNS-templated AgNCs was observed at 670 nm.More significantly,a bifunctional blocker was introduced not only to reduce the background red-emitting fluorescence but also to play as an internal green-emitting AgNCs nucleation sequence.On the one hand,the blocker could increase the signal-to-noise-ratio of the constructed biosensor,and on the other hand,the blocker also helped to prepare ratiometric HCR-AgNCs assay with self-calibrating ability to strengthen its reproducibility.Compared with the traditional HCR-AgNCs sensors,the developed ratiometric assay based on the bifunctional-blocker-aided HCR has higher reliability,which is important for the fabrication of biosensors in various fields for practical biosensing applications.
基金supported by the National Natural Science Foundation of China(22122403,22274042,22234003)the Natural Science Foundation of Hunan Province(2021JJ10012)。
文摘Accurate signal amplification in living cells is highly important in biomedical research and medical diagnostics.Benefiting from its enzyme-free,efficient isothermal signal amplification ability,hybridization chain reaction(HCR)plays an important role in intracellular signal amplification;however,HCR fails the accurate signal amplification in the situation when the properties of some biological targets and analogues are too similar.Particularly,their signal amplification accuracy for mature mi RNAs is unsatisfactory due to the signal interference of precursor micro RNAs(abbreviated as pre-mi RNAs),which also contain the sequence of mature mi RNAs.Herein,we develop the first example of size-selective hybridization chain reaction probe for accurate signal amplification,which achieved accurate and sensitive biosensing of mature mi RNAs in living cancer cells.Our probe,termed as q Tcage,consists of a DNA nanocage for size-selective responsive to mature mi RNAs,as well as a quadrivalent tetrahedral DNA structure for HCR signal amplification.Benefiting from the size-selectivity of DNA nanocage,shorter mature mi RNAs(19–23 nt)rather than longer pre-mi RNAs(60–70 nt)could enter the cavity to release triggers strand,which activates HCR reaction for fluorescence signal recovery.The probe efficiently reduces signal interference of pre-mi RNAs and improves the imaging sensitivity for intracellular mature mi RNAs,which was successfully applied for mature mi RNAs imaging during drug treatment.Overall,this strategy provides the hybridization chain reaction with the feature of size-selective ability,which holds promise for further accurate signal amplification in biological processes study and clinical diagnostics.
基金supported by the National Natural Science Foundation of China(22304062)the Zhejiang Provincial Natural Science Foundation of China(LTGY24B050002)+1 种基金the Program for Science and Technology of Jiaxing(2023AY40028)the Baiqing Foundation of Jiaxing University(CD70621010).
文摘Although immobilization-free and label-free electrochemical DNA(E-DNA)biosensors have engaged tremendous interest due to their superior properties,such as easy operation,time-saving and cost-saving,most of them are fabricated in homogeneous modes and usually produce high background current.In the present work,we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction(HCR)for one-pot nucleic acid detection with zero background.The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins,resulting in the generation of HCR products with multibranched arms,which can be captured onto the electrode viaπ-πstacking interactions between multibranched arms and reduced graphene oxide(rGO).Prior to the incubation process with an electrode,two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes.Thus,an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized.This strategy also presents additional merits of simplicity and cheap cost,since probe immobilization,signal tag labeling,and multiple incubation processes are avoided.Therefore,the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.
基金supported by the National Basic Research Program of China(2015CB932600,2013CB933000)the National Natural Science Foundation of China(21505101,21375042,21405054, 21404097)1000 Young Talent(to Fan Xia) and Zhejiang Provincial Natural Science Foundation of China(LQ16B050003)
文摘The fabrication of sensitive sensors with high selectivity is highly desirable for the detection of some important biomarkers,such as nucleic acids,proteins,small molecules and ions.DNA hybridization chain reaction(HCR) and DNA supersandwich self-assembly(SSA) are two prevalent enzyme-free signal amplification strategies to improve sensitivity of the sensors.In this review,we firstly describe the characteristics about DNA HCR and DNA SSA,and then summarize the advances in the one-dimensional DNA nanostructures assisted by HCR and SSA.This review has been divided into three parts according to the two signal amplification methods and highlights recent progress in these two strategies to improve the detection sensitivity of proteins,nucleic acids,small molecules and ions.
基金supported by the National Key Research and Development Program of China (No. 2017YFA0205302)the Natural Science Foundation of Jiangsu Province-Major Project (No. BK20212012)+2 种基金the National Natural Science Foundation of China (No. 21874071)the “Six Talents Peak” Foundation of Jiangsu Province (No. SWYY-046)the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, No. YX030003)。
文摘Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases.Herein,a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR),hybridization chain reaction (HCR) and enzyme catalytic amplification.Four DNA hairpins(H1,H2,H3,H4) are employed to form an ultralong double-strand DNA (dsDNA) structure,which is initiated by target miRNA-21.As H3 and H4 are labeled with horseradish peroxidase (HRP),numerous HRPs are loaded on the long dsDNA,producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H_(2)O_(2)) and 3,3,5,5-tetramethylbenzidine (TMB) reaction strategy.Compared with single signal amplification,the triple signal amplification strategy shows higher electrochemical response,wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity,reproducibility and stability.Taking advantage of the triple signal amplification strategy,the proposed electrochemical biosensor can detect miRNA-21 in 10 He La cell lysates,suggesting that it is a promising method for fruitful assay in clinical diagnosis.
基金supported by the National Natural Science Foundation of China(21535006,21405123)
文摘Assembling and ordering nanomaterials into desirable patterns are considerably significant,since the properties of nanomaterials depend not only on the size and shape,but also on the spatial arrangement among the collective building blocks.In this work,the DNA self-assembly technology of hybridization chain reaction(HCR) provided a convenient method to yield long double-strand DNA(dsDNA) to install gold nanoparticles(AuNPs) into one dimensional assembly along the skeleton of dsDNA.Interestingly,the tunable length of AuNPs assemblies along dsDNA chain could be achieved by adjusting the reaction time of HCR,which is based on the formation of covalent bond between Au and the-SH group of DNA.Compared with weak light scattering of single AuNP,these AuNPs assemblies could be clearly imaged under the dark field microscopy,indicating that the light scattering was greatly improved after assembling.
基金supported by the Natural Science Foundation of China(Nos.21877030,21735002 and 21778016).
文摘Accurate and sensitive detection of caner cells is of significant importance for early diagnosis and treat-ment of cancer.Here,we developed an extracellular ATP-dctivated hybridization chain reaction(HCR)amplification strategy to meet this purpose.This strategy relies on three DNA probes,Apt-trigger,H1-AТP aptamer duplex and hairpin H2.The Apt-trigger probe consists of two com sequence for specific recognition of the target cells.and a trigger sequence for the HCR assembly.Theроnents:an aptamer duplex structure of H1-ATP aptamer causes the tochold in hairpin H1 to be hidden,preventing the strand-ent displacement reaction between haipin H1 and Apt-trigger.Upon activation with ATP the ATP aptamer will blnd to ATP to dissoci iate from hairpin H1,thus leading to an Apt-trigger-induced strand-displacement reaction and subsequent HCR with hairpin H2 on the target cell surface.Benefiting from aptamer recogni-tion and ATP-activated HCR amplification,this strategy can not only perform sensitive quantitative anal-ysis with a detection limit of 25 cells in 200 ul.of binding buffer,but also show desirable specificity and accuracy for identifylng target cells from control cells and mixed cell samples,Imporantly,this method retains stable and good perfor mance for target cell detection in 10%fetal bovine ser rum,den onstrating great potential for clinical diagnosis in complex biological matrices.Furthermore,this strategy can be adapted to detect various types of cancer cells by changing the corresponding aptamer sequence.
基金This work was supported by the National Natural Science Foundation of China(nos.21735002 and 21874036)the Opening Foundation of the State Key Laboratory of Chemo/Biosensing and Chemometrics,Hunan University(no.2019013)+1 种基金the Changsha Municipal Natural Science Foundation(no.kq2007021)the Natural Science Foundation for Distinguished Young Scholars of Hunan Province(no.2021JJ10011).
文摘DNAzyme has emerged as a promising gene silencer for therapeutic applications.However,DNAzymebased gene therapy against tumors is still challenged by undesired cytotoxicity in normal cells.Herein,we explored the potential application of hybridization chain reaction(HCR)for endogenous miRNA-activated DNAzyme-based gene silencing,which performing amplified signal and cellular onsite therapy in one system.The DNA probes,called HCR-tDz circuits,are designed by combining HCR and therapeutic DNAzyme(tDz).The miRNA-21 is selected as the diagnostic target for its distinctive overexpression in cancerous cells,and the HCR is employed to amplify the miR-21 recognition event as well as the tDz activation.The massively repeated DNAzyme activated by miR-21-triggered HCR is utilized to catalytically cleave early growth response-1 mRNA and implement a cellular gene-silencing strategy.Results show that HCR-tDz nanostructure enables amplified miR-21 imaging in living cells.Furthermore,this nanosystem can effectively inhibit the proliferation of cancer cells with high specificity,thus resulting in sensitive diagnostic signaling and accurate therapeutic effects.
基金We thank for the funding:National Key R&D Program of China(grant numbers 2018 YFC0310800,2018YFC0310803)COMRA Project DY135-B2-12+3 种基金the National Natural Science Foundation of China(grant numbers 41525011,91751205,11774225)the Recruitment Program of Global Experts(Program for Young Professionals),and the Natural Science Foundation of Shanghai(grant no.18ZR1419800)This is also a contribution to the Center for Ocean Mega-Science,Chinese Academy of Sciences,the Senior User Project of RV KEXUE(KEXUE2019GZ06)the International Center for Deep-Life Investigation(IC-DLI).We thank Gunter Wegener for providing the ANME enrichment sample.
文摘Fluorescence in situ hybridization(FISH)is a canonical tool commonly used in environmental microbiology research to visualize targeted cells.However,the problems of low signal intensity and false-positive signals impede its widespread application.Alternatively,the signal intensity can be amplified by incorporating Hybridization Chain Reaction(HCR)with FISH,while the specificity can be improved through protocol modification and proper counterstaining.Here we optimized the HCR-FISH protocol for studying microbes in environmental samples,particularly marine sediments.Firstly,five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methano-coccoides methylutens,and two sets displayed high hybridization efficiency and specificity.Secondly,we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals.Various detachment methods,extraction methods and formulas of hybridization buffer were tested using sediment samples.Thirdly,an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles,providing a reliable reference for FISH imaging.In summary,our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.
文摘The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol （EMB） resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization （RDBH） in addition to evaluating the clinical value with application of PCR-RDBH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates （65.9%） showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation （38/91, 41.8% ） and the ATA mutation （16/91, 17.6% ） were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing fcr one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.
基金supported by the National Natural Science Foundation of China,No.81360562
文摘Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan（GV3）, Dazhui（GV14）, Zusanli（ST36） and Ciliao（BL32） and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ（ROCKⅡ） of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.
基金This study was supported by the Key Research and Development Program of China(No.2018YFC0213400)the National Natural Science Foundation of China(Nos.21737002,21976119,and 22176126)。
文摘An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(Exo Ⅲ-ATC)and hybridization chain reaction(HCR)synergistic triple signal amplification.The MrGO-AuNPs is a superior carrier for capture DNA(cDNA)and acts as magnetic media for automatic separation and adsorption.This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode.Additionally,the three DNA hairpins(cDNA,methylene blue(MB)labeled HP1 and HP2)further contribute to biosensor specificity and selectivity.When cDNA captures Hgt,it activates Exo Ⅲ-ATC due to the formation of a sticky end in the DNA stem via thymine-Hig-thymidine(T-Hg^(2+)-T),this leads to the hydrolysis of self-folded DNA by Exo Ⅲ-ATC to form"key"DNA(kDNA).The kDNA subsequently initiates HCR,resulting in massive super-sandwich structures(kDNA-[HP1/HP2])carrying signaling molecules on MrGO-AuNPs,and this overall structure serves as a signal probe(SP).Leveraging magnetic adsorption,the SP was automatically adsorbed onto the magneto-glass carbon electrode(MGCE),generating an amplified signal.This biosensor's detection limit(LOD)was 3.14 pmol/L,far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA.The biosensor also showed excellent selectivity when challenged by interfering ions,and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring.
基金financial supported from the National Natural Science Foundation of China(No.22174115)the Graduate Education and Teaching Reform Research Project of Chongqing(No.yjg223038)the Fundamental Research Funds for the Central Universities(No.SWU-XDJH202321)。
文摘Natural enzymes,such as horseradish peroxidase(HRP),are a class of important biocatalysts with the high specificity,but their catalytic efficiency is usually unsatisfactory.Thus,the higher catalytic efficiency induced by the confinement effect is promising in optical sensing systems.In this work,a dark-field light scattering sensing platform was fabricated by the confinement effect of HRP from hybridization chain reaction(HCR)and then released to solution by the toehold-mediated strand displacement reaction(TSDR).Then,HRP catalyzed the 3,3,5,5-tetramethylbenzidine(TMB)to TMB^(2+)with the assistance of hydrogen peroxide,which etched the gold nanorods(Au NRs)with the weakened light scattering.The single-particle assay was established based on the decreased light scattering intensity of AuNRs under dark-field microscope.The proposed assay revealed excellent analytical performance within a linear range from 25 pmol/L to 600 pmol/L,and a low limit of detection of 3.12 pmol/L.Additionally,it also manifested satisfactory recovery of mi RNA-21 in human serum samples.The high sensitivity,excellent specificity,and universal applicability make this sensing platform promising for disease diagnosis.
基金supported by the National Natural Science Foundation of China(No.22004003)the Natural Science Foundation of Anhui Province for Distinguished Young Scholars(No.2008085J11)+2 种基金the Open Project Program of Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science(No.M2024-5)MOE,the Open Project of Engineering Research Center of Biofilm Water Purification and Utilization Technology of Ministry of Education(No.BWPU2023KF06)the Natural Science Research Project of Anhui Province Education Department(No.2023AH051116).
文摘Circulating microRNAs(miRNAs)play a pivotal role in the occurrence and development of acute myocardial infarction(AMI),and precise detection of them holds significant clinical implications.The development of luminol-based luminophores in the field of electrochemiluminescence(ECL)for miRNA detection has been significant,while their effectiveness is hindered by the instability of co-reactant hydrogen peroxide(H_(2)O_(2)).In this work,an iron single-atom catalyst(Fe-PNC)was employed for catalyzing the luminol-O_(2) ECL system to achieve ultra-sensitive detection of myocardial miRNA.Target miRNA triggers a hybridization chain reaction(HCR),resulting in the generation of a DNA product featuring multiple sticky ends that facilitate the attachment of Fe-PNC probes to the electrode surface.The Fe-PNC catalyst exhibits high promise and efficiency for the oxygen reduction reaction(ORR)in electrochemical energy conversion systems.The resulting ECL biosensor allowed ultrasensitive detection of myocardial miRNA with a low detection limit of 0.42 fM and a wide linear range from 1 fM to 1.0 nM.Additionally,it demonstrates exceptional performance when evaluated using serum samples collected from patients with AMI.This work expands the application of single-atom catalysis in ECL sensing and introduces novel perspectives for utilizing ECL in disease diagnosis.
基金the financial support of Natural Science Foundation of Fujian Province(No.2020J01545)National Natural Science Foundation of China(No.21874019)+3 种基金United Fujian Provincial Health and Education Project for Tackling the Key Research,China(No.WKJ2016-2-30)Fujian Science and Technology Innovation Joint Found Project(No.2019Y9008)Science and Technology Plan Guided Project of Fujian Provincial Science and Technology Department(No.2020Y0022)Young Topnotch Talent Project of Colleges and Universities of Fujian Province(No.3002360301).
文摘Exosomal miRNAs,as potential biomarkers in liquid biopsy for cancer early diagnosis,have aroused widespread concern.Herein,an electrochemical biosensor based on DNA“nano-bridge”was designed and applied to detect exosomal microRNA-21(miR-21)derived from breast cancer cells.In brief,the target miR-21 can specifically open the hairpin probe 1(HP1)labeled on the gold electrode(GE)surface through strand displacement reaction.Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction(HCR)between two kinetically trapped hairpin probes:HP2 immobilized on the GE surface and biotin labeled HP3 in solution.Cascade HCR leads to the formation of DNA“nano-bridge”tethered to the GE surface with a great deal of“piers”.Upon addition of avidin-modified horseradish peroxidase(HRP),numerous HRP were bound to the formed“nano-bridge”through biotin-avidin interaction to arouse tremendous current signal.In theory,only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted.Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L,as well as little cross-hybridization even at the single-base-mismatched level.Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line.To our knowledge,this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection.In a word,the high sensitivity,selectivity,low cost make the proposed method hold great potential application for early point-of-care(POC)diagnostics of cancer.
基金We gratefully acknowledge the National Natural Science Foundation of China(21361162002,21635005)Priority development areas of The National Research Foundation for the Doctoral Program of Higher Education of China(20130091130005).
文摘To dissolve the bottleneck problem of life and biomedical science in detection of biomolecules with low abundance and acquisition of ultraweak biological signals,highly sensitive analytical methods coupling with the specificity of biological recognition events have been quickly developed by the exploring of signal amplification strategies.These strategies have extensively been introduced into the development of highly sensitive immunosensing methods by combining with highly specific immunological recognition.They can be divided into two groups.One group of strategies attempts to transfer the immunological recognition event into large number of reporter probes or signal probes for signal readout by employing nano/micro-materials as vehicles for multi-labeling and/or molecular biological amplification for increasing the abundance of the signal molecules.The other uses nanomaterials or enzyme mimics as catalytic tools/tags to obtain enhanced detection signal.This review focuses on the significant advances in design of signal amplification strategies for highly sensitive immunosensing.
