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...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 T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.展开更多
Currently,the technology for detecting antibiotics is developing rapidly,with good sensitivity and selectivity.However,some detection methods still have issues with cumbersome pretreatment and high experimental costs....Currently,the technology for detecting antibiotics is developing rapidly,with good sensitivity and selectivity.However,some detection methods still have issues with cumbersome pretreatment and high experimental costs.Herein,A“turn-off”bifunctional DNA tweezer was constructed through the design of DNA sequence and selfassembly technology for the dual detection of Enrofloxacin(ENR)and Kanamycin(KANA)in a food matrix.The operation is simple,which increases the detection speed and reduces the detection cost.The two ends of the bifunctional DNA tweezers are connected with specific split aptamer sequences,and the split aptamer forms a specific ternary complex with the target.The tweezers“clip”ENR and KANA at their respective ends,causing a structural change.Under the optimal experimental conditions,the linear range of detection ENR is 0.2 ng/mL-1000 ng/mL with an LOD of 0.18 ng/mL,the linear range of detection KANA is 0.5 ng/mL-1000 ng/mL with an LOD of 0.36 ng/mL.The bifunctional DNA tweezers involved provide a new idea for the construction of DNA nanomachines,which is also suitable for the recognition and detection of other small molecules.展开更多
The sandwich-type lateral fl ow assays relying on dual aptamers with high sensitivity and specifi city have been broadly explored.However,it is unlikely to match a pair of specifi c aptamers that can bind a small mole...The sandwich-type lateral fl ow assays relying on dual aptamers with high sensitivity and specifi city have been broadly explored.However,it is unlikely to match a pair of specifi c aptamers that can bind a small molecular target(e.g.,cocaine)simultaneously due to the steric hindrance.In response,we herein introduced the strategy of“one divides into two”into the construction of sandwich-type lateral fl ow strip assay(LFSA).Specifi cally,we split a single cocaine-recognizing aptamer into two segments,either of which was conjugated with gold nanoparticles(AuNPs)or labeled with biotin,serving as signal probe and capture probe,respectively.Upon the presence of the target molecule,a ternary sandwich complex comprised of the two halves of the aptamer and the target formed.Such sandwich-type LFSA exhibited an excellent nonlinear logarithmic response in the range from 10μmol/L to 5 mmol/L with R^(2)=0.9994.The sensitive on-site detection of cocaine in artifi cial biological samples including urine and sweat was achieved within 15 min,with the visual limit of detection as low as 50μmol/L for urine and 200μmol/L for sweat,and the recovery rates of 83.6-107.4%.展开更多
An improved tum-on aptasensor for thrombin detection using split aptamer fragments and graphene oxide （GO） was reported. The thrombin-binding aptamer （Aptl5） was split into two parts for target recognition, an 8-b...An improved tum-on aptasensor for thrombin detection using split aptamer fragments and graphene oxide （GO） was reported. The thrombin-binding aptamer （Aptl5） was split into two parts for target recognition, an 8-base se- quence labeled with fluorescein （FAM-Apt-A） and a 7-base oligonucleotide sequence （Apt-B）. In the absence of target protein, the fluorescence of FAM-Apt-A/Apt-B was quenched by GO through n-n stacking between GO and single-stranded DNA. However, when thrombin was introduced into the system, a target-induced G-quadruplex forms with two split aptamer fragments and thrombin. The fluorescence recovered due to weak interaction between G-quadruplex and GO. Compared to the strategy using intact aptamer, probe concentration was lowered, and an improved sensitivity was obtained. Moreover, heating process to avoid unfavorable secondary structure was avoided due to the use of shorter split aptamer fragments.展开更多
T-2 toxin is a highly poisonous trichothecene and a well-known contaminant in cereal grains and cereal-based products,hence it is essential to design a simple,reliable,and precise detection mechanism for ensuring food...T-2 toxin is a highly poisonous trichothecene and a well-known contaminant in cereal grains and cereal-based products,hence it is essential to design a simple,reliable,and precise detection mechanism for ensuring food safety and human health.In response to this imperative,we innovatively designed an unreported split T-2 toxin aptamer probe,and the cooperative binding mechanism of the split aptamer to T-2 was first investigated.Based on these,a dual-mode fluorescence(FL)and fluorescence polarization(FP)aptasensor was developed for T-2 toxin detection in peanut and beer samples.The results showed an excellent detection property of the aptasensor,indicating a linear range of 0.1-20 nM and 0.2-50 nM at a detection limit of 0.1 nM,0.12 nM for FL and FP signals,respectively.In actual sample assays,recoveries of 101.7%-115.2%,109.8%-121.6%for FL and FP mode,respectively,were reached in peanuts,whereas recoveries ranged from 81.0%-124.0%,94.1%-107.8%for FL and FP mode in beer.This designed dual-mode biosensor not only showed excellent performances but also verified each other and proved the reliability of binary split aptamer molecular probes.展开更多
基金supported by the Natural Science Foundation of China(Nos.21877030,21735002,21778016 and 21521063)。
文摘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 T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.
基金partly funded by the National Natural Science Foundation of China(32372423 and 32302190)Natural Science Foundation of Jiangsu Province(BK20221073)+2 种基金the S&T Plan Project of Jiangsu Provincial(BE2022324)the National First-class Discipline Program of Food Science and Technology(JUFSTR20180303)Jiangsu Specially Appointed Professor Program.
文摘Currently,the technology for detecting antibiotics is developing rapidly,with good sensitivity and selectivity.However,some detection methods still have issues with cumbersome pretreatment and high experimental costs.Herein,A“turn-off”bifunctional DNA tweezer was constructed through the design of DNA sequence and selfassembly technology for the dual detection of Enrofloxacin(ENR)and Kanamycin(KANA)in a food matrix.The operation is simple,which increases the detection speed and reduces the detection cost.The two ends of the bifunctional DNA tweezers are connected with specific split aptamer sequences,and the split aptamer forms a specific ternary complex with the target.The tweezers“clip”ENR and KANA at their respective ends,causing a structural change.Under the optimal experimental conditions,the linear range of detection ENR is 0.2 ng/mL-1000 ng/mL with an LOD of 0.18 ng/mL,the linear range of detection KANA is 0.5 ng/mL-1000 ng/mL with an LOD of 0.36 ng/mL.The bifunctional DNA tweezers involved provide a new idea for the construction of DNA nanomachines,which is also suitable for the recognition and detection of other small molecules.
基金supported by the National Natural Science Foundation of China(22090050,22122410,21874121)the National Key Research and Development Program of China(2018YFE0206900)+1 种基金Hubei Provincial Natural Science Foundation of China(2020CFA037)Zhejiang Provincial Natural Science Foundation of China(LD21B050001)
文摘The sandwich-type lateral fl ow assays relying on dual aptamers with high sensitivity and specifi city have been broadly explored.However,it is unlikely to match a pair of specifi c aptamers that can bind a small molecular target(e.g.,cocaine)simultaneously due to the steric hindrance.In response,we herein introduced the strategy of“one divides into two”into the construction of sandwich-type lateral fl ow strip assay(LFSA).Specifi cally,we split a single cocaine-recognizing aptamer into two segments,either of which was conjugated with gold nanoparticles(AuNPs)or labeled with biotin,serving as signal probe and capture probe,respectively.Upon the presence of the target molecule,a ternary sandwich complex comprised of the two halves of the aptamer and the target formed.Such sandwich-type LFSA exhibited an excellent nonlinear logarithmic response in the range from 10μmol/L to 5 mmol/L with R^(2)=0.9994.The sensitive on-site detection of cocaine in artifi cial biological samples including urine and sweat was achieved within 15 min,with the visual limit of detection as low as 50μmol/L for urine and 200μmol/L for sweat,and the recovery rates of 83.6-107.4%.
文摘An improved tum-on aptasensor for thrombin detection using split aptamer fragments and graphene oxide （GO） was reported. The thrombin-binding aptamer （Aptl5） was split into two parts for target recognition, an 8-base se- quence labeled with fluorescein （FAM-Apt-A） and a 7-base oligonucleotide sequence （Apt-B）. In the absence of target protein, the fluorescence of FAM-Apt-A/Apt-B was quenched by GO through n-n stacking between GO and single-stranded DNA. However, when thrombin was introduced into the system, a target-induced G-quadruplex forms with two split aptamer fragments and thrombin. The fluorescence recovered due to weak interaction between G-quadruplex and GO. Compared to the strategy using intact aptamer, probe concentration was lowered, and an improved sensitivity was obtained. Moreover, heating process to avoid unfavorable secondary structure was avoided due to the use of shorter split aptamer fragments.
基金funded by National Natural Science Foundation of China[32372417,32302198]Natural Science Foundation of Jiangsu Province[BK20210882,BK20201452].
文摘T-2 toxin is a highly poisonous trichothecene and a well-known contaminant in cereal grains and cereal-based products,hence it is essential to design a simple,reliable,and precise detection mechanism for ensuring food safety and human health.In response to this imperative,we innovatively designed an unreported split T-2 toxin aptamer probe,and the cooperative binding mechanism of the split aptamer to T-2 was first investigated.Based on these,a dual-mode fluorescence(FL)and fluorescence polarization(FP)aptasensor was developed for T-2 toxin detection in peanut and beer samples.The results showed an excellent detection property of the aptasensor,indicating a linear range of 0.1-20 nM and 0.2-50 nM at a detection limit of 0.1 nM,0.12 nM for FL and FP signals,respectively.In actual sample assays,recoveries of 101.7%-115.2%,109.8%-121.6%for FL and FP mode,respectively,were reached in peanuts,whereas recoveries ranged from 81.0%-124.0%,94.1%-107.8%for FL and FP mode in beer.This designed dual-mode biosensor not only showed excellent performances but also verified each other and proved the reliability of binary split aptamer molecular probes.
