Single-molecule detection is one of the fundamental challenges of modern biology.Such experiments often use labels that can be expensive,difficult to produce,and for small analytes,might perturb the molecular events b...Single-molecule detection is one of the fundamental challenges of modern biology.Such experiments often use labels that can be expensive,difficult to produce,and for small analytes,might perturb the molecular events being studied.Analyte size plays an important role in determining detectability.Here we use laser-frequency locking in the context of sensing to improve the signal-to-noise ratio of microtoroid optical resonators to the extent that single nanoparticles 2.5 nm in radius,and 15.5 kDa molecules are detected in aqueous solution,thereby bringing these detectors to the size limits needed for detecting the key macromolecules of the cell.Our results,covering several orders of magnitude of particle radius(100 nm to 2 nm),agree with the‘reactive’model prediction for the frequency shift of the resonator upon particle binding.This confirms that the main contribution of the frequency shift for the resonator upon particle binding is an increase in the effective path length due to part of the evanescent field coupling into the adsorbed particle.We anticipate that our results will enable many applications,including more sensitive medical diagnostics and fundamental studies of single receptor–ligand and protein–protein interactions in real time.展开更多
The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that...The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that OI-RD is a label-free method which can not only distinguish the concentration difference of oligonucleotides before and after the hybridization but also detect the hybridization of short oligonucleotides. At present the OI-RD method can detect 0.39 μmol/L 20-base oligonucleotide or less. These results suggest that the OI-RD method is a promising and potential technique for label-free detection of biological microarrays.展开更多
Mouse-Immunoglobulin G(mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence refl...Mouse-Immunoglobulin G(mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence reflectivity difference(OI-RD) method.Two detection signals,consisting of an imaginary part(Im{Δp-Δs}) and a real part(Re{Δp-Δs}) of OI-RD,were obtained simultaneously.The detection results of hybridization by OI-RD were in accord with that of traditional fluorescent scans.In particular,we label-freely detected the washed mouse-IgG microarray with a series of concentrations and acquired a linear correlation between OI-RD intensities and the protein concentrations in logarithmic coordinates.The detection sensitivity of OI-RD can reach 14 fg.These experimental results suggest that the OI-RD method has potential applications in proteomics and clinical diagnosis.展开更多
This manuscript reports on the first two-photon,label-free,metabolic imaging of biological tissues in vivo at histological resolution on an extremely compact,fiber-optic endomicroscopy platform.This system provides ne...This manuscript reports on the first two-photon,label-free,metabolic imaging of biological tissues in vivo at histological resolution on an extremely compact,fiber-optic endomicroscopy platform.This system provides new opportunities for performing noninvasive and functional histological imaging of internal organs in vivo,in situ and in real time.As a routine clinical procedure,traditional histology has made significant impacts on medicine.However,the procedure is invasive and time consuming,suffers random sampling errors,and cannot provide in vivo functional information.The technology reported here features an extremely compact and flexible fiber-optic probe~2 mm in diameter,enabling direct access to internal organs.Unprecedented two-photon imaging quality comparable to a large bench-top laser scanning microscope was achieved through technological innovations in double-clad fiber optics and miniature objective lenses(among many others).In addition to real-time label-free visualization of biological tissues in situ with subcellular histological detail,we demonstrated for the first time in vivo two-photon endomicroscopic metabolic imaging on a functioning mouse kidney model.Such breakthroughs in nonlinear endoscopic imaging capability present numerous promising opportunities for paradigm-shifting applications in both clinical diagnosis and basic research.展开更多
The achievement of functional nanomodules for subcellular label-free measurement has long been pursued in order to fully understand cellular functions.Here,a compact label-free nanosensor based on a fiber taper and zi...The achievement of functional nanomodules for subcellular label-free measurement has long been pursued in order to fully understand cellular functions.Here,a compact label-free nanosensor based on a fiber taper and zinc oxide nanogratings is designed and applied for the early monitoring of apoptosis in individual living cells.Because of its nanoscale dimensions,mechanical flexibility,and minimal cytotoxicity to cells,the sensing module can be loaded in cells for long term in situ tracking with high sensitivity.A gradual increase in the nuclear refractive index during the apoptosis process is observed,revealing the increase in molecular density and the decrease in cell volume.The strategy used in our study not only contributes to the understanding of internal environmental variations during cellular apoptosis but also provides a new platform for nonfluorescent fiber devices for investigation of cellular events and understanding fundamental cell biochemical engineering.展开更多
Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect ...Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively d展开更多
Optical microscope is one of the most popular characterization techniques for general purposes in many fields. It is distinguishedfrom the vacuum or tip-based imaging techniques for its flexibility, low cost, and fast...Optical microscope is one of the most popular characterization techniques for general purposes in many fields. It is distinguishedfrom the vacuum or tip-based imaging techniques for its flexibility, low cost, and fast speed. However, its resolutionlimits the functionality of current optical imaging performance. While microspheres have been demonstrated forimproving the observation power of optical microscope, they are directly deposited on the sample surface and thus theapplications are greatly limited. We develop a remote-mode microsphere nano-imaging platform which can scan freelyand in real-time across the sample surfaces. It greatly increases the observation power and successfully characterizesvarious practical samples with the smallest feature size down to 23 nm. This method offers many unique advantages,such as enabling the detection to be non-invasive, dynamic, real-time, and label-free, as well as leading to more functionalitiesin ambient air and liquid environments, which extends the nano-scale observation power to a broad scope inour life.展开更多
A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A（OTA） was developed using the specific aptamer of OTA（OSA） as recognition dement, an aggregation-induced emission（AIE） m...A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A（OTA） was developed using the specific aptamer of OTA（OSA） as recognition dement, an aggregation-induced emission（AIE） molecule（a 9,10-distyrylanthracene with two ammonium groups, DSAI） as a fluorescent probe, and graphene oxide（GO） as a quencher. In the absence of OTA, the AIE probe DSAI and OSA complex（DSAI/OSA） is adsorbed on the GO surface, and the fluorescence of DSAI will be quenched efficiently via the fluorescence resonance energy transfer（FRET） from DSAI to GO. Upon the OTA addition, a more stable complex（OSA-OTA） is formed and released from GO. Meanwhile, DSAI and OSA-OTA can form a new complex（DSAI/OSA-OTA）, then the fluorescent signal of DSAI recovers gradually. Therefore, by introducing GO and DSAI, the fluorescence signal of DSAI can be easily turned from ＂off＂ to ＂on＂ after the addition of OTA, and the ultrasensitive detection of OTA by monitoring the change of the fluorescence signal of DSAI can be readily realized. The detection limit of the assay can reach 0.324 nmol/L with a linear detection range of 10-200 nmol/L. And the aptasensor exhibits high selectivity for OTA against other analogues. Moreover, it has been successfully applied for the detection of OTA in red wine samples.展开更多
In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, wh...In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T（Th T） as a signal transduction probe（STP）. In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to Th T which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.展开更多
Xiao-Xu-Ming decoction has been widely used to treat stroke and sequelae of stroke. We have previously shown that the active fractions of Xiao-Xu-Ming decoction attenuate cerebral ischemic injury. However, the global ...Xiao-Xu-Ming decoction has been widely used to treat stroke and sequelae of stroke. We have previously shown that the active fractions of Xiao-Xu-Ming decoction attenuate cerebral ischemic injury. However, the global protein profile and signaling conduction pathways regulated by Xiao-Xu-Ming decoction are still unclear. This study established a two-vessel occlusion rat model by bilateral common carotid artery occlusion. Rats were intragastrically administered 50 or 150 mg/kg Xiao-Xu-Ming decoction for 4 consecutive weeks. Learning and memory abilities were measured with Morris water maze. Motor ability was detected with prehensile test. Coordination ability was examined using the inclined screen test. Neuronal plasticity was observed by immunofluorescent staining. Differentially expressed proteins of rat hippocampus were analyzed by label-free quantitative proteomics. Real time-polymerase chain reaction and western blot assay were used to identify the changes in proteins. Results showed that Xiao-Xu-Ming decoction dramatically alleviated learning and memory deficits, and motor and coordination dysfunction, and increased the expression of microtubule-associated protein 2. Xiao-Xu-Ming decoction extract remarkably decreased 13 upregulated proteins and increased 39 downregulated proteins. The regulated proteins were mainly involved in oxidation reduction process, intracellular signaling cascade process, and protein catabolic process. The signaling pathways were mainly involved in ubiquitin mediated proteolysis and the phosphatidylinositol signaling system. Furthermore, there was an interaction among Rab2 a, Ptpn1, Ppm1 e, Cdk18, Gorasp2, Eps15, Capza2, Syngap1 and Mt-nd1. Protein analyses confirmed the changes in expression of MTND1. The current findings provide new insights into the molecular mechanisms of Xiao-Xu-Ming decoction extract's effects on chronic cerebral hypoperfusion.展开更多
文摘Single-molecule detection is one of the fundamental challenges of modern biology.Such experiments often use labels that can be expensive,difficult to produce,and for small analytes,might perturb the molecular events being studied.Analyte size plays an important role in determining detectability.Here we use laser-frequency locking in the context of sensing to improve the signal-to-noise ratio of microtoroid optical resonators to the extent that single nanoparticles 2.5 nm in radius,and 15.5 kDa molecules are detected in aqueous solution,thereby bringing these detectors to the size limits needed for detecting the key macromolecules of the cell.Our results,covering several orders of magnitude of particle radius(100 nm to 2 nm),agree with the‘reactive’model prediction for the frequency shift of the resonator upon particle binding.This confirms that the main contribution of the frequency shift for the resonator upon particle binding is an increase in the effective path length due to part of the evanescent field coupling into the adsorbed particle.We anticipate that our results will enable many applications,including more sensitive medical diagnostics and fundamental studies of single receptor–ligand and protein–protein interactions in real time.
基金supported by the National Basic Research Program of China (Grant No. 2007CB935700)
文摘The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that OI-RD is a label-free method which can not only distinguish the concentration difference of oligonucleotides before and after the hybridization but also detect the hybridization of short oligonucleotides. At present the OI-RD method can detect 0.39 μmol/L 20-base oligonucleotide or less. These results suggest that the OI-RD method is a promising and potential technique for label-free detection of biological microarrays.
基金supported by the National Basic Research Program of China (Grant No. 2007CB935700)
文摘Mouse-Immunoglobulin G(mouse-IgG) with different concentrations in a range from 1000 to 0.0128 μg/mL and a specific hybridization with goat anti-mouse IgG were detected successfully by using an oblique-incidence reflectivity difference(OI-RD) method.Two detection signals,consisting of an imaginary part(Im{Δp-Δs}) and a real part(Re{Δp-Δs}) of OI-RD,were obtained simultaneously.The detection results of hybridization by OI-RD were in accord with that of traditional fluorescent scans.In particular,we label-freely detected the washed mouse-IgG microarray with a series of concentrations and acquired a linear correlation between OI-RD intensities and the protein concentrations in logarithmic coordinates.The detection sensitivity of OI-RD can reach 14 fg.These experimental results suggest that the OI-RD method has potential applications in proteomics and clinical diagnosis.
基金supported by the National Institutes of Health under a Grant R01CA153023(XL)the National Science Foundation under Grant CBET-1430040(XL)the Individual Biomedical Research Award(XL)from The Hartwell Foundation.
文摘This manuscript reports on the first two-photon,label-free,metabolic imaging of biological tissues in vivo at histological resolution on an extremely compact,fiber-optic endomicroscopy platform.This system provides new opportunities for performing noninvasive and functional histological imaging of internal organs in vivo,in situ and in real time.As a routine clinical procedure,traditional histology has made significant impacts on medicine.However,the procedure is invasive and time consuming,suffers random sampling errors,and cannot provide in vivo functional information.The technology reported here features an extremely compact and flexible fiber-optic probe~2 mm in diameter,enabling direct access to internal organs.Unprecedented two-photon imaging quality comparable to a large bench-top laser scanning microscope was achieved through technological innovations in double-clad fiber optics and miniature objective lenses(among many others).In addition to real-time label-free visualization of biological tissues in situ with subcellular histological detail,we demonstrated for the first time in vivo two-photon endomicroscopic metabolic imaging on a functioning mouse kidney model.Such breakthroughs in nonlinear endoscopic imaging capability present numerous promising opportunities for paradigm-shifting applications in both clinical diagnosis and basic research.
基金sponsored by the National Natural Science Foundation of China (Nos. 61925502 and 62135007)
文摘The achievement of functional nanomodules for subcellular label-free measurement has long been pursued in order to fully understand cellular functions.Here,a compact label-free nanosensor based on a fiber taper and zinc oxide nanogratings is designed and applied for the early monitoring of apoptosis in individual living cells.Because of its nanoscale dimensions,mechanical flexibility,and minimal cytotoxicity to cells,the sensing module can be loaded in cells for long term in situ tracking with high sensitivity.A gradual increase in the nuclear refractive index during the apoptosis process is observed,revealing the increase in molecular density and the decrease in cell volume.The strategy used in our study not only contributes to the understanding of internal environmental variations during cellular apoptosis but also provides a new platform for nonfluorescent fiber devices for investigation of cellular events and understanding fundamental cell biochemical engineering.
基金supported by the grants from Shanghai Shuguang Plan Project,No.18SG15(to SC)Shanghai Outstanding Young Scholars Project+2 种基金Shanghai Talent Development Project,No.2019044(to SC)Medical-engineering cross fund of Shanghai Jiao Tong University,No.YG2022QN009(to QZ)the National Natural Science Foundation of China,No.82201558(to QZ)。
文摘Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively d
文摘Optical microscope is one of the most popular characterization techniques for general purposes in many fields. It is distinguishedfrom the vacuum or tip-based imaging techniques for its flexibility, low cost, and fast speed. However, its resolutionlimits the functionality of current optical imaging performance. While microspheres have been demonstrated forimproving the observation power of optical microscope, they are directly deposited on the sample surface and thus theapplications are greatly limited. We develop a remote-mode microsphere nano-imaging platform which can scan freelyand in real-time across the sample surfaces. It greatly increases the observation power and successfully characterizesvarious practical samples with the smallest feature size down to 23 nm. This method offers many unique advantages,such as enabling the detection to be non-invasive, dynamic, real-time, and label-free, as well as leading to more functionalitiesin ambient air and liquid environments, which extends the nano-scale observation power to a broad scope inour life.
文摘A label-free fluorescent aptasensor for specific and ultrasensitive monitoring ochratoxin A（OTA） was developed using the specific aptamer of OTA（OSA） as recognition dement, an aggregation-induced emission（AIE） molecule（a 9,10-distyrylanthracene with two ammonium groups, DSAI） as a fluorescent probe, and graphene oxide（GO） as a quencher. In the absence of OTA, the AIE probe DSAI and OSA complex（DSAI/OSA） is adsorbed on the GO surface, and the fluorescence of DSAI will be quenched efficiently via the fluorescence resonance energy transfer（FRET） from DSAI to GO. Upon the OTA addition, a more stable complex（OSA-OTA） is formed and released from GO. Meanwhile, DSAI and OSA-OTA can form a new complex（DSAI/OSA-OTA）, then the fluorescent signal of DSAI recovers gradually. Therefore, by introducing GO and DSAI, the fluorescence signal of DSAI can be easily turned from ＂off＂ to ＂on＂ after the addition of OTA, and the ultrasensitive detection of OTA by monitoring the change of the fluorescence signal of DSAI can be readily realized. The detection limit of the assay can reach 0.324 nmol/L with a linear detection range of 10-200 nmol/L. And the aptasensor exhibits high selectivity for OTA against other analogues. Moreover, it has been successfully applied for the detection of OTA in red wine samples.
基金supported by National Natural Science Foundation of China (Nos. 21205142, 31370104)The Research Innovation Program for Graduates of Central South University (No. 2016zzts580)
文摘In this assay, a label-free fluorescent sensing platform based on triple-helix molecular switch（THMS） and G-quadruplex was developed for the detection of tetracycline. We demonstrated this approach by using THMS, which consists of a central section with a shortened 8-mer aptamer sequence with high affinity to tetracycline and flanked by two arm segments. G-rich oligonucleotide can specifically bind to thioflavin T（Th T） as a signal transduction probe（STP）. In the absence of tetracycline, THMS remains stable, the fluorescence of background is low. By the addition of target tetracycline, the aptamer-target binding results in the formation of a structured aptamer-target complex, which disassembles the THMS and releases the STP. The free STP self-assembles into G-quadruplex and specifically binds to Th T which generates a obvious fluorescence enhancement. Using the triple-helix molecular switch, the developed aptamer-based fluorescent sensing platform showed a linear relationship with the concentration of tetracycline ranging from 0.2 to 20.0 nmol/L. The detection limit of tetracycline was determined to be970.0 pmol/L. The assay avoids complicated modifications or chemical labeling, making it simple and cost-effective. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.
基金supported in part by the National Natural Science Foundation of China,No.81473383(to YHW)the Significant New-Drugs Creation of Science and Technology Major Projects in China,No.2018ZX09711001-003-019(to YHW)the Innovation Fund for Graduate of Beijing Union Medical College of China,No.2017-1007-02(to XC)
文摘Xiao-Xu-Ming decoction has been widely used to treat stroke and sequelae of stroke. We have previously shown that the active fractions of Xiao-Xu-Ming decoction attenuate cerebral ischemic injury. However, the global protein profile and signaling conduction pathways regulated by Xiao-Xu-Ming decoction are still unclear. This study established a two-vessel occlusion rat model by bilateral common carotid artery occlusion. Rats were intragastrically administered 50 or 150 mg/kg Xiao-Xu-Ming decoction for 4 consecutive weeks. Learning and memory abilities were measured with Morris water maze. Motor ability was detected with prehensile test. Coordination ability was examined using the inclined screen test. Neuronal plasticity was observed by immunofluorescent staining. Differentially expressed proteins of rat hippocampus were analyzed by label-free quantitative proteomics. Real time-polymerase chain reaction and western blot assay were used to identify the changes in proteins. Results showed that Xiao-Xu-Ming decoction dramatically alleviated learning and memory deficits, and motor and coordination dysfunction, and increased the expression of microtubule-associated protein 2. Xiao-Xu-Ming decoction extract remarkably decreased 13 upregulated proteins and increased 39 downregulated proteins. The regulated proteins were mainly involved in oxidation reduction process, intracellular signaling cascade process, and protein catabolic process. The signaling pathways were mainly involved in ubiquitin mediated proteolysis and the phosphatidylinositol signaling system. Furthermore, there was an interaction among Rab2 a, Ptpn1, Ppm1 e, Cdk18, Gorasp2, Eps15, Capza2, Syngap1 and Mt-nd1. Protein analyses confirmed the changes in expression of MTND1. The current findings provide new insights into the molecular mechanisms of Xiao-Xu-Ming decoction extract's effects on chronic cerebral hypoperfusion.
