Pub Date : 2024-07-01DOI: 10.1021/acs.analchem.4c01270
Ruben Szabo, Gyongyi Gyemant, Cynthia Nagy, Melinda Andrasi, Attila Gaspar
As has recently been shown, Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more "native-like" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.
{"title":"Taylor-Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins.","authors":"Ruben Szabo, Gyongyi Gyemant, Cynthia Nagy, Melinda Andrasi, Attila Gaspar","doi":"10.1021/acs.analchem.4c01270","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01270","url":null,"abstract":"<p><p>As has recently been shown, Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more \"native-like\" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-30DOI: 10.1021/acs.analchem.3c05857
Jianli Liu, Yumian Li, Tailong Chen, Fa Zhang, Fan Xu
Single-molecule localization microscopy (SMLM) is a versatile tool for realizing nanoscale imaging with visible light and providing unprecedented opportunities to observe bioprocesses. The integration of machine learning with SMLM enhances data analysis by improving efficiency and accuracy. This tutorial aims to provide a comprehensive overview of the data analysis process and theoretical aspects of SMLM, while also highlighting the typical applications of machine learning in this field. By leveraging advanced analytical techniques, SMLM is becoming a powerful quantitative analysis tool for biological research.
{"title":"Machine Learning for Single-Molecule Localization Microscopy: From Data Analysis to Quantification.","authors":"Jianli Liu, Yumian Li, Tailong Chen, Fa Zhang, Fan Xu","doi":"10.1021/acs.analchem.3c05857","DOIUrl":"https://doi.org/10.1021/acs.analchem.3c05857","url":null,"abstract":"<p><p>Single-molecule localization microscopy (SMLM) is a versatile tool for realizing nanoscale imaging with visible light and providing unprecedented opportunities to observe bioprocesses. The integration of machine learning with SMLM enhances data analysis by improving efficiency and accuracy. This tutorial aims to provide a comprehensive overview of the data analysis process and theoretical aspects of SMLM, while also highlighting the typical applications of machine learning in this field. By leveraging advanced analytical techniques, SMLM is becoming a powerful quantitative analysis tool for biological research.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-30DOI: 10.1021/acs.analchem.4c00274
Laura Dhellemmes, Laurent Leclercq, Lisa Lichtenauer, Alisa Höchsmann, Michael Leitner, Andreas Ebner, Michel Martin, Christian Neusüß, Hervé Cottet
Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.
要提高毛细管电泳（CE）的分离效率，需要系统地研究电场（或溶质线速度）对板高的影响，以便更好地了解控制峰展的关键参数。即使是聚(二烯丙基二甲基氯化铵)（PDADMAC）/聚(苯乙烯磺酸钠)（PSS）连续多离子聚合物层（SMIL）涂层（可实现高效、可重复的蛋白质分离），板高也会随迁移速度的增加而增加，从而限制了高电场在 CE 中的使用。一般认为，溶质在毛细管壁上的吸附是峰值分散的主要来源，这也是板高增加的原因。然而，在相同条件下进行的泰勒分散分析和 CE 实验表明，其他现象也可能起作用。蛋白质吸附动力学缓慢，吸附位点较少，这已被确定为特定蛋白质峰值增宽的来源。表面电荷不均匀性也被确定为局部电渗波动对板高度的贡献。提出了一个模型，并将其应用于部分 PDADMAC/聚环氧乙烷毛细管涂层以及 PDADMAC/PSS SMIL 涂层。通过原子力显微镜的形貌和识别成像，确定了 PDADMAC/PSS SMIL 表面的粗糙度和电荷分布。
{"title":"Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.","authors":"Laura Dhellemmes, Laurent Leclercq, Lisa Lichtenauer, Alisa Höchsmann, Michael Leitner, Andreas Ebner, Michel Martin, Christian Neusüß, Hervé Cottet","doi":"10.1021/acs.analchem.4c00274","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c00274","url":null,"abstract":"<p><p>Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-29DOI: 10.1021/acs.analchem.4c00419
Simone Bjørstorp, Joan Malmstrøm
One of the most widely used techniques for the quantification of small interfering ribonucleic acid (siRNA) is the ultraviolet (UV) spectroscopy method. However, due to uncertainties in the extinction coefficient affecting the accuracy of the method and a sample preparation including several dilution steps, the purpose of this study was to explore the possibility of determining the content of siRNA by a platform method using quantitative 31P nuclear magnetic resonance (31P-qNMR) and the internal standard method. In this paper, acquisition time, selection of a suitable internal certified reference material, signal selection used for quantification, relaxation delay, and precision are discussed. In addition, the robustness of the method and the ability to apply this platform method to both drug substance (DS) and drug product samples is also discussed. Quantifications of siRNA determined by the 31P-qNMR platform method were on average 98.5%w/w when adjusting for the sodium and water contents. The data confirmed the applicability of 31P-qNMR in siRNA content determinations. The quantifications were compared to quantifications determined by the traditional UV spectroscopy method by F- and t-tests. The statistical tests showed that the platform 31P-qNMR method provided more accurate results (mass balance close to 100% w/w) compared to the traditional UV spectroscopy method when analyzing DS samples.
{"title":"Quantitative 31P NMR Spectroscopy Platform Method for the Assay of Oligonucleotides as Pure Drug Substances and in Drug Product Formulations Using the Internal Standard Method","authors":"Simone Bjørstorp, Joan Malmstrøm","doi":"10.1021/acs.analchem.4c00419","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c00419","url":null,"abstract":"One of the most widely used techniques for the quantification of small interfering ribonucleic acid (siRNA) is the ultraviolet (UV) spectroscopy method. However, due to uncertainties in the extinction coefficient affecting the accuracy of the method and a sample preparation including several dilution steps, the purpose of this study was to explore the possibility of determining the content of siRNA by a platform method using quantitative <sup>31</sup>P nuclear magnetic resonance (<sup>31</sup>P-qNMR) and the internal standard method. In this paper, acquisition time, selection of a suitable internal certified reference material, signal selection used for quantification, relaxation delay, and precision are discussed. In addition, the robustness of the method and the ability to apply this platform method to both drug substance (DS) and drug product samples is also discussed. Quantifications of siRNA determined by the <sup>31</sup>P-qNMR platform method were on average 98.5%w/w when adjusting for the sodium and water contents. The data confirmed the applicability of <sup>31</sup>P-qNMR in siRNA content determinations. The quantifications were compared to quantifications determined by the traditional UV spectroscopy method by <i>F</i>- and <i>t</i>-tests. The statistical tests showed that the platform <sup>31</sup>P-qNMR method provided more accurate results (mass balance close to 100% w/w) compared to the traditional UV spectroscopy method when analyzing DS samples.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand–protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep–Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1–2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from Salvia miltiorrhiza extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a KD value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.
{"title":"One-Step Synthesis and Oriented Immobilization of Strep-Tag II Fused PDGFRβ for Screening Intracellular Domain-Targeted Ligands","authors":"Chengliang Wang, Yanqiu Gu, Chun Chen, Yanting Li, Ling Li, Yifeng Chai, Zhengjin Jiang, Xiaofei Chen, Yongfang Yuan","doi":"10.1021/acs.analchem.4c02067","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c02067","url":null,"abstract":"Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand–protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep–Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1–2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from <i>Salvia miltiorrhiza</i> extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a <i>K</i><sub>D</sub> value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463612","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-29DOI: 10.1021/acs.analchem.3c05875
Mark I Grimes, Matthew Cheeks, Jennifer Smith, Fabio Zurlo, Mick D Mantle
Protein-based biopharmaceutical drugs, such as monoclonal antibodies, account for the majority of the best-selling drugs globally in recent years. For bioprocesses, key performance indicators are the concentration and aggregate level for the product being produced. In water NMR (wNMR), the use of the water transverse relaxation rate [R2(1H2O)] has been previously used to determine protein concentration and aggregate level; however, it cannot be used to separate between them without using an additional technique. This work shows that it is possible to "decouple" these two key characteristics by recording the water diffusion coefficient [D(1H2O)] in conjunction with R2(1H2O), even in the event of overlap in either D(1H2O) or R2(1H2O). This method is demonstrated on three different systems, following appropriate D(1H2O) or R2(1H2O) calibration data acquisition for a protein of interest. Our method highlights the potential use of benchtop NMR as an at-line process analytical technique.
{"title":"Decoupling Protein Concentration and Aggregate Content Using Diffusion and Water NMR.","authors":"Mark I Grimes, Matthew Cheeks, Jennifer Smith, Fabio Zurlo, Mick D Mantle","doi":"10.1021/acs.analchem.3c05875","DOIUrl":"https://doi.org/10.1021/acs.analchem.3c05875","url":null,"abstract":"<p><p>Protein-based biopharmaceutical drugs, such as monoclonal antibodies, account for the majority of the best-selling drugs globally in recent years. For bioprocesses, key performance indicators are the concentration and aggregate level for the product being produced. In water NMR (<i>w</i>NMR), the use of the water transverse relaxation rate [<i>R</i><sub>2</sub>(<sup>1</sup>H<sub>2</sub>O)] has been previously used to determine protein concentration and aggregate level; however, it cannot be used to separate between them without using an additional technique. This work shows that it is possible to \"decouple\" these two key characteristics by recording the water diffusion coefficient [<i>D</i>(<sup>1</sup>H<sub>2</sub>O)] in conjunction with <i>R</i><sub>2</sub>(<sup>1</sup>H<sub>2</sub>O), even in the event of overlap in either <i>D</i>(<sup>1</sup>H<sub>2</sub>O) or <i>R</i><sub>2</sub>(<sup>1</sup>H<sub>2</sub>O). This method is demonstrated on three different systems, following appropriate <i>D</i>(<sup>1</sup>H<sub>2</sub>O) or <i>R</i><sub>2</sub>(<sup>1</sup>H<sub>2</sub>O) calibration data acquisition for a protein of interest. Our method highlights the potential use of benchtop NMR as an at-line process analytical technique.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141464235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-29DOI: 10.1021/acs.analchem.4c01265
Mengdi Guo, Zhibin Yi, Huo Li, Yang Liu, Liping Ding, Sergey P. Babailov, Chunhong Xiong, Ganhui Huang, Jinsheng Zhang
Detecting harmful pathogens in food is not only a crucial aspect of food quality management but also an effective way to ensure public health. In this paper, a complete nuclear magnetic resonance biosensor based on a novel gadolinium (Gd)-targeting molecular probe was developed for the detection of Salmonella in milk. First, streptavidin was conjugated to the activated macromolecular polyaspartic acid (PASP) via an amide reaction to generate SA–PASP. Subsequently, the strong chelating and adsorption properties of PASP toward the lanthanide metal gadolinium ions were exploited to generate the magnetic complex (SA–PASP–Gd). Finally, the magnetic complex was linked to biotinylated antibodies to obtain the bioprobe and achieve the capture of Salmonella. Under optimal experimental conditions, the sensor we have constructed can achieve a rapid detection of Salmonella within 1.5 h, with a detection limit of 7.1 × 103 cfu mL–1.
{"title":"NMR Immunosensor Based on a Targeted Gadolinium Nanoprobe for Detecting Salmonella in Milk","authors":"Mengdi Guo, Zhibin Yi, Huo Li, Yang Liu, Liping Ding, Sergey P. Babailov, Chunhong Xiong, Ganhui Huang, Jinsheng Zhang","doi":"10.1021/acs.analchem.4c01265","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01265","url":null,"abstract":"Detecting harmful pathogens in food is not only a crucial aspect of food quality management but also an effective way to ensure public health. In this paper, a complete nuclear magnetic resonance biosensor based on a novel gadolinium (Gd)-targeting molecular probe was developed for the detection of Salmonella in milk. First, streptavidin was conjugated to the activated macromolecular polyaspartic acid (PASP) via an amide reaction to generate SA–PASP. Subsequently, the strong chelating and adsorption properties of PASP toward the lanthanide metal gadolinium ions were exploited to generate the magnetic complex (SA–PASP–Gd). Finally, the magnetic complex was linked to biotinylated antibodies to obtain the bioprobe and achieve the capture of Salmonella. Under optimal experimental conditions, the sensor we have constructed can achieve a rapid detection of Salmonella within 1.5 h, with a detection limit of 7.1 × 10<sup>3</sup> cfu mL<sup>–1</sup>.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-29DOI: 10.1021/acs.analchem.4c02489
Yutian Zhang, Can Hu, Yashi Yin, Kejing Ren, Yingsi He, Yanru Gao, Heyou Han, Chengzhou Zhu, Wenjing Wang
Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I0 – I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.
{"title":"CRISPR/Cas12a-Responsive Smart DNA Hydrogel for Sensitive Electrochemiluminescence Detection of the Huanglongbing Outer Membrane Protein Gene","authors":"Yutian Zhang, Can Hu, Yashi Yin, Kejing Ren, Yingsi He, Yanru Gao, Heyou Han, Chengzhou Zhu, Wenjing Wang","doi":"10.1021/acs.analchem.4c02489","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c02489","url":null,"abstract":"Citrus Huanglongbing (HLB) is known as the cancer of citrus, where <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the <i>C</i>Las outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the <i>trans</i>-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (<i>I</i><sub>0</sub> – <i>I</i>) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463629","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-28DOI: 10.1021/acs.analchem.4c02404
Yanxue Wei, Yueli Hu, Chengchao Zhang, Rui Liu, Yi Lv
DNA walking machines have achieved significant breakthroughs in areas such as biosensing, bioimaging, and early cancer diagnosis, facilitated by the self-assembly of DNA or its combination with other materials, such as magnetic beads and metal nanoparticles. However, current DNA walking machine strategies are constantly challenged by inadequate analytical sensitivity, while sophisticated signal amplification procedures are often indispensable. Single-particle inductively coupled plasma mass spectrometry (SP-ICPMS) provides superior sensitivity and can effectively discriminate between background noise and detected signals due to the large number of metal atoms in a nanoparticle and the concentrating effect of single nanoparticle detection. In this study, we present a novel approach utilizing single nanoparticle counting and duplex-specific nuclease (DSN)-assisted signal amplification to construct a 3D DNA walking machine for detecting the aggressive prostate cancer (PCa) biomarker miRNA-200c. The proposed strategy showed an improvement in sensitivity with a detection limit (LOD) of 0.93 pM (28 amol) and was successfully applied in human serum samples. To the best of our knowledge, this is the first report of the DNA walking machine with single nanoparticle counting study.
通过 DNA 的自组装或与磁珠和金属纳米粒子等其他材料的结合，DNA 步进机在生物传感、生物成像和早期癌症诊断等领域取得了重大突破。然而，目前的 DNA 步进机策略一直面临着分析灵敏度不足的挑战，而复杂的信号放大程序往往又是必不可少的。单颗粒电感耦合等离子体质谱法（SP-ICPMS）具有极高的灵敏度，由于纳米颗粒中含有大量金属原子，且单个纳米颗粒检测具有聚光效应，因此能有效区分背景噪声和检测到的信号。在这项研究中，我们提出了一种新方法，利用单纳米粒子计数和双工特异性核酸酶（DSN）辅助信号放大来构建三维 DNA 步行机，用于检测侵袭性前列腺癌（PCa）生物标记物 miRNA-200c。所提出的策略提高了灵敏度，检测限 (LOD) 为 0.93 pM（28 amol），并成功应用于人类血清样本。据我们所知，这是第一份关于 DNA 步行机与单纳米粒子计数研究的报告。
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Pub Date : 2024-06-28DOI: 10.1021/acs.analchem.4c01465
Jia Chen, Qi Liu, Yongchun Fu, Juan Xiang
Low-mass soluble β-amyloid peptide oligomers (LSAβOs) play a crucial role in the pathogenesis of Alzheimer’s disease. However, these oligomers exhibit heterogeneity in terms of structure, stability, and stoichiometry, and their abundance in biofluids is low, making accurate identification challenging. In this study, we developed a DNA nanocage-assisted method for selective sizing and sensitive quantification of LSAβOs in serum. Using LSAβO less than 10 kDa (LSAβO10kD) and less than 30 kDa (LSAβO30kD) as models, the size-matching rules between DNA nanocages and LSAβOs were investigated, and two appropriate nanocages were selected for the detection of two LSAβOs, respectively. Both nanocages were functionalized by encapsulating oligomer’s aptamer and a complementary sequence within their cavities. Once the LSAβO entered the corresponding nanocage cavity, the complementary sequence was released, triggering a hybridization chain reaction on an electrochemical sensing platform. The system achieved size-selective discrimination of LSAβO10kD with a linear range of 10–150 pM and LSAβO30kD with a linear range of 15–150 pM. Real sample testing confirmed the applicability of the method for blood-based diagnosis. The DNA nanocage-assisted electrochemical analysis platform provides an accurate, highly selective, and sensitive approach for oligomer analysis, which is significant for amyloid protein research and related disease diagnosis.
{"title":"DNA Nanocage-Assisted Size-Selective Recognition and Quantification toward Low-Mass Soluble β-Amyloid Oligomers","authors":"Jia Chen, Qi Liu, Yongchun Fu, Juan Xiang","doi":"10.1021/acs.analchem.4c01465","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01465","url":null,"abstract":"Low-mass soluble β-amyloid peptide oligomers (LSAβOs) play a crucial role in the pathogenesis of Alzheimer’s disease. However, these oligomers exhibit heterogeneity in terms of structure, stability, and stoichiometry, and their abundance in biofluids is low, making accurate identification challenging. In this study, we developed a DNA nanocage-assisted method for selective sizing and sensitive quantification of LSAβOs in serum. Using LSAβO less than 10 kDa (LSAβO<sub>10kD</sub>) and less than 30 kDa (LSAβO<sub>30kD</sub>) as models, the size-matching rules between DNA nanocages and LSAβOs were investigated, and two appropriate nanocages were selected for the detection of two LSAβOs, respectively. Both nanocages were functionalized by encapsulating oligomer’s aptamer and a complementary sequence within their cavities. Once the LSAβO entered the corresponding nanocage cavity, the complementary sequence was released, triggering a hybridization chain reaction on an electrochemical sensing platform. The system achieved size-selective discrimination of LSAβO<sub>10kD</sub> with a linear range of 10–150 pM and LSAβO<sub>30kD</sub> with a linear range of 15–150 pM. Real sample testing confirmed the applicability of the method for blood-based diagnosis. The DNA nanocage-assisted electrochemical analysis platform provides an accurate, highly selective, and sensitive approach for oligomer analysis, which is significant for amyloid protein research and related disease diagnosis.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463678","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"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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