A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease III-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

Portable biosensor for quantitative detection of melamine based on CRISPRCas12a and a personal glucose meterEI北大核心CSCD

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. In this paper, a portable biosensor based on the trans-cleavage activity of CRISPR-Cas12a and a personal glucose meter has been developed for quantitative, sensitive and specific detection of melamine. The presence of the target melamine binds to the aptamer, leading to the release of locker DNA. And then, the leasing locker DNA activates the trans-cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA modified electrode, releasing a short DNA fragment labeled with sucrase in the resulting solution. The sucrase could further catalyze sucrose to glucose, which could be detected by the PGM. Under the optimized conditions, the increase of PGM signal was relative with the concentration of melamine ranging from 0.1 to 2.5 μmol/L and the limit of detection (LOD) was 37 nmol/L. Moreover, the portable biosensor has strong specificity and can be used for the quantitative detection of melamine in milk samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

Determination of uracil in the culture fluid of Glomerella cingulata and infected hosts by high-performance liquid chromatographyEI北大核心CSCD

A CRISPR-Cas system holds great promise as a next-generation biosensing technology for molecular diagnostics. In this paper, a portable biosensor based on the trans-cleavage activity of CRISPR-Cas12a and a personal glucose meter has been developed for quantitative, sensitive and specific detection of melamine. The presence of the target melamine binds to the aptamer, leading to the release of locker DNA. And then, the leasing locker DNA activates the trans-cleavage activity of CRISPR-Cas12a to cleave the single-strand DNA (ssDNA) linker on sucrase-ssDNA modified electrode, releasing a short DNA fragment labeled with sucrase in the resulting solution. The sucrase could further catalyze sucrose to glucose, which could be detected by the PGM. Under the optimized conditions, the increase of PGM signal was relative with the concentration of melamine ranging from 0.1 to 2.5 μmol/L and the limit of detection (LOD) was 37 nmol/L. Moreover, the portable biosensor has strong specificity and can be used for the quantitative detection of melamine in milk samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

A fluorescent aptasensing strategy for adenosine triphosphate detection using tris(bipyridine)ruthenium(Ⅱ) complex containing six cyclodextrin units

A sensitive label-free fluorescent aptasensing strategy for the detection of adenosine triphosphate(ATP)has been developed with a metallocyclodextrin, tris(bipyridine)ruthenium(Ⅱ) complex containing six cyclodextrin units(6CD-Ru), which exhibited much stronger emission signal compared to the parent compound Ru(bpy)_3Cl_2. Furthermore, the emission spectrum showed that the ATP-aptamer(ss DNA)could increase the fluorescence intensity of 6CD-Ru dramatically, attributed to the interaction between aptamer and cyclodextrin, which could provide protection to the ruthenium core from the quenching of emission by oxygen in the solution. With the addition of ATP, the interaction between aptamer and cyclodextrins on 6CD-Ru was diminished, since the ATP/aptamer complex had the priority to be formed,leading to the corresponding reduction of fluorescence intensity, which could be utilized to detect ATP quantitatively. A linear relationship was displayed between the fluorescence and the logarithm of ATP concentrations in the range from 1 nmol/L to 1μmmol/L with the detection limit of 0.5 nmol/L(S/N = 3).The proposed fluorescent aptasensing strategy exhibited high sensitivity and specificity, without any labeling or amplification procedures, and it could also be applied for the detection of many other aptamer-specific targets.     

Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology

This work develops a fluorescence approach for sensitive detection of DNA methyltransferase activity based on endonuclease and rolling circle amplification （RCA） technique. In the presence of DNA adenine methylation （Dam） MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn 1. The products cleaved by restriction endonuclease Dpn I then function as a signal primer to initiate RCA reaction by hybridizing with the circular DNA template. Each RCA product containing thousands of repeated sequences might hybridize with a large number of molecular beacons （detection probes）, resulting in an enhanced fluorescence signal. In the absence of Dam MTase, neither methylation/cleavage nor RCA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a dynamic range from 0.5 U/mL to 30 U/mL and a detection limit of 0.18 U/mL. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.     

Homogeneous DNA Detection Based on Fluorescence Quenching by Nanoparticles in Single-step Format :Target-Induced Configuration Transform

A new strategy for homogeneous detection of DNA hybridization in single-step format was developed based on fluorescence quenching by gold nanoparticles. The gold nanoparticle is functionalized with 5’-thiolated 48-base oligonucleotide (probe sequence), whose 3’-terminus is labeled with fluorescein (FAM), a negatively charged fluorescence dye. The oligonucleotide adopts an extended configuration due to the electrostatic repulsion between negatively charged gold nanoparticle and the FAM-attached probe sequence. After addition of the complementary target sequence, specific DNA hybridization induces a conformation change of the probe from an extended structure to an arch-like configuration, which brings the fluorophore and the gold nanoparticle in close proximity. The fluorescence is efficiently quenched by gold nanoparticles. The fluorescence quenching efficiency is related to the target concentration, which allows the quantitative detection for target sequence in a sample. A linear detection range from 1.6 to 209.4 nmol/L was obtained under the optimized experimental conditions with a detection limit of 0.1 nmol/L. In the assay system, the gold nanoparticles act as both nanoscaffolds and nanoquenchers. Furthermore, the proposed strategy, in which only two DNA sequences are involved, is not only different from the traditional molecular beacons or reverse molecular beacons but also different from the commonly used sandwich hybridization methods. In addition, the DNA hybridization detection was achieved in homogenous solution in a single-step format, which allows real-time detection and quantification with other advantages such as easy operation and elimination of washing steps.     

A Triode-like Enzyme-free Catalytic Circuit with Junction Fuel

Target detection circuits have been previously designed,which are propelled by conventional PCR,isothermal amplification and strand-displacement reaction.These detection circuits obtain the target signal via the replication of the target strand,the aggregation of the signal particles or the branch migration.Here we constructed a tri-ode-like enzyme-free catalyst strand-displacement circuit for target DNA detection.The target strand triggered die reaction and released the fluorescence signal strand circularly through branch migration.However,the main challenge of strand-displacement reaction is the signal leakage.Therefore,we designed a double strand structure"junction fuel",which was used to increase the binding energy across the displacement process.Ultimately,the leakage of the system obtained stable inhabitation due to the junction fuel strand.The limit of detection of the system was as low as 0.11 nmol/L and the gain of the system was as high as 28-fold(the concentration of target was 50 nmol/L).Furthermore,the process of the system was visualized vividly in the reaction curve through the kinetic simulation implemented,which suggests that the combination of the kinetic simulation and the experiment exhibits a promising prospect towards the use of strand-displacement circuit in analytical,diagnostic application and synthetic biology.     

Sensitive and enzyme-free detection for single nucleotide polymorphism using microbead-assisted toehold-mediated strand displacement reaction

This report described a free-enzyme, convenient and inexpensive genotyping biosensor capable of detecting single nucleotide polymorphism at normal temperature based on the combination of toeholdmediated strand displacement reaction(toehold-SDR) and microbead-capture technique. The biosensor consists of a pre-hybridized strand formed by a reporter probe and a capture probe. In the presence of a mutant sequence, there is no toehold-mediated strand displacement and the reporter probe cannot be released from the pre-hybridized strand. Microbeads capture the fluorescent pre-hybridized strand through biotin–streptavidin interaction, so microbeads give out significant fluorescence signal, while there is no fluorescence in the solution. However, in the presence of a matched target, the strand displacement is effectively initiated and the reporter probe is released from pre-hybridized strand. After adding microbeads, the solution produces bright fluorescence, while microbeads have no obvious signal.Genotypes are identified conveniently according to the fluorescence intensity of the solution. The method provides a simple and inexpensive strategy to detect point mutation. Moreover, this biosensor shows the linear relationship in the range of 1–40 nmol/L and reaches a detection limit of 0.3 nmol/L.     

DNA-silver Nanocluster Binary Probes for Ratiometric Fluorescent Detection of HPV-related DNA

Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas tlie red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. Tlie ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 ninol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in hiunan serum, illustrating its potential promise for clinical diagnosis.     

Double Magnetic Separation-assisted Fluorescence Method for Sensitive Detection of Ochratoxin A

A double magnetic separation-assisted fluorescence method was developed to rapidly detect ochratoxin A(OTA). The OTA aptamer functionalized magnetic nanomaterial(Fe3O4-Aptanier) and complementary DNA conjugated nitrogen-doped graphene quantum dots(NGQDs-cDNA) were used in this assay. Aptamer could hybridize with cDNA, which induced tlie NGQDs-cDNA to bind onto Fe3O4-Aptamer, and resulted in the fluorescence quenching of NGQDs. After the addition of OTA, the NGQDs-cDNA could release into the solution, and resulted in the recovery of fluorescence signal of NGQDs consequently. By utilizing the magnetic separation, the unbonded NGQDs-cDNA and residual Fe3O4-Aptamer were removed, which significantly increased the fluorescence signal intensity. OTA could be detected in the linear range of 10 nmol/L to 2000 nmol/L, with a limit of detection as 0.66 mnol/L. The advantages of this method include simple operation, good selectivity and high sensitivity, and this method can be used for the rapid detection of ochratoxin A in wheat and com.     

DNA tetrahedron-based split aptamer probes for reliable imaging of ATP in living cells

Accurate detection and imaging of adenosine triphosphate(ATP) expression levels in living cells is of great value for understanding cell metabolism, physiological activities, and pathologic mechanisms. Here, we developed a DNA tetrahedron-based split aptamer probe(TD probe) for ratiometric fluorescence imaging of ATP in living cells. The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b) to a DNA tetrahedron assembled by four DNA oligonucleotides(T1, T2, T3 and ...     

A pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration

We present here a p H-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This p H-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3′-end and an extension sequence at the 5′-end with 5-carboxytetramethylrhodamine(TAMRA) label(denoted as strand A). The other(strand I), being competent to work on the formation of i-motif structure, contained four stretches of the cytosine(C) rich domain and was labeled with a Black Hole Quencher 2(BHQ2) at the 3′-end. At neutral or slightly alkaline p H, strand I was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe(I-AAP). Because of proximityinduced energy transfer, the I-AAP was in a "signal off" state. The slightly acidic p H enforced the strand I to form an intramolecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.     

An electrochemical aptasensor based on cu2+-induced signal amplification strategy for the detection of ochratoxin aEI北大核心CSCD

A novel electrochemical aptasensor based on a Cu2+-induced signal amplification strategy was constructed for the rapid, sensitive and specific detection of ochratoxin A （OTA）. The OTA aptamer with poly （T） was hybridized with the captured DNA probe on the electrode surface. In the presence of Cu2+ and ascorbic acid, the end of poly（T） was used as a template to in situ grow copper nanoclusters （Cu NCs）. In the absence of targeted OTA, the gold electrodes after decorating Cu NCs were immersed into an acidic environment to release Cu2+. After enriching Cu2+ at a potential of − 1.6 V, the strongest current value of copper was recorded by measuring differential pulse voltammetry （DPV）. In the presence of OTA, the OTA aptamer was tightly bound to the target OTA. The OTA aptamer broke away from the electrode to reduce the growth of Cu NCs, resulting in lower DPV current response. This proposed method was employed to detect OTA with linear range from 0.1 to 50.0 ng/mL, and the detection limit was 41.2 pg/mL. The Cu2+-induced electrochemical aptasensor can be further applied in the analysis of target OTA in coffee solution samples. © 2023, Youke Publishing Co.,Ltd. All rights reserved.     

Cascaded DNA circuits-programmed self-assembly of spherical nucleic acids for high signal amplification

Signal amplification is an important issue in DNA nanotechnology and molecular diagnostics. In this work, we report a strategy for the catalytic self-assembly of spherical nucleic acids(SNAs) programmed by two-layer cascaded DNA circuits through integrating an entropy-driven catalytic network, a catalytic hairpin assembly circuit, and a facile SNA assembly-based reporter system. This integrated system could implement ～100,000-fold signal amplification in the presence of 1 p M of input target.Possessing powerful amplification ability of nucleic acid signal, our strategy should be of great potential in fabricating more robust dynamic networks to be applied for signal transduction, DNA computing, and nucleic acid-based diagnostics.     

Selection of a DNA aptamer for the development of fluorescent aptasensor for carbaryl detection

An improved ssDNA library immobilized systematic evolution of ligands by enrichment(SELEX) was applied to select aptamers against carbaryl.After nine selection rounds,a highly enriched ssDNA pool was obtained.The Apta3 was demonstrated as the optimal aptame r.In order to facilitate the modification of aptamer,the Apta3 was further truncated with the dissociation constant(K_d) of 0.3 64 ± 0.055 μmol/L and a fluorescent aptasensor was developed.The linear range for carbaryl was from 100 nmol/L to1500 nmol/L,with the limit of detection was as low as 15.23 nmol/L.Besides,the biosensor was validated for the carbaryl spiked real samples,and the recoveries were between 97.7% and 107.3%.     

TNF-α responsive DNA star trigon formation from four hairpin probes and the analytical application

Tumor necrosis factor-alpha(TNF-α) is a type of critical pro-inflammatory cytokines,which participates in numerous cellular signal pathways and is regarded as a critical protein biomarker for inflammatory based diseases.In this contribution,we have developed a strategy to fabricate multiple DNA star trigon structures with fluorescence signals from four hairpin probes which are detonated by a single molecule of TNF-α.This process causes significant enhancement of fluorescence and a sensitive and selective biosensor for TNF-α assay is constructed.This method is able to achieve the limit of detection(LOD) at 5 pg/mL(0.285 pM).Moreover,some other advantages such as fast response,high selectivity and convenient operation promise the potential use of this method for TNF-α measurement in point of care testing application.Upon further development,this strategy can also be converted to detect other analytes such as small molecules,nucleic acids and other proteins.     

Interactions between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin TMPyP4 and DNA G-quadruplex of telomeric repeated sequence TTAGGG

The binding properties between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the parallel DNA G-quadruplex (G4) of telomeric repeated sequence 5′-TTAGGG-3′ have been characterized by means of circular dichroism,steady-state absorption,steady-state fluorescence and picosecond time-resolved fluorescence spectroscopies. The binding constant and the saturated binding number were determined as 1.29×106 (mol/L)-1 and 3,respectively,according to steady-state absorption spec-troscopy. Based on the findings by the use of time-resolved fluorescence spectroscopic technique,it is deduced that TMPyP4 binds to a DNA G-quadruplex with both the thread-intercalating and end-stacking modes and at the saturated binding state,one TMPyP4 molecule intercalates into the intervals of G-tetrads while the other two stack to the ends of the DNA G-quadruplex.