Pub Date : 2024-06-27DOI: 10.1021/acs.analchem.4c01700
Xiaoling Zhang, Ning Hu, Yunjiao Wang, Yun Zhao, Deqiang Wang
Zero-depth interfacial nanopores, which are formed by two crossed nanoscale channels at their intersection interface, have been proposed to increase the spatial resolution of solid-state nanopores. However, research on zero-depth interfacial nanopores is still in its early stages. Although it has been shown that the current passing through an interfacial nanopore is largely independent of the membrane thickness, existing studies have not fully considered the impact of membrane thickness on other ion transport characteristics within these nanopores. In this paper, we investigate the electrokinetic ion transport phenomenon in the zero-depth interfacial nanopores, especially focusing on the influence of membrane thickness on the ion transport phenomenon. Our model incorporates the Poisson-Nernst-Planck equations and the Navier-Stokes equations, featuring a pH-regulated surface charge density. We find that when the thickness of the nanochannels is close to the interface size of the formed interfacial nanopore, the phenomenon of ion transport in the interfacial nanopore is similar to that in a conventional cylindrical nanopore. However, when the thickness of the nanochannels is much greater than the interface size of the formed interfacial nanopore, several distinct phenomena occur. The surface charge density on the inner walls of the interfacial nanopores has a small peak at the interface of the two crossing nanochannels, and the anion concentration changes greatly between the two nanochannels; that is, a much greater anion concentration forms in the nanochannel near the anode side than in the nanochannel near the cathode side. When the surface charge is nonzero, the electric field within the interfacial nanopore creates three extreme points, and the directions of the local electric fields are opposite at the ends of the membrane.
{"title":"Effect of Membrane Thickness on Ion Transport in pH-Regulated Zero-Depth Interfacial Nanopores.","authors":"Xiaoling Zhang, Ning Hu, Yunjiao Wang, Yun Zhao, Deqiang Wang","doi":"10.1021/acs.analchem.4c01700","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01700","url":null,"abstract":"<p><p>Zero-depth interfacial nanopores, which are formed by two crossed nanoscale channels at their intersection interface, have been proposed to increase the spatial resolution of solid-state nanopores. However, research on zero-depth interfacial nanopores is still in its early stages. Although it has been shown that the current passing through an interfacial nanopore is largely independent of the membrane thickness, existing studies have not fully considered the impact of membrane thickness on other ion transport characteristics within these nanopores. In this paper, we investigate the electrokinetic ion transport phenomenon in the zero-depth interfacial nanopores, especially focusing on the influence of membrane thickness on the ion transport phenomenon. Our model incorporates the Poisson-Nernst-Planck equations and the Navier-Stokes equations, featuring a pH-regulated surface charge density. We find that when the thickness of the nanochannels is close to the interface size of the formed interfacial nanopore, the phenomenon of ion transport in the interfacial nanopore is similar to that in a conventional cylindrical nanopore. However, when the thickness of the nanochannels is much greater than the interface size of the formed interfacial nanopore, several distinct phenomena occur. The surface charge density on the inner walls of the interfacial nanopores has a small peak at the interface of the two crossing nanochannels, and the anion concentration changes greatly between the two nanochannels; that is, a much greater anion concentration forms in the nanochannel near the anode side than in the nanochannel near the cathode side. When the surface charge is nonzero, the electric field within the interfacial nanopore creates three extreme points, and the directions of the local electric fields are opposite at the ends of the membrane.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453684","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 : 2024-06-27DOI: 10.1021/acs.analchem.4c01371
Felicia Auer, Andras Guttman
Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel–buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10–8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50–200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel–buffer system, as demonstrated in this paper.
{"title":"In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis","authors":"Felicia Auer, Andras Guttman","doi":"10.1021/acs.analchem.4c01371","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01371","url":null,"abstract":"Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel–buffer system, the oppositely migrating positively charged propidium ligand <i>in migratio</i> complexes with the SDS-proteins, therefore, supports <i>in situ</i> labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10<sup>–8</sup> M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50–200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel–buffer system, as demonstrated in this paper.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463634","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}
Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding stain for a bacterial viability assessment. It was found that live bacteria were able to exclude GelRed, which however could easily penetrate dead ones and be absorbed nonspecifically on the bacterial periplasm. Cations were used to reduce the nonspecific adsorption and greatly increase the red fluorescence ratio of dead to live bacteria. Combined with SYTO 9 (a membrane-permeable dye) for double-staining, a ratiometric fluorescent method was established. Using Escherichia coli O157:H7 as a bacteria model, the ratiometric fluorescent method can probe dead bacteria as low as 0.1%. A linear correlation between the ratiometric fluorescence and the theoretical ratio of dead bacteria was acquired, with a correlation coefficient R2 of 0.97. Advantages in sensitivity, accuracy, and safety of the GelRed/SYTO9-based ratiometric fluorescent method against traditional methods were demonstrated. The established method was successfully applied to the assessment of germicidal efficacy of different heat treatments. It was found that even 50 °C treatment could lead to the death of minor bacteria. The as-developed method has many potential applications in microbial researches, and we believe it could be expanded to the viability assessment of mammalian cells.
{"title":"Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing.","authors":"Shengbin He, Yajing Chen, Jingtong Wang, Jian Sun, Xinyi Zhang, Quanzhi Chen","doi":"10.1021/acs.analchem.4c01737","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01737","url":null,"abstract":"<p><p>Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding stain for a bacterial viability assessment. It was found that live bacteria were able to exclude GelRed, which however could easily penetrate dead ones and be absorbed nonspecifically on the bacterial periplasm. Cations were used to reduce the nonspecific adsorption and greatly increase the red fluorescence ratio of dead to live bacteria. Combined with SYTO 9 (a membrane-permeable dye) for double-staining, a ratiometric fluorescent method was established. Using <i>Escherichia coli</i> O157:H7 as a bacteria model, the ratiometric fluorescent method can probe dead bacteria as low as 0.1%. A linear correlation between the ratiometric fluorescence and the theoretical ratio of dead bacteria was acquired, with a correlation coefficient <i>R</i><sup>2</sup> of 0.97. Advantages in sensitivity, accuracy, and safety of the GelRed/SYTO9-based ratiometric fluorescent method against traditional methods were demonstrated. The established method was successfully applied to the assessment of germicidal efficacy of different heat treatments. It was found that even 50 °C treatment could lead to the death of minor bacteria. The as-developed method has many potential applications in microbial researches, and we believe it could be expanded to the viability assessment of mammalian cells.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453691","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 : 2024-06-27DOI: 10.1021/acs.analchem.4c00962
Gaihua Cao, Nannan Yang, Jun Yang, Jiali Li, Lin Wang, Fuping Nie, Danqun Huo, Changjun Hou
Lumpy skin disease virus (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·μL-1. The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the Capripoxvirus, such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.
{"title":"Label-Free and DNAzyme-Mediated Biosensor with a High Signal-to-Noise Ratio for a Lumpy Skin Disease Virus Assay.","authors":"Gaihua Cao, Nannan Yang, Jun Yang, Jiali Li, Lin Wang, Fuping Nie, Danqun Huo, Changjun Hou","doi":"10.1021/acs.analchem.4c00962","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c00962","url":null,"abstract":"<p><p><i>Lumpy skin disease virus</i> (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·μL<sup>-1</sup>. The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the <i>Capripoxvirus</i>, such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453688","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}
Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system’s quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.
{"title":"Internal Standard Addition System for Online Breath Analysis","authors":"Cedric Wüthrich, Timon Käser, Renato Zenobi, Stamatios Giannoukos","doi":"10.1021/acs.analchem.4c01924","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01924","url":null,"abstract":"Breath analysis with secondary electrospray ionization (SESI) coupled to mass spectrometry (MS) is a sensitive method for breath metabolomics. To enable quantitative assessments using SESI-MS, a system was developed to introduce controlled amounts of gases into breath samples and carry out standard addition experiments. The system combines gas standard generation through controlled evaporation, humidification, breath dilution, and standard injection with the help of mass-flow controllers. The system can also dilute breath, which affects the signal of the detected components. This response can be used to filter out contaminating compounds in an untargeted metabolomics workflow. The system’s quantitative capabilities have been shown through standard addition of pyridine and butyric acid into breath in real time. This system can improve the quality and robustness of breath data.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":7.4,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141463573","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}
Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.
{"title":"Fluorescence Lifetime Super-Resolution Imaging Unveil the Dynamic Relationship between Mitochondrial Membrane Potential and Cristae Structure Using the Förster Resonance Energy Transfer Strategy.","authors":"Fei Peng, Xiangnan Ai, Jing Sun, Xichuan Ge, Meiqi Li, Peng Xi, Baoxiang Gao","doi":"10.1021/acs.analchem.4c01905","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01905","url":null,"abstract":"<p><p>Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453686","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 : 2024-06-26DOI: 10.1021/acs.analchem.4c01353
Qingqing Zou, Qianqian Zhang, Bin Du, Hongqiang Wang, Xiaohai Yang, Qing Wang, Kemin Wang
Overexpression of receptor tyrosine kinases (RTKs) or binding to ligands can lead to the formation of specific unliganded and liganded RTK dimers, and these two RTK dimers are potential targets for preventing tumor metastasis. Traditional RTK dimer inhibitor analysis was mostly based on end point assays, which required cumbersome cell handling and behavior monitoring. There are still challenges in developing intuitive process-based analytical methods to study RTK dimer inhibitors, especially those used to visually distinguish between unliganded and liganded RTK dimer inhibitors. Herein, taking the mesenchymal-epithelial transition factor (MET) receptor, an intuitive method for evaluating MET inhibitors has been developed based on atomic force microscopy (AFM) lifetime analysis. The time interval between the start of the force and the bond break point was regarded as the bond lifetime, which could reflect the stability of the MET dimer. The results showed that there was a significant difference in the lifetime (τ) of unliganded MET dimers (τ1 = 207.87 ± 4.69 ms) and liganded MET dimers (τ2 = 330.58 ± 15.60 ms) induced by the hepatocyte growth factor, and aptamer SL1 could decrease τ1 and τ2, suggesting that SL1 could inhibit both unliganded and liganded MET dimers. However, heparin only decreased τ2, suggesting that it could inhibit only the liganded MET dimer. AFM-based lifetime analysis methods could monitor RTK dimer status rather than provide overall average results, allowing for intuitive process-based analysis and evaluation of RTK dimers and related inhibitors at the single-molecule level. This study provides a novel complementary strategy for simple and intuitive RTK inhibitor research.
受体酪氨酸激酶(RTK)的过度表达或与配体的结合会导致形成特定的未配体和配体RTK二聚体,而这两种RTK二聚体是防止肿瘤转移的潜在靶点。传统的 RTK 二聚体抑制剂分析大多基于终点检测,需要繁琐的细胞处理和行为监测。在开发直观的基于过程的分析方法来研究 RTK 二聚体抑制剂方面仍存在挑战,尤其是用于直观区分未配体和配体 RTK 二聚体抑制剂的方法。在此,我们以间充质-上皮转化因子(MET)受体为例,开发了一种基于原子力显微镜(AFM)寿命分析的直观方法来评估MET抑制剂。从受力开始到键断点之间的时间间隔被视为键寿命,它可以反映 MET 二聚体的稳定性。结果表明,在肝细胞生长因子的诱导下,未加载的 MET 二聚体(τ1 = 207.87 ± 4.69 ms)和加载的 MET 二聚体(τ2 = 330.58 ± 15.60 ms)的寿命(τ)存在显著差异。然而,肝素只能降低τ2,表明它只能抑制配位的 MET 二聚体。基于原子力显微镜的寿命分析方法可以监测 RTK 二聚体的状态,而不是提供总体平均结果,从而可以在单分子水平上对 RTK 二聚体和相关抑制剂进行直观的基于过程的分析和评估。这项研究为简单直观的 RTK 抑制剂研究提供了一种新颖的补充策略。
{"title":"Atomic Force Microscopy Lifetime Analysis: An Intuitive Method for Evaluating Receptor Tyrosine Kinase Dimer-Targeting Inhibitors.","authors":"Qingqing Zou, Qianqian Zhang, Bin Du, Hongqiang Wang, Xiaohai Yang, Qing Wang, Kemin Wang","doi":"10.1021/acs.analchem.4c01353","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01353","url":null,"abstract":"<p><p>Overexpression of receptor tyrosine kinases (RTKs) or binding to ligands can lead to the formation of specific unliganded and liganded RTK dimers, and these two RTK dimers are potential targets for preventing tumor metastasis. Traditional RTK dimer inhibitor analysis was mostly based on end point assays, which required cumbersome cell handling and behavior monitoring. There are still challenges in developing intuitive process-based analytical methods to study RTK dimer inhibitors, especially those used to visually distinguish between unliganded and liganded RTK dimer inhibitors. Herein, taking the mesenchymal-epithelial transition factor (MET) receptor, an intuitive method for evaluating MET inhibitors has been developed based on atomic force microscopy (AFM) lifetime analysis. The time interval between the start of the force and the bond break point was regarded as the bond lifetime, which could reflect the stability of the MET dimer. The results showed that there was a significant difference in the lifetime (τ) of unliganded MET dimers (τ<sub>1</sub> = 207.87 ± 4.69 ms) and liganded MET dimers (τ<sub>2</sub> = 330.58 ± 15.60 ms) induced by the hepatocyte growth factor, and aptamer SL1 could decrease τ<sub>1</sub> and τ<sub>2</sub>, suggesting that SL1 could inhibit both unliganded and liganded MET dimers. However, heparin only decreased τ<sub>2</sub>, suggesting that it could inhibit only the liganded MET dimer. AFM-based lifetime analysis methods could monitor RTK dimer status rather than provide overall average results, allowing for intuitive process-based analysis and evaluation of RTK dimers and related inhibitors at the single-molecule level. This study provides a novel complementary strategy for simple and intuitive RTK inhibitor research.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453683","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 : 2024-06-26DOI: 10.1021/acs.analchem.4c02273
Jinge Zhao, Limin Zhang, Jingtian Cao, Yao Yu, Bokai Ma, Yujiu Jiang, Junfeng Han, Weizhi Wang
Peptide self-assemblies could leverage their specificity, stability, biocompatibility, and electrochemical activity to create functionalized interfaces for molecular sensing and detection. However, the dynamics within these interfaces are complex, with competing forces, including those maintaining peptide structures, recognizing analytes, and facilitating signal transmission. Such competition could lead to nonspecific interference, compromising the detection sensitivity and accuracy. In this study, a series of peptides with precise structures and controllable electron transfer capabilities were designed. Through examining their stacking patterns, the interplay between the peptides' hierarchical structures, their ability to recognize targets, and their conductivity were clarified. Among these, the EP5 peptide assembly was identified for its ability to form controllable electronic tunnels facilitated by π-stacking induced β-sheets. EP5 could enhance the long-range conductivity, minimize nonspecific interference, and exhibit targeted recognition capabilities. Based on EP5, an electrochemical sensing interface toward the disease marker PD-L1 (programmed cell death ligand 1) was developed, suitable for both whole blood assay and in vivo companion diagnosis. It opens a new avenue for crafting electrochemical detection interfaces with specificity, sensitivity, and compatibility.
{"title":"Sequence-Prescribed β-Sheet for Enhanced Electron Tunneling: Boosting Interface Recognition and Electrochemical Measurement.","authors":"Jinge Zhao, Limin Zhang, Jingtian Cao, Yao Yu, Bokai Ma, Yujiu Jiang, Junfeng Han, Weizhi Wang","doi":"10.1021/acs.analchem.4c02273","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c02273","url":null,"abstract":"<p><p>Peptide self-assemblies could leverage their specificity, stability, biocompatibility, and electrochemical activity to create functionalized interfaces for molecular sensing and detection. However, the dynamics within these interfaces are complex, with competing forces, including those maintaining peptide structures, recognizing analytes, and facilitating signal transmission. Such competition could lead to nonspecific interference, compromising the detection sensitivity and accuracy. In this study, a series of peptides with precise structures and controllable electron transfer capabilities were designed. Through examining their stacking patterns, the interplay between the peptides' hierarchical structures, their ability to recognize targets, and their conductivity were clarified. Among these, the EP<sup>5</sup> peptide assembly was identified for its ability to form controllable electronic tunnels facilitated by π-stacking induced β-sheets. EP<sup>5</sup> could enhance the long-range conductivity, minimize nonspecific interference, and exhibit targeted recognition capabilities. Based on EP<sup>5</sup>, an electrochemical sensing interface toward the disease marker PD-L1 (programmed cell death ligand 1) was developed, suitable for both whole blood assay and <i>in vivo</i> companion diagnosis. It opens a new avenue for crafting electrochemical detection interfaces with specificity, sensitivity, and compatibility.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141453692","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}
A high-sensitivity fiber-optic photoacoustic sensor with pressure compensation is proposed to analyze the decomposition component SO2 in high-pressure gas insulation equipment. The multiple influence mechanism of pressure on photoacoustic excitation and cantilever detection has been theoretically analyzed and verified. In the high-pressure environment, the excited photoacoustic signal is enhanced, which compensates for the loss of sensitivity of the cantilever. A fiber-optic F-P cantilever is utilized to simultaneously measure static pressure and dynamic photoacoustic wave, and a spectral demodulation method based on white light interference is applied to calculate the optical path difference of the F-P interferometer (FPI). The real-time pressure is judged through the linear relationship between the average optical path difference of FPI and the pressure, which gives the proposed fiber-optic photoacoustic sensor the inherent advantages of being uncharged and resistant to electromagnetic interference. The average optical path difference of FPI is positively related to pressure, with a responsivity of 0.6 μm/atm, which is based on changes in the refractive index of gas. In the range of 1-4 atm, the SO2 sensor has a higher detection sensitivity at high-pressure, which benefits from the pressure compensation effect. With the pressure environment of gas insulation equipment at 4 atm as the application background, the SO2 gas is tested. The detection limit is 20 ppb with an averaging time of 400 s.
{"title":"Pressure-Compensated Fiber-Optic Photoacoustic Sensors for Trace SO<sub>2</sub> Analysis in Gas Insulation Equipment.","authors":"Xinyu Zhao, Yajie Zhang, Xiao Han, Hongchao Qi, Fengxiang Ma, Ke Chen","doi":"10.1021/acs.analchem.4c01480","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01480","url":null,"abstract":"<p><p>A high-sensitivity fiber-optic photoacoustic sensor with pressure compensation is proposed to analyze the decomposition component SO<sub>2</sub> in high-pressure gas insulation equipment. The multiple influence mechanism of pressure on photoacoustic excitation and cantilever detection has been theoretically analyzed and verified. In the high-pressure environment, the excited photoacoustic signal is enhanced, which compensates for the loss of sensitivity of the cantilever. A fiber-optic F-P cantilever is utilized to simultaneously measure static pressure and dynamic photoacoustic wave, and a spectral demodulation method based on white light interference is applied to calculate the optical path difference of the F-P interferometer (FPI). The real-time pressure is judged through the linear relationship between the average optical path difference of FPI and the pressure, which gives the proposed fiber-optic photoacoustic sensor the inherent advantages of being uncharged and resistant to electromagnetic interference. The average optical path difference of FPI is positively related to pressure, with a responsivity of 0.6 μm/atm, which is based on changes in the refractive index of gas. In the range of 1-4 atm, the SO<sub>2</sub> sensor has a higher detection sensitivity at high-pressure, which benefits from the pressure compensation effect. With the pressure environment of gas insulation equipment at 4 atm as the application background, the SO<sub>2</sub> gas is tested. The detection limit is 20 ppb with an averaging time of 400 s.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448988","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}
Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.
{"title":"Integration of Hybridization Chain Reaction and Protein-Binding Amplification for Long-Term Imaging of Intracellular mRNA: Avoiding Signal Fluctuation.","authors":"Yibo Zhou, Huiqiu Shi, Xinchao Xia, Sheng Yang, Junbin Li, Zhihe Qing, Jing Zheng, Ronghua Yang","doi":"10.1021/acs.analchem.4c01992","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c01992","url":null,"abstract":"<p><p>Amplified nanoprobes based on hybridization chain reaction (HCR) have been widely developed for the detection of intracellular low abundance mRNA. However, the formed chain-like assembly decorated with fluorophore would be degraded rapidly by endogenous enzyme, resulting in failure of the long-term fluorescence imaging. To address this issue, herein, a composite signal-amplifying strategy that integrates HCR into protein-binding signal amplification (HPSA) was communicated for the in situ imaging of mRNA by avoiding signal fluctuation. Different from conventional HCR-based nanoprobes (HCR-nanoprobe), the HCR was used as the signal-triggered mode and the amplifying signal generated from in situ fluorophore-protein binding in cells, which can maintain high stability of the signal for a long time. As a proof-of-principle, a nanobeacon based on HPSA (HPSA-nanobeacon) was constructed to detect TK1 mRNA. Taking advantage of the double signal-amplifying mode, the endogenous TK1 mRNA was sensitively detected and the fluorescence signal was maintained for more than 8 h in HepG2 cells. The attempt in this work provides a new option to the current signal-amplifying strategy for sensing nucleic acid targets with high stability, significantly enhancing the acquisition of intracellular molecular information.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141448987","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}