Phospholipase A_(2)(PLA_(2))is a critical biomarker of many diseases.Among various fluorescent probes,the ratiometric ones have shown great promise because they can provide built-in calibration to avoid interference f...Phospholipase A_(2)(PLA_(2))is a critical biomarker of many diseases.Among various fluorescent probes,the ratiometric ones have shown great promise because they can provide built-in calibration to avoid interference from analyte-independent factors by simultaneously monitoring two or more fluorescence peaks.Herein,we develop a biomimetic membrane-coated ratiometric fluorescent nanoprobe for the detection of PLA_(2).In this design,silica nanoparticles modified with fluorescein isothiocyanate(FITC)are functionalized with a layer of biomimetic membrane that is loaded with Dil dye.Therefore,when the nanocomposite is illuminated by 488 nm incident light,in addition to the fluorescence of FITC centered at 522 nm,that of Dil peaked at 568 nm originating from the energy transfer from FITC to Dil can also be monitored,forming a ratiometric fluorescent nanoprobe.When the target PLA_(2)is added,FITC and Dil will no longer be in close proximity because it hydrolyzes the biomimetic membrane,cutting off the energy flow between the two dyes.Consequently,the ratiometric fluorescent signal of the nanoprobe is altered to quantitatively reflect the content of PLA_(2).This study provides an effective approach to PLA_(2)detection,and the biomimetic membrane-mediated ratiometric design is promising in developing a new generation of biosensors.展开更多
Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require sp...Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require specialized equipment or proprietary reagents/consumables.Herein,we report a microchamber-free and spherical nucleic acid(SNA)-amplified digital flow cytometric bead assay(dFBA)for ultrasensitive protein and exosome analysis with simple workflows,easily accessible instruments/reagents,and high discriminating ability towards the fluorescence-positive and fluorescence-negative beads.In this dFBA,microbeads are employed as independent carriers to anchor the single target molecule-initiated signal amplification reaction,avoiding the use of sealed droplets or microwell microchambers.Meanwhile,antibody-functionalized SNAs(FSNAs)with a high density of DNA probes act as a bridge for efficiently amplified target-to-DNA signal conversion,which allows the use of DNA-based rolling circle amplification(RCA)as the fluorescence signal amplification technique to quantify non-nucleic acid targets.Even a single target-induced on-bead RCA and fluorescence enriching are sufficient to make the target-loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer(FCM).This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers,enlarging the toolbox of digital bioassays for clinical applications.展开更多
MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging...MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging due to their small size and low abundance.Herein,we have developed a nanoparticle-confined DNA walker strategy for the specific detection of miRNA.In the existence of the target miRNA;the on-particle DNA walking reaction will be initiated,providing a fluorescence-positive nanoparticle.Otherwise,the nanoparticle would be fluorescence-negative.Utilizing the total internal reflection fluorescent microscope(TIRFM)to digitally count the fluorescence-positive nanoparticles,the proposed method possesses a detection limit of 0.2 pmol/L miRNA and can accurately distinguish the single-base mismatched target.This design combines the merits of the DNA walker for signal amplification and the TIRFM for highly sensitive detection,paving a new way for the digital counting-based analysis of exosomal miRNAs.展开更多
T4 polynucleotide kinase(T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determina...T4 polynucleotide kinase(T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle,the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope(TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.展开更多
Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies.How to eliminate the false-positive interference from the...Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies.How to eliminate the false-positive interference from the abundant normal mRNA is still a big challenge.Herein,we have proposed an LNA(locked nucleic acid)-assisted high-specificity strategy which can selectively guide the RNase H to cleave only the wildtype mRNA(wtRNA)while the mutant mRNA(mutRNA)will remain intact.The intact mutRNA can be amplified and detected by real-time reverse transcription(RT)-PCR but the disconnected wtRNA will be not replicated at all.Based on the highly selective depletion of wtRNA,this elegant design effectively avoids the false-positive interference from the high background of normal mRNA and thus can guarantee the accurate and reliable detection of rare mutRNA in real biomedical samples.Besides for the excellent specificity,ultrahigh sensitivity is also achieved for this proposed assay,which allows the quantification of mutRNA at single molecule and single cell level.Due to its easy design,high sensitivity and specificity,the established LNA probe-assisted RT-PCR strategy provides a powerful tool for studying the function of mutRNA at the single cell level and for the mutRNA-associated liquid biopsy.展开更多
In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loo...In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of mi RNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a mi RNA/DNA hybrid and a single strand(ss)DNA,the ss DNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the mi RNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of ds DNA can produced specific fluorescence signal for mi RNA detection.The released mi RNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L mi RNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different mi RNA targets and labeling them with different fluorophores,multiplexed mi RNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.展开更多
基金supported by the National Natural Science Foundation of China(21904083)the Program for Changjiang Scholars and Innovative Research Team in University(IRT_15R43)+1 种基金the Fundamental Research Funds for the Central Universities(GK202201009,GK202101001,and GK202305001)the Innovation Capability Support Program of Shaanxi Province(2021TD-42).
文摘Phospholipase A_(2)(PLA_(2))is a critical biomarker of many diseases.Among various fluorescent probes,the ratiometric ones have shown great promise because they can provide built-in calibration to avoid interference from analyte-independent factors by simultaneously monitoring two or more fluorescence peaks.Herein,we develop a biomimetic membrane-coated ratiometric fluorescent nanoprobe for the detection of PLA_(2).In this design,silica nanoparticles modified with fluorescein isothiocyanate(FITC)are functionalized with a layer of biomimetic membrane that is loaded with Dil dye.Therefore,when the nanocomposite is illuminated by 488 nm incident light,in addition to the fluorescence of FITC centered at 522 nm,that of Dil peaked at 568 nm originating from the energy transfer from FITC to Dil can also be monitored,forming a ratiometric fluorescent nanoprobe.When the target PLA_(2)is added,FITC and Dil will no longer be in close proximity because it hydrolyzes the biomimetic membrane,cutting off the energy flow between the two dyes.Consequently,the ratiometric fluorescent signal of the nanoprobe is altered to quantitatively reflect the content of PLA_(2).This study provides an effective approach to PLA_(2)detection,and the biomimetic membrane-mediated ratiometric design is promising in developing a new generation of biosensors.
基金supported by the National Natural Science Foundation of China(22074088,21622507)the Program for Changjiang Scholars and Innovative Research Team in University(IRT_15R43)+1 种基金the Fundamental Research Funds for the Central Universities(GK202101001,GK202206040)Innovation Capability Support Program of Shaanxi(2021TD-42).
文摘Comprehensive Summary,Most conventional digital bioassays rely on the use of fully-sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction,which require specialized equipment or proprietary reagents/consumables.Herein,we report a microchamber-free and spherical nucleic acid(SNA)-amplified digital flow cytometric bead assay(dFBA)for ultrasensitive protein and exosome analysis with simple workflows,easily accessible instruments/reagents,and high discriminating ability towards the fluorescence-positive and fluorescence-negative beads.In this dFBA,microbeads are employed as independent carriers to anchor the single target molecule-initiated signal amplification reaction,avoiding the use of sealed droplets or microwell microchambers.Meanwhile,antibody-functionalized SNAs(FSNAs)with a high density of DNA probes act as a bridge for efficiently amplified target-to-DNA signal conversion,which allows the use of DNA-based rolling circle amplification(RCA)as the fluorescence signal amplification technique to quantify non-nucleic acid targets.Even a single target-induced on-bead RCA and fluorescence enriching are sufficient to make the target-loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer(FCM).This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers,enlarging the toolbox of digital bioassays for clinical applications.
基金the National Natural Science Foundation of China(Nos.22074088 and 21904083)the Program for Changjiang Scholars and Innovative Research Team in University(IRT_15R43)the Fundamental Research Funds for the Central Universities(Nos.GK202003038,GK201901003,2020TS089,GK202101001).
文摘MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging due to their small size and low abundance.Herein,we have developed a nanoparticle-confined DNA walker strategy for the specific detection of miRNA.In the existence of the target miRNA;the on-particle DNA walking reaction will be initiated,providing a fluorescence-positive nanoparticle.Otherwise,the nanoparticle would be fluorescence-negative.Utilizing the total internal reflection fluorescent microscope(TIRFM)to digitally count the fluorescence-positive nanoparticles,the proposed method possesses a detection limit of 0.2 pmol/L miRNA and can accurately distinguish the single-base mismatched target.This design combines the merits of the DNA walker for signal amplification and the TIRFM for highly sensitive detection,paving a new way for the digital counting-based analysis of exosomal miRNAs.
基金supported by the National Natural Science Foundation of China (Nos. 22074088, 21622507, 21904083)the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R43)+1 种基金the Innovation Capability Support Program of Shaanxi (No. 2021TD-42)the Fundamental Research Funds for the Central Universities (Nos. GK202101001 and GK202201009)。
文摘T4 polynucleotide kinase(T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle,the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope(TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.
基金supported by the National Natural Science Foundation of China(Nos.21622507 and 21575086)the Program for Changjiang Scholars and Innovative Re-search Team in University(No.IRT_15R43)the Fundamental Research Funds for the Central Universities(No.GK201802016)。
文摘Accurate quantitation of site-specific mRNA mutation in single cells or in peripheral blood is of great significance for both biological and biomedical studies.How to eliminate the false-positive interference from the abundant normal mRNA is still a big challenge.Herein,we have proposed an LNA(locked nucleic acid)-assisted high-specificity strategy which can selectively guide the RNase H to cleave only the wildtype mRNA(wtRNA)while the mutant mRNA(mutRNA)will remain intact.The intact mutRNA can be amplified and detected by real-time reverse transcription(RT)-PCR but the disconnected wtRNA will be not replicated at all.Based on the highly selective depletion of wtRNA,this elegant design effectively avoids the false-positive interference from the high background of normal mRNA and thus can guarantee the accurate and reliable detection of rare mutRNA in real biomedical samples.Besides for the excellent specificity,ultrahigh sensitivity is also achieved for this proposed assay,which allows the quantification of mutRNA at single molecule and single cell level.Due to its easy design,high sensitivity and specificity,the established LNA probe-assisted RT-PCR strategy provides a powerful tool for studying the function of mutRNA at the single cell level and for the mutRNA-associated liquid biopsy.
基金the National Natural Science Foundation of China(21335005,21472120)the Fundamental Research Funds for the Central Universities(GK201501003,GK201303003)the Excellent Doctor Innovation Project of Shaanxi Normal University
文摘In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of mi RNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a mi RNA/DNA hybrid and a single strand(ss)DNA,the ss DNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the mi RNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of ds DNA can produced specific fluorescence signal for mi RNA detection.The released mi RNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L mi RNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different mi RNA targets and labeling them with different fluorophores,multiplexed mi RNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.
