Advances in single-cell proteomics have enabled the investigation of the distinctive proteomic makeup of individual cells, significantly impacting biomedical research. However, most existing approaches involve complex sample preparation workflows and are sensitive to potential sample loss, which limits their applicability. In this paper, we reported an advanced workflow for easy-to-use single-cell proteome analysis using an in situ simplified strategy, named “in situ simplified single-cell proteomics (IS-SCP)”. This workflow was developed following a comprehensive evaluation of reagent mix, volume, and reaction conditions, notably including the utilization of a cleavable surfactant, n-decyl-disulfide-β-D-maltoside (DSSM). In comparison to previous workflows that require multiple steps in sample preparation, the IS-SCP workflow simplifies the single-cell proteome pretreatment to a single step of adding single-cell samples into a mixed reagent, which increases the repeatability and depth of single-cell proteome analysis. The IS-SCP workflow was applied to the proteomic analysis of single mammalian tumor cells, specifically HeLa and A549 cells, resulting in the quantification of an average of 3021 and 3289 protein groups, respectively. These results showed the potential of this workflow for investigating cellular heterogeneity at a deep single-cell level.
{"title":"IS-SCP: enhanced single-cell proteomics using an in situ simplified strategy","authors":"Zhuo Yang, Yi-Rong Jiang, Qin-Qin Xu, Jian-Bo Chen, Jian-Zhang Pan, Xin Di, Qun Fang","doi":"10.1039/d5an00149h","DOIUrl":"https://doi.org/10.1039/d5an00149h","url":null,"abstract":"Advances in single-cell proteomics have enabled the investigation of the distinctive proteomic makeup of individual cells, significantly impacting biomedical research. However, most existing approaches involve complex sample preparation workflows and are sensitive to potential sample loss, which limits their applicability. In this paper, we reported an advanced workflow for easy-to-use single-cell proteome analysis using an <em>in situ</em> simplified strategy, named “<em>in situ</em> simplified single-cell proteomics (IS-SCP)”. This workflow was developed following a comprehensive evaluation of reagent mix, volume, and reaction conditions, notably including the utilization of a cleavable surfactant, <em>n</em>-decyl-disulfide-β-<small>D</small>-maltoside (DSSM). In comparison to previous workflows that require multiple steps in sample preparation, the IS-SCP workflow simplifies the single-cell proteome pretreatment to a single step of adding single-cell samples into a mixed reagent, which increases the repeatability and depth of single-cell proteome analysis. The IS-SCP workflow was applied to the proteomic analysis of single mammalian tumor cells, specifically HeLa and A549 cells, resulting in the quantification of an average of 3021 and 3289 protein groups, respectively. These results showed the potential of this workflow for investigating cellular heterogeneity at a deep single-cell level.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"34 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zhuoer Lu, Xiaoxiao Feng, Bin Fu, Xiaojin Mo, Ting Zhang, Liming Wei, Zhonghua Li, Haojie Lu
Echinococcosis is a serious and potentially fatal parasitic zoonosis, which can be divided into two subtypes in humans including cystic echinococcosis (CE) and alveolar echinococcosis (AE). It poses a great threat to patients’ lives, making timely diagnosis and subtype discrimination crucial. AE is easily confused with hepatocellular carcinoma (HCC) due to their highly similar features, so differential diagnosis is also imperative. In this work, the galactosylation level of serum IgG was analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) in a cohort comprised of patients, including 100 diagnosed with CE, 27 with AE and 29 with HCC. The relative quantification of IgG digalactosyl (G2), monogalactosyl (G1), and agalactosylated (G0) N-glycans with the formula G0/(G1+G2*2) (IgG Gal-ratio) was obtained and found to effectively distinguish between echinococcosis patients and healthy controls, CE and AE, respectively. Meanwhile, the IgG Gal-ratio was evidently related to different types of CE (from CE1 to CE5) and the follow-up CE disease progress. Furthermore, the IgG Gal-ratio shown the potential differential diagnosis of AE and HCC. Thus, the results demonstrate that IgG Gal-ratio has the potential to be a biomarker for diagnosis and discrimination of echinococcosis, which also needs to be verified in further study.
{"title":"Serum IgG galactosylation as a potential biomarker for diagnosis of echinococcosis","authors":"Zhuoer Lu, Xiaoxiao Feng, Bin Fu, Xiaojin Mo, Ting Zhang, Liming Wei, Zhonghua Li, Haojie Lu","doi":"10.1039/d4an01578a","DOIUrl":"https://doi.org/10.1039/d4an01578a","url":null,"abstract":"Echinococcosis is a serious and potentially fatal parasitic zoonosis, which can be divided into two subtypes in humans including cystic echinococcosis (CE) and alveolar echinococcosis (AE). It poses a great threat to patients’ lives, making timely diagnosis and subtype discrimination crucial. AE is easily confused with hepatocellular carcinoma (HCC) due to their highly similar features, so differential diagnosis is also imperative. In this work, the galactosylation level of serum IgG was analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) in a cohort comprised of patients, including 100 diagnosed with CE, 27 with AE and 29 with HCC. The relative quantification of IgG digalactosyl (G2), monogalactosyl (G1), and agalactosylated (G0) N-glycans with the formula G0/(G1+G2*2) (IgG Gal-ratio) was obtained and found to effectively distinguish between echinococcosis patients and healthy controls, CE and AE, respectively. Meanwhile, the IgG Gal-ratio was evidently related to different types of CE (from CE1 to CE5) and the follow-up CE disease progress. Furthermore, the IgG Gal-ratio shown the potential differential diagnosis of AE and HCC. Thus, the results demonstrate that IgG Gal-ratio has the potential to be a biomarker for diagnosis and discrimination of echinococcosis, which also needs to be verified in further study.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"94 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Emeline Hanozin, Conner C. Harper, Jacob S. Jordan, Zachary M. Miller, Evan R. Williams
Charge detection mass spectrometry (CDMS) measurements of individual ions using either Orbitrap or electrostatic ion trap-based instruments have heretofore been performed under ultra-high vacuum conditions (10−9 Torr or lower). The rationale for this expensive and often cumbersome requirement is that these measurements need to be performed in an environment where collisions with background gas do not adversely affect the measurements. Here, the use of an electrostatic trap that accepts a broad range of ion energies and a dynamic ion signal analysis method enables accurate CDMS mass measurements at pressures as high as 1 × 10−6 Torr, multiple orders of magnitude higher than previously demonstrated. Consistent, accurate masses were obtained for pentameric antibody complexes (∼800 kDa), adeno-associated viruses (∼4.8 MDa), and both ∼50 and ∼100 nm diameter polystyrene nanoparticles (∼35 MDa and ∼330 MDa, respectively) at pressures ranging from 1 × 10−8 Torr to 1 × 10−6 Torr. The relationships between ion mass, trap pressure, ion lifetimes, individual ion energies and survival rates were investigated over a 1 s trapping period. Larger ions are more robust to higher pressures. While the trapping lifetimes of smaller ions decrease with increasing pressure, enough survive long enough for accurate mass measurements to be made. Some ions are lost because collisional dampening decreases their energies below the minimum stability threshold of the trap, but others with sufficient energy are still lost due to collision-induced scattering that moves the ions too far from the central trapping axis.
{"title":"High performance charge detection mass spectrometry without ultra-high vacuum","authors":"Emeline Hanozin, Conner C. Harper, Jacob S. Jordan, Zachary M. Miller, Evan R. Williams","doi":"10.1039/d5an00019j","DOIUrl":"https://doi.org/10.1039/d5an00019j","url":null,"abstract":"Charge detection mass spectrometry (CDMS) measurements of individual ions using either Orbitrap or electrostatic ion trap-based instruments have heretofore been performed under ultra-high vacuum conditions (10<small><sup>−9</sup></small> Torr or lower). The rationale for this expensive and often cumbersome requirement is that these measurements need to be performed in an environment where collisions with background gas do not adversely affect the measurements. Here, the use of an electrostatic trap that accepts a broad range of ion energies and a dynamic ion signal analysis method enables accurate CDMS mass measurements at pressures as high as 1 × 10<small><sup>−6</sup></small> Torr, multiple orders of magnitude higher than previously demonstrated. Consistent, accurate masses were obtained for pentameric antibody complexes (∼800 kDa), adeno-associated viruses (∼4.8 MDa), and both ∼50 and ∼100 nm diameter polystyrene nanoparticles (∼35 MDa and ∼330 MDa, respectively) at pressures ranging from 1 × 10<small><sup>−8</sup></small> Torr to 1 × 10<small><sup>−6</sup></small> Torr. The relationships between ion mass, trap pressure, ion lifetimes, individual ion energies and survival rates were investigated over a 1 s trapping period. Larger ions are more robust to higher pressures. While the trapping lifetimes of smaller ions decrease with increasing pressure, enough survive long enough for accurate mass measurements to be made. Some ions are lost because collisional dampening decreases their energies below the minimum stability threshold of the trap, but others with sufficient energy are still lost due to collision-induced scattering that moves the ions too far from the central trapping axis.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"93 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677879","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vancomycin (Van) is the preferred drug for clinically treating severe infections caused by Gram-positive bacteria, especially in intensive care unit (ICU). However, due to safety concerns, strict management and administration of Van are required in clinical practice. Hence, there is a need to develop a rapid and accurate method for determining the concentration of Van. In this study, a cyanostilbene-based fluorescent probe, Cy-KAA, is designed for the rapid, highly sensitive and selective detection of Van. In the probe, L-Lys-D-Ala-D-Ala polypeptide moiety can interact with Van, resulting in a rapid fluorescence response that reaches a plateau within 2 minutes. Cy-KAA can be employed for the detection of Van in clinical ICU samples, demonstrating strong correlation with the traditional HPLC method (consistency up to 0.99). These findings highlight the high accuracy and potential utility of Cy-KAA as a powerful tool for real-time monitoring of Van levels in clinical practice, thereby providing valuable guidance for precise drug administration.
{"title":"Rapid and Sensitive Determination of Vancomycin by AIE-Active Fluorescent Probe for Clinical Monitoring","authors":"Yige Lin, yujie Wang, Fang Fan, Guoyue Shi","doi":"10.1039/d4an01588f","DOIUrl":"https://doi.org/10.1039/d4an01588f","url":null,"abstract":"Vancomycin (Van) is the preferred drug for clinically treating severe infections caused by Gram-positive bacteria, especially in intensive care unit (ICU). However, due to safety concerns, strict management and administration of Van are required in clinical practice. Hence, there is a need to develop a rapid and accurate method for determining the concentration of Van. In this study, a cyanostilbene-based fluorescent probe, Cy-KAA, is designed for the rapid, highly sensitive and selective detection of Van. In the probe, L-Lys-D-Ala-D-Ala polypeptide moiety can interact with Van, resulting in a rapid fluorescence response that reaches a plateau within 2 minutes. Cy-KAA can be employed for the detection of Van in clinical ICU samples, demonstrating strong correlation with the traditional HPLC method (consistency up to 0.99). These findings highlight the high accuracy and potential utility of Cy-KAA as a powerful tool for real-time monitoring of Van levels in clinical practice, thereby providing valuable guidance for precise drug administration.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"27 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143672407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yu Liu, Gongli Wei, Yutian Jiao, Baocai Xu, Li Zhao
A highly sensitive sensing system for heparin has been developed using a synthetic fluorescent dye, diphenylalanine-modified dansyl (FF-Dns), in combination with the cationic surfactant octadecyltrimethylammonium chloride (OTAC). At low concentrations, FF-Dns and OTAC self-assemble, leading to a reduction in the fluorescence intensity of the dansyl group. However, upon the introduction of heparin, the electrostatic binding between OTAC and heparin disrupts the self-assembly, effectively releasing FF-Dns and restoring its fluorescence. This system shows exceptional selectivity for heparin over a range of potential interfering substances. The detection limit for heparin was determined to be 31.7 ng/mL, with a linear response range of 0 to 4 μg/mL. In addition, a “turn-on” fluorescence-based assay platform using FF-Dns/OTAC was successfully established in 0.2% serum with a detection limit of 30.57 ng/mL and a linear span of 0 to 5 μg/mL. In addition, the FF-Dns/OTAC/heparin mixture was identified as a potential sensing platform for protamine, exploiting the strong binding affinity between protamine and heparin.
{"title":"Sensitive fluorescent detection of heparin based on the assembly of a cationic surfactant and an anionic dye","authors":"Yu Liu, Gongli Wei, Yutian Jiao, Baocai Xu, Li Zhao","doi":"10.1039/d5an00154d","DOIUrl":"https://doi.org/10.1039/d5an00154d","url":null,"abstract":"A highly sensitive sensing system for heparin has been developed using a synthetic fluorescent dye, diphenylalanine-modified dansyl (FF-Dns), in combination with the cationic surfactant octadecyltrimethylammonium chloride (OTAC). At low concentrations, FF-Dns and OTAC self-assemble, leading to a reduction in the fluorescence intensity of the dansyl group. However, upon the introduction of heparin, the electrostatic binding between OTAC and heparin disrupts the self-assembly, effectively releasing FF-Dns and restoring its fluorescence. This system shows exceptional selectivity for heparin over a range of potential interfering substances. The detection limit for heparin was determined to be 31.7 ng/mL, with a linear response range of 0 to 4 μg/mL. In addition, a “turn-on” fluorescence-based assay platform using FF-Dns/OTAC was successfully established in 0.2% serum with a detection limit of 30.57 ng/mL and a linear span of 0 to 5 μg/mL. In addition, the FF-Dns/OTAC/heparin mixture was identified as a potential sensing platform for protamine, exploiting the strong binding affinity between protamine and heparin.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"125 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143666773","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xueting wang, Xiaorong Sun, Zhongfang Hu, Guang-Li Wang
This study explores Bi₂WO₆ nanoflowers as novel haloperoxidase (HPO) mimetics and their application in analytical science, aiming to develop an efficient colorimetric method for methotrexate (MTX) detection. Bi₂WO₆ nanoflowers were synthesized via a modified hydrothermal method and exhibited bromoperoxidase- and iodoperoxidase-like activities, catalyzing the bromination of phenol red (PR) and iodination of thymol blue (TB). After optimizing reaction conditions, the kinetic parameters, including the Michaelis-Menten constant (Km) and maximum reaction velocity (Vmax), exceeded those of most of the reported HPO nanozymes. Investigation of the catalytic mechanism identified singlet oxygen (1O2) as a reactive intermediate. Leveraging the inhibitory effect of MTX on Bi₂WO₆-based nanozymes, a colorimetric assay for MTX was developed, demonstrating excellent detection performance in terms of a wide linear range and a low detection limit. Furthermore, the developed assay exhibited reliable performance in detecting actual samples. This study validates Bi₂WO₆ nanoflowers as efficient HPO nanozymes and provides a reliable approach for the rapid and simple detection of MTX.
{"title":"Colorimetric detection of methotrexate leveraging the halogen peroxidase-mimicking activity of Bi₂WO₆ nanoflowers","authors":"Xueting wang, Xiaorong Sun, Zhongfang Hu, Guang-Li Wang","doi":"10.1039/d5an00138b","DOIUrl":"https://doi.org/10.1039/d5an00138b","url":null,"abstract":"This study explores Bi₂WO₆ nanoflowers as novel haloperoxidase (HPO) mimetics and their application in analytical science, aiming to develop an efficient colorimetric method for methotrexate (MTX) detection. Bi₂WO₆ nanoflowers were synthesized via a modified hydrothermal method and exhibited bromoperoxidase- and iodoperoxidase-like activities, catalyzing the bromination of phenol red (PR) and iodination of thymol blue (TB). After optimizing reaction conditions, the kinetic parameters, including the Michaelis-Menten constant (Km) and maximum reaction velocity (Vmax), exceeded those of most of the reported HPO nanozymes. Investigation of the catalytic mechanism identified singlet oxygen (1O2) as a reactive intermediate. Leveraging the inhibitory effect of MTX on Bi₂WO₆-based nanozymes, a colorimetric assay for MTX was developed, demonstrating excellent detection performance in terms of a wide linear range and a low detection limit. Furthermore, the developed assay exhibited reliable performance in detecting actual samples. This study validates Bi₂WO₆ nanoflowers as efficient HPO nanozymes and provides a reliable approach for the rapid and simple detection of MTX.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"15 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143666774","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jie Wu, Fei Li, Jing-Wen Zhou, Hongmei Li, Zilong Wang, Xian-Ming Guo, Yue-Jiao Zhang, Lin Zhang, Pei Liang, Shisheng Zheng, Jian-Feng Li
Rapid and reliable detection of chemical warfare agents (CWAs) is essential for military defense and counter-terrorism operations. Although Raman spectroscopy provides a non-destructive method for on-site detection, existing methods are difficult to cope with complex spectral overlap and concentration changes when analyzing mixtures containing trace components and highly complex mixtures. Based on the idea of convolutional neural network and multi-layer perceptron, this study proposes a qualitative and quantitative analysis algorithm of Raman spectroscopy based on deep learning (RS-MLP). The feature reference library is built from pure substance spectral features, while multi-head attention adaptively captures mixture weights. The MLP-Mixer then performs hierarchical feature matching for qualitative identification and quantitative analysis. The recognition rate of spectral data for the four types of combinations used for validation reached 100%, with an average root mean square error (RMSE) of less than 0.473% for the concentration prediction of three components. Furthermore, the model exhibited robust performance even under conditions of highly overlapping spectra. At the same time, the interpretability of the model is also enhanced. The model has excellent accuracy and robustness in component identification and concentration identification in complex mixtures, and provides a practical solution for rapid and non-contact detection of persistent chemicals in complex environments.
{"title":"Raman Spectroscopy Algorithm Based on Convolutional Neural Network and Multilayer Perceptron: Qualitative and Quantitative Analysis of Chemical Warfare Agent Simulants","authors":"Jie Wu, Fei Li, Jing-Wen Zhou, Hongmei Li, Zilong Wang, Xian-Ming Guo, Yue-Jiao Zhang, Lin Zhang, Pei Liang, Shisheng Zheng, Jian-Feng Li","doi":"10.1039/d5an00075k","DOIUrl":"https://doi.org/10.1039/d5an00075k","url":null,"abstract":"Rapid and reliable detection of chemical warfare agents (CWAs) is essential for military defense and counter-terrorism operations. Although Raman spectroscopy provides a non-destructive method for on-site detection, existing methods are difficult to cope with complex spectral overlap and concentration changes when analyzing mixtures containing trace components and highly complex mixtures. Based on the idea of convolutional neural network and multi-layer perceptron, this study proposes a qualitative and quantitative analysis algorithm of Raman spectroscopy based on deep learning (RS-MLP). The feature reference library is built from pure substance spectral features, while multi-head attention adaptively captures mixture weights. The MLP-Mixer then performs hierarchical feature matching for qualitative identification and quantitative analysis. The recognition rate of spectral data for the four types of combinations used for validation reached 100%, with an average root mean square error (RMSE) of less than 0.473% for the concentration prediction of three components. Furthermore, the model exhibited robust performance even under conditions of highly overlapping spectra. At the same time, the interpretability of the model is also enhanced. The model has excellent accuracy and robustness in component identification and concentration identification in complex mixtures, and provides a practical solution for rapid and non-contact detection of persistent chemicals in complex environments.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"14 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143660885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
V. S. Manu, Marco Tonelli, Ann Bell Bailey, Alok Sharma, Tim S. Bugni, Gianluigi Veglia
Approximately 40% of bacterial and mammalian metabolites contain nitrogen-based chemical moieties such as amides, amines, and imines. The identification and quantification of these groups via 2D 1H-15N heteronuclear NMR spectroscopy have broadened the catalog of NMR-detected metabolites. However, these NMR experiments necessitate broadband ra-diofrequency (RF) pulses for inversion and refocusing operations to encompass the full range of 15N chemical shifts, a chal-lenge that becomes increasingly apparent at high and ultra-high magnetic fields. Here, we show that a newly AI-designed broadband 15N universal 180o pulse for both inversion and refocusing incorporated in the 2D 1H, 15N heteronuclear single quantum coherence (2D 1H-15N BB-HSQC) experiment significantly enhances spectral sensitivity. We demonstrate the ad-vantage of the new technique by analyzing the crude extract of Micromonospora sp. WMMC264, a microbial strain that produces siderophores for iron absorption from the environment. The implementation of the AI-designed pulse in the 2D 1H-15N BB-HSQC experiment will contribute to advancing the analysis of nitrogen-containing metabolites in biological fluids and cell extracts.
{"title":"Detection of 15N-labeled Metabolites in Microbial Extracts using AI-Designed Broadband pulses for 1H, 15N Heteronuclear NMR Spectroscopy","authors":"V. S. Manu, Marco Tonelli, Ann Bell Bailey, Alok Sharma, Tim S. Bugni, Gianluigi Veglia","doi":"10.1039/d5an00074b","DOIUrl":"https://doi.org/10.1039/d5an00074b","url":null,"abstract":"Approximately 40% of bacterial and mammalian metabolites contain nitrogen-based chemical moieties such as amides, amines, and imines. The identification and quantification of these groups via 2D 1H-15N heteronuclear NMR spectroscopy have broadened the catalog of NMR-detected metabolites. However, these NMR experiments necessitate broadband ra-diofrequency (RF) pulses for inversion and refocusing operations to encompass the full range of 15N chemical shifts, a chal-lenge that becomes increasingly apparent at high and ultra-high magnetic fields. Here, we show that a newly AI-designed broadband 15N universal 180o pulse for both inversion and refocusing incorporated in the 2D 1H, 15N heteronuclear single quantum coherence (2D 1H-15N BB-HSQC) experiment significantly enhances spectral sensitivity. We demonstrate the ad-vantage of the new technique by analyzing the crude extract of Micromonospora sp. WMMC264, a microbial strain that produces siderophores for iron absorption from the environment. The implementation of the AI-designed pulse in the 2D 1H-15N BB-HSQC experiment will contribute to advancing the analysis of nitrogen-containing metabolites in biological fluids and cell extracts.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"8 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143666775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Margot Sanchez, Thomas Paris, Anthony Martinez, Gaëtan Assemat, Serge Akoka
Quantitative NMR experiments, especially on low-abundance nuclei such as 13C, can be extremely time-consuming due to the various constraints to ensure the quality of the results: high number of scans and long recovery delay. We propose the combination of the DEFT pulse sequence and the R2D2 method, hereafter referred to as R2D3. The addition of the DEFT to the R2D2 method reduces the quantitative limitations imposed by partial saturation. The parameters influencing the accuracy were evaluated with simulations and the quantitative performance of R2D3 were assessed by observing the trueness and precision on three different samples. The effect of different processing - the number of added rows and apodization - are also discussed. A precision of 1% or less was obtained in almost all the cases, showing that the R2D3 approach can drastically decrease the experimental time, while retaining the key aspects of a quantitative experiment. A high time gain factor can be achieved, close to that of INEPT and without its drawbacks, when trueness is less critical than precision. The R2D3 method will particularly benefit qNMR applications based on observing heteronuclei and analysing large sample series.
{"title":"The R2D3 approach to fast quantitative NMR: maintaining accuracy and reducing experimental time","authors":"Margot Sanchez, Thomas Paris, Anthony Martinez, Gaëtan Assemat, Serge Akoka","doi":"10.1039/d4an01369g","DOIUrl":"https://doi.org/10.1039/d4an01369g","url":null,"abstract":"Quantitative NMR experiments, especially on low-abundance nuclei such as 13C, can be extremely time-consuming due to the various constraints to ensure the quality of the results: high number of scans and long recovery delay. We propose the combination of the DEFT pulse sequence and the R2D2 method, hereafter referred to as R2D3. The addition of the DEFT to the R2D2 method reduces the quantitative limitations imposed by partial saturation. The parameters influencing the accuracy were evaluated with simulations and the quantitative performance of R2D3 were assessed by observing the trueness and precision on three different samples. The effect of different processing - the number of added rows and apodization - are also discussed. A precision of 1% or less was obtained in almost all the cases, showing that the R2D3 approach can drastically decrease the experimental time, while retaining the key aspects of a quantitative experiment. A high time gain factor can be achieved, close to that of INEPT and without its drawbacks, when trueness is less critical than precision. The R2D3 method will particularly benefit qNMR applications based on observing heteronuclei and analysing large sample series.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"90 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143660883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sijia Yang, Lei Ren, Ningke Fan, Shuhao Wang, Bo Shen, Zhongmei Liu, Xinmin Li, Shijia Ding
microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNAs detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.
{"title":"CRISPR-Cas12a with split crRNA for the direct and sensitive detection of microRNA","authors":"Sijia Yang, Lei Ren, Ningke Fan, Shuhao Wang, Bo Shen, Zhongmei Liu, Xinmin Li, Shijia Ding","doi":"10.1039/d5an00142k","DOIUrl":"https://doi.org/10.1039/d5an00142k","url":null,"abstract":"microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNAs detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.","PeriodicalId":63,"journal":{"name":"Analyst","volume":"91 1","pages":""},"PeriodicalIF":4.2,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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