Circulating tumor cells (CTCs) serve as important biomarkers in the liquid biopsy of hepatocellular carcinoma (HCC). Herein, a homogeneous dual fluorescence indicators aptasensing strategy is described for CTCs in HCC, with the core assistance of a steric hindrance-mediated enzymatic reaction. CTCs in the sample could specifically bind to a 5′-biotin-modified glypican-3 (GPC3) aptamer and remove the steric hindrance formed by the biotin–streptavidin system. This influences the efficiency of the terminal deoxynucleotidyl transferase enzymatic reaction. Then, methylene blue (MB) was introduced to react with the main product poly cytosine (polyC) chain, and trivalent cerium ion (Ce3+) was added to react with the byproduct pyrophosphate to form fluorescent pyrophosphate cerium coordination polymeric nanoparticles. Finally, the CTCs were quantified by dual fluorescence indicators analysis. Under optimized conditions, the linear range was 5 to 104 cells/mL, and the limits of detection reached 2 cells/mL. Then, 40 clinical samples (15 healthy and 25 HCC patients) were analyzed. The receiver operating characteristic curve analysis revealed an area under the curve of 0.96, a sensitivity of 92%, and a specificity of 100%. Therefore, this study established a sensitive and accurate CTCs sensing system for clinical HCC patients, promoting early tumor diagnosis.
{"title":"Steric Hindrance-Mediated Enzymatic Reaction Enable Homogeneous Dual Fluorescence Indicators Aptasensing of Hepatocellular Carcinoma CTCs","authors":"Pengjun Jiang, Zixuan Zhan, Yufu Peng, Chengyong Wu, Yue Wang, Longfei Wu, Shiya Shi, Binwu Ying, Yonggang Wei*, Piaopiao Chen* and Jie Chen*, ","doi":"10.1021/acs.analchem.4c01624","DOIUrl":"10.1021/acs.analchem.4c01624","url":null,"abstract":"<p >Circulating tumor cells (CTCs) serve as important biomarkers in the liquid biopsy of hepatocellular carcinoma (HCC). Herein, a homogeneous dual fluorescence indicators aptasensing strategy is described for CTCs in HCC, with the core assistance of a steric hindrance-mediated enzymatic reaction. CTCs in the sample could specifically bind to a 5′-biotin-modified glypican-3 (GPC3) aptamer and remove the steric hindrance formed by the biotin–streptavidin system. This influences the efficiency of the terminal deoxynucleotidyl transferase enzymatic reaction. Then, methylene blue (MB) was introduced to react with the main product poly cytosine (polyC) chain, and trivalent cerium ion (Ce<sup>3+</sup>) was added to react with the byproduct pyrophosphate to form fluorescent pyrophosphate cerium coordination polymeric nanoparticles. Finally, the CTCs were quantified by dual fluorescence indicators analysis. Under optimized conditions, the linear range was 5 to 10<sup>4</sup> cells/mL, and the limits of detection reached 2 cells/mL. Then, 40 clinical samples (15 healthy and 25 HCC patients) were analyzed. The receiver operating characteristic curve analysis revealed an area under the curve of 0.96, a sensitivity of 92%, and a specificity of 100%. Therefore, this study established a sensitive and accurate CTCs sensing system for clinical HCC patients, promoting early tumor diagnosis.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141441760","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 : 2024-06-22DOI: 10.1021/acs.analchem.4c00556
Satoshi Hashimoto, and , Koichi Matsuo*,
The elucidation of protein–membrane interactions is pivotal for comprehending the mechanisms underlying diverse biological phenomena and membrane-related diseases. In this investigation, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy, utilizing synchrotron radiation (SR), was employed to dynamically observe membrane interaction processes involving water-soluble proteins at the secondary-structure level. The study utilized a time-resolved (TR) T-shaped microfluidic cell, facilitating the rapid and efficient mixing of protein and membrane solutions. This system was instrumental in acquiring measurements of the time-resolved circular dichroism (TRCD) spectra of β-lactoglobulin (bLG) during its interaction with lysoDMPG micelles. The results indicate that bLG undergoes a β–α conformation change, leading to the formation of the membrane-interacting state (M-state), with structural alterations occurring in more than two steps. Global fitting analysis, employing biexponential functions with all of the TRCD spectral data sets, yielded two distinct rate constants (0.18 ± 0.01 and 0.06 ± 0.003/s) and revealed a unique spectrum corresponding to an intermediate state (I-state). Secondary-structure analysis of bLG in its native (N-, I-, and M-states) highlighted that structural changes from the N- to I-states predominantly occurred in the N- and C-terminal regions, which were prominently exposed to the membrane. Meanwhile, transitions from the I- to M-states extended into the inner barrel regions of bLG. Further examination of the physical properties of α-helical segments, such as effective charge and hydrophobicity, revealed that the N- to I- and I- to M-state transitions, which are ascribed to first- and second-rate constants, respectively, are primarily driven by electrostatic and hydrophobic interactions, respectively. These findings underscore the capability of the TR-VUVCD system as a robust tool for characterizing protein–membrane interactions at the molecular level.
阐明蛋白质与膜的相互作用对于理解各种生物现象和膜相关疾病的内在机制至关重要。在这项研究中,利用同步辐射(SR)的真空-紫外圆二色性(VUVCD)光谱技术,在二级结构水平上动态观察了涉及水溶性蛋白质的膜相互作用过程。该研究利用了一个时间分辨(TR)T 型微流体池,有助于快速有效地混合蛋白质和膜溶液。该系统有助于获取β-乳球蛋白(bLG)与溶菌二甲基亚砜胶束相互作用时的时间分辨圆二色性(TRCD)光谱。结果表明,bLG经历了β-α构象变化,最终形成膜相互作用态(M态),其结构变化发生在两个以上的步骤中。利用双指数函数对所有 TRCD 光谱数据集进行全局拟合分析,得出了两个不同的速率常数(0.18 ± 0.01 和 0.06 ± 0.003/s),并揭示了与中间状态(I-state)相对应的独特光谱。对原生态(N态、I态和M态)bLG的二级结构分析显示,从N态到I态的结构变化主要发生在N端和C端区域,这两个区域主要暴露在膜上。同时,从 I 态到 M 态的转变延伸到了 bLG 的内桶区。对α螺旋段的物理性质(如有效电荷和疏水性)的进一步研究表明,N-到I-和I-到M-状态的转变分别归因于一速率常数和二速率常数,主要分别由静电和疏水相互作用驱动。这些发现强调了 TR-VUVCD 系统作为在分子水平表征蛋白质-膜相互作用的强大工具的能力。
{"title":"Dynamic Observation of the Membrane Interaction Processes of β-Lactoglobulin by Time-Resolved Vacuum-Ultraviolet Circular Dichroism","authors":"Satoshi Hashimoto, and , Koichi Matsuo*, ","doi":"10.1021/acs.analchem.4c00556","DOIUrl":"10.1021/acs.analchem.4c00556","url":null,"abstract":"<p >The elucidation of protein–membrane interactions is pivotal for comprehending the mechanisms underlying diverse biological phenomena and membrane-related diseases. In this investigation, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy, utilizing synchrotron radiation (SR), was employed to dynamically observe membrane interaction processes involving water-soluble proteins at the secondary-structure level. The study utilized a time-resolved (TR) T-shaped microfluidic cell, facilitating the rapid and efficient mixing of protein and membrane solutions. This system was instrumental in acquiring measurements of the time-resolved circular dichroism (TRCD) spectra of β-lactoglobulin (bLG) during its interaction with lysoDMPG micelles. The results indicate that bLG undergoes a β–α conformation change, leading to the formation of the membrane-interacting state (M-state), with structural alterations occurring in more than two steps. Global fitting analysis, employing biexponential functions with all of the TRCD spectral data sets, yielded two distinct rate constants (0.18 ± 0.01 and 0.06 ± 0.003/s) and revealed a unique spectrum corresponding to an intermediate state (I-state). Secondary-structure analysis of bLG in its native (N-, I-, and M-states) highlighted that structural changes from the N- to I-states predominantly occurred in the N- and C-terminal regions, which were prominently exposed to the membrane. Meanwhile, transitions from the I- to M-states extended into the inner barrel regions of bLG. Further examination of the physical properties of α-helical segments, such as effective charge and hydrophobicity, revealed that the N- to I- and I- to M-state transitions, which are ascribed to first- and second-rate constants, respectively, are primarily driven by electrostatic and hydrophobic interactions, respectively. These findings underscore the capability of the TR-VUVCD system as a robust tool for characterizing protein–membrane interactions at the molecular level.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141439886","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}
Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 105–107 particles/mL, along with a detection limit of 1.1 × 104 particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.
肿瘤源性细胞外囊泡 (TEV) 含有丰富的细胞信息,有望成为无创癌症诊断的生物标记物。然而,由于TEVs的丰度较低,且可能受到大量来自正常细胞的EVs的干扰,因此准确测量TEVs面临着挑战。本文开发了一种用于 EV 膜识别的适配体-近接-激活的滚动圈扩增(RCA)方法,并结合单颗粒电感耦合等离子体质谱法(sp-ICP-MS)对 TEV 进行定量。当 DNA 标记的超小型金纳米粒子(AuNP)探针与 RCA 形成的长链结合时,它们会聚集形成大颗粒。值得注意的是,小 AuNPs 在 sp-ICP-MS 中几乎不产生脉冲信号,因此可以免清洗的方式检测 TEV。通过利用适配体的强结合亲和力、识别 EpCAM 和 PD-L1 的双适配体以及 sp-ICP-MS 技术,该方法在追踪 TEV 方面具有显著的灵敏度和选择性。在优化条件下,本方法的 sp-ICP-MS 脉冲信号频率与 TEV 浓度在 105-107 个/毫升的范围内呈良好的线性关系,检测限为 1.1 × 104 个/毫升。sp-ICP-MS 的脉冲信号与机器学习算法相结合,可用于区分癌症患者和健康供体,准确率达 100%。由于其操作简单快捷,灵敏度和准确度极高,这种方法在生命科学和个性化医疗的各种应用中具有巨大潜力。
{"title":"Ultrasensitive and Wash-Free Detection of Tumor Extracellular Vesicles by Aptamer-Proximity-Ligation-Activated Rolling Circle Amplification Coupled to Single Particle ICP-MS","authors":"Xue-Wei Zhang, Gong-Xiang Qi, Shuai Chen*, Yong-Liang Yu* and Jian-Hua Wang, ","doi":"10.1021/acs.analchem.4c02066","DOIUrl":"10.1021/acs.analchem.4c02066","url":null,"abstract":"<p >Tumor-derived extracellular vesicles (TEVs) are rich in cellular information and hold great promise as a biomarker for noninvasive cancer diagnosis. However, accurate measurement of TEVs presents challenges due to their low abundance and potential interference from a high number of EVs derived from normal cells. Herein, an aptamer-proximity-ligation-activated rolling circle amplification (RCA) method for EV membrane recognition, coupled with single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) for the quantification of TEVs, is developed. When DNA-labeled ultrasmall gold nanoparticle (AuNP) probes bind to the long chains formed by RCA, they aggregate to form large particles. Notably, small AuNPs scarcely produce pulse signals in sp-ICP-MS, thereby detecting TEVs in a wash-free manner. By leveraging the strong binding affinity of aptamers, dual aptamers for EpCAM and PD-L1 recognition, and the sp-ICP-MS technique, this method offers remarkable sensitivity and selectivity in tracing TEVs. Under optimized conditions, the present method shows a favorable linear relationship between the pulse signal frequency of sp-ICP-MS and TEV concentration within the range of 10<sup>5</sup>–10<sup>7</sup> particles/mL, along with a detection limit of 1.1 × 10<sup>4</sup> particles/mL. The pulse signals from sp-ICP-MS combined with machine learning algorithms are used to discriminate cancer patients from healthy donors with 100% accuracy. Due to its simple and fast operation and excellent sensitivity and accuracy, this approach holds significant potential for diverse applications in life sciences and personalized medicine.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430785","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 : 2024-06-21DOI: 10.1021/acs.analchem.4c00968
Alexander M. Leshansky*, Boris Y. Rubinstein, Itzhak Fouxon, Diethelm Johannsmann, Marta Sadowska and Zbigniew Adamczyk,
Quartz crystal microbalance with dissipation monitoring (QCM-D) has become a major tool enabling accurate investigation of the adsorption kinetics of nanometric objects such as DNA fragments, polypeptides, proteins, viruses, liposomes, polymer, and metal nanoparticles. However, in liquids, a quantitative analysis of the experimental results is often intricate because of the complex interplay of hydrodynamic and adhesion forces varying with the physicochemical properties of adsorbates and functionalized QCM-D sensors. In the present paper, we dissect the role of hydrodynamics for the analytically tractable case of stiff contact, whereas the adsorbed rigid particles oscillate with the resonator without rotation. Under the assumption of the low surface coverage, we theoretically study the excess shear force exerted on the resonator, which has two contributions: (i) the fluid-mediated force due to flow disturbance created by the particle and (ii) the force exerted on the particle by the fluid and transmitted to the sensor via contact. The theoretical analysis enables an accurate interpretation of the QCM-D impedance measurements. It is demonstrated inter alia that for particles of the size comparable with protein molecules, the hydrodynamic force dominates over the inertial force and that the apparent mass derived from QCM independently of the overtone is about 10 times the Sauerbrey (inertial) mass. The theoretical results show excellent agreement with the results of experiments and advanced numerical simulations for a wide range of particle sizes and oscillation frequencies.
具有耗散监测功能的石英晶体微天平(QCM-D)已成为精确研究 DNA 片段、多肽、蛋白质、病毒、脂质体、聚合物和金属纳米颗粒等纳米物体吸附动力学的重要工具。然而,在液体中,由于吸附剂和功能化 QCM-D 传感器的理化性质不同,流体动力和粘附力的相互作用也各不相同,因此对实验结果进行定量分析往往十分复杂。在本文中,我们剖析了流体力学在可分析的刚性接触情况下的作用,而吸附的刚性颗粒则随谐振器摆动而不旋转。在低表面覆盖率的假设下,我们从理论上研究了施加在谐振器上的过量剪切力,它有两个贡献:(i) 颗粒产生的流动扰动导致的流体介导力和 (ii) 流体施加在颗粒上并通过接触传递到传感器的力。理论分析有助于准确解释 QCM-D 阻抗测量结果。除其他外,研究还证明,对于与蛋白质分子大小相当的颗粒,流体动力比惯性力占主导地位,QCM 独立于泛音得出的表观质量约为 Sauerbrey(惯性)质量的 10 倍。理论结果表明,对于各种颗粒大小和振荡频率,实验结果和先进的数值模拟结果都非常吻合。
{"title":"Quartz Crystal Microbalance Frequency Response to Discrete Adsorbates in Liquids","authors":"Alexander M. Leshansky*, Boris Y. Rubinstein, Itzhak Fouxon, Diethelm Johannsmann, Marta Sadowska and Zbigniew Adamczyk, ","doi":"10.1021/acs.analchem.4c00968","DOIUrl":"10.1021/acs.analchem.4c00968","url":null,"abstract":"<p >Quartz crystal microbalance with dissipation monitoring (QCM-D) has become a major tool enabling accurate investigation of the adsorption kinetics of nanometric objects such as DNA fragments, polypeptides, proteins, viruses, liposomes, polymer, and metal nanoparticles. However, in liquids, a quantitative analysis of the experimental results is often intricate because of the complex interplay of hydrodynamic and adhesion forces varying with the physicochemical properties of adsorbates and functionalized QCM-D sensors. In the present paper, we dissect the role of hydrodynamics for the analytically tractable case of stiff contact, whereas the adsorbed rigid particles oscillate with the resonator without rotation. Under the assumption of the low surface coverage, we theoretically study the excess shear force exerted on the resonator, which has two contributions: (i) the fluid-mediated force due to flow disturbance created by the particle and (ii) the force exerted on the particle by the fluid and transmitted to the sensor via contact. The theoretical analysis enables an accurate interpretation of the QCM-D impedance measurements. It is demonstrated inter alia that for particles of the size comparable with protein molecules, the hydrodynamic force dominates over the inertial force and that the apparent mass derived from QCM independently of the overtone is about 10 times the Sauerbrey (inertial) mass. The theoretical results show excellent agreement with the results of experiments and advanced numerical simulations for a wide range of particle sizes and oscillation frequencies.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.analchem.4c00968","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141436430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-21DOI: 10.1021/acs.analchem.4c01176
Baoting Dou, Keming Wang, Yan Chen and Po Wang*,
The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the CuΙ-catalyzed azide–alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.
{"title":"Programmable DNA Nanomachine Integrated with Electrochemically Controlled Atom Transfer Radical Polymerization for Antibody Detection at Picomolar Level","authors":"Baoting Dou, Keming Wang, Yan Chen and Po Wang*, ","doi":"10.1021/acs.analchem.4c01176","DOIUrl":"10.1021/acs.analchem.4c01176","url":null,"abstract":"<p >The quantitative detection of antibodies is crucial for the diagnosis of infectious and autoimmune diseases, while the traditional methods experience high background signal noise and restricted signal gain. In this work, we have developed a highly efficient electrochemical biosensor by constructing a programmable DNA nanomachine integrated with electrochemically controlled atom transfer radical polymerization (eATRP). The sensor works by binding the target antidigoxin antibody (anti-Dig) to the epitope of the recognization probe, which then initiates the cascaded strand displacement reaction on a magnetic bead, leading to the capture of cupric oxide (CuO) nanoparticles through magnetic separation. After CuO was dissolved, the eATRP initiators were attached to the electrode based on the Cu<sup>Ι</sup>-catalyzed azide–alkyne cycloaddition. The subsequent eATRP reaction results in the formation of long electroactive polymers (poly-FcMMA), producing an amplified current response for sensitive detection of anti-Dig. This method achieved a detection limit at clinically relevant picomolar concentration in human serum, offering a sensitive, convenient, and cost-effective tool for detecting various biomarkers in a wide range of applications.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430870","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 : 2024-06-21DOI: 10.1021/acs.analchem.4c01576
Xixuan Wu, and , Wei Jia*,
Precision mapping of selenium at structural and position levels poses significant challenges in selenium-containing polysaccharide identification. Due to the absence of reference spectra, database-centric approaches are still limited in the discovery of selenium binding sites and distinction among different isomeric structures. A multilayer annotation strategy, AnnoSePS, is proposed for achieving the identification of seleno-substituent and the unbiased profiling of polysaccharides. Applying Snoop-triggered multiple reaction monitoring (Snoop-MRM) identified multidimensional monosaccharides in selenium-containing polysaccharides. Galactose, galacturonic acid, and glucose were the predominant monosaccharides with a molar ratio of 25.19, 19.45, and 11.72, respectively. Selenium present in seleno-rhamnose was found to substitute the hydroxyl group located at C-1 positions through the formation of a Se–H bond. Ions C6H9O3Se–, C6H7O3Se–, C5H5O3Se–, C4H5O2Se–, C3H5O2Se–, C2H3O2Se–, and CHOSe– were defined as the characteristic fragments of seleno-rhamnose. The agglomerative hierarchical clustering algorithm is applied to group spectra from each run based on the characteristic information. Preferential fragmentation patterns in mass spectrometry are revealed by training a probabilistic model. A list of candidate oligosaccharides is generated by step-by-step browsing through the transition pairs for all reference spectra and applying the transitions (addition, insertion, removal, and substitution) to reference structures. Combining time course analyses revealed the linkage composition of selenium-containing oligosaccharides. Glycosidic linkages were annotated based on a synthesis-driven approach. T-Galactose (16.67 ± 5.23%) and T-Galacturonic acid (11.54 ± 4.66%) were the predominant linkage residues. As the database-independent mapping strategy, AnnoSePS makes it possible to comprehensively interrogate spectral data and dissect the fine structure of selenium-containing polysaccharides.
{"title":"Multilayer Annotation Strategy AnnoSePS: Disentangling the Intricate Structure of Selenium-Containing Polysaccharides Based on Preferential Fragmentation Patterns","authors":"Xixuan Wu, and , Wei Jia*, ","doi":"10.1021/acs.analchem.4c01576","DOIUrl":"10.1021/acs.analchem.4c01576","url":null,"abstract":"<p >Precision mapping of selenium at structural and position levels poses significant challenges in selenium-containing polysaccharide identification. Due to the absence of reference spectra, database-centric approaches are still limited in the discovery of selenium binding sites and distinction among different isomeric structures. A multilayer annotation strategy, AnnoSePS, is proposed for achieving the identification of seleno-substituent and the unbiased profiling of polysaccharides. Applying Snoop-triggered multiple reaction monitoring (Snoop-MRM) identified multidimensional monosaccharides in selenium-containing polysaccharides. Galactose, galacturonic acid, and glucose were the predominant monosaccharides with a molar ratio of 25.19, 19.45, and 11.72, respectively. Selenium present in seleno-rhamnose was found to substitute the hydroxyl group located at C-1 positions through the formation of a Se–H bond. Ions C<sub>6</sub>H<sub>9</sub>O<sub>3</sub>Se<sup>–</sup>, C<sub>6</sub>H<sub>7</sub>O<sub>3</sub>Se<sup>–</sup>, C<sub>5</sub>H<sub>5</sub>O<sub>3</sub>Se<sup>–</sup>, C<sub>4</sub>H<sub>5</sub>O<sub>2</sub>Se<sup>–</sup>, C<sub>3</sub>H<sub>5</sub>O<sub>2</sub>Se<sup>–</sup>, C<sub>2</sub>H<sub>3</sub>O<sub>2</sub>Se<sup>–</sup>, and CHOSe<sup>–</sup> were defined as the characteristic fragments of seleno-rhamnose. The agglomerative hierarchical clustering algorithm is applied to group spectra from each run based on the characteristic information. Preferential fragmentation patterns in mass spectrometry are revealed by training a probabilistic model. A list of candidate oligosaccharides is generated by step-by-step browsing through the transition pairs for all reference spectra and applying the transitions (addition, insertion, removal, and substitution) to reference structures. Combining time course analyses revealed the linkage composition of selenium-containing oligosaccharides. Glycosidic linkages were annotated based on a synthesis-driven approach. T-Galactose (16.67 ± 5.23%) and T-Galacturonic acid (11.54 ± 4.66%) were the predominant linkage residues. As the database-independent mapping strategy, AnnoSePS makes it possible to comprehensively interrogate spectral data and dissect the fine structure of selenium-containing polysaccharides.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430868","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 : 2024-06-21DOI: 10.1021/acs.analchem.4c01890
Ran Chen, Pavithra Pathirathna, Ryan J. Balla, Jiyeon Kim and Shigeru Amemiya*,
The nuclear pore complex (NPC) is a proteinaceous nanopore that solely and selectively regulates the molecular transport between the cytoplasm and nucleus of a eukaryotic cell. The ∼50 nm-diameter pore of the NPC perforates the double-membrane nuclear envelope to mediate both passive and facilitated molecular transport, thereby playing paramount biological and biomedical roles. Herein, we visualize single NPCs by scanning electrochemical microscopy (SECM). The high spatial resolution is accomplished by employing ∼25 nm-diameter ion-selective nanopipets to monitor the passive transport of tetrabutylammonium at individual NPCs. SECM images are quantitatively analyzed by employing the finite element method to confirm that this work represents the highest-resolution nanoscale SECM imaging of biological samples. Significantly, we apply the powerful imaging technique to address the long-debated origin of the central plug of the NPC. Nanoscale SECM imaging demonstrates that unplugged NPCs are more permeable to the small probe ion than are plugged NPCs. This result supports the hypothesis that the central plug is not an intrinsic transporter, but is an impermeable macromolecule, e.g., a ribonucleoprotein, trapped in the nanopore. Moreover, this result also supports the transport mechanism where the NPC is divided into the central pathway for RNA export and the peripheral pathway for protein import to efficiently mediate the bidirectional traffic.
{"title":"Nanoscale Quantitative Imaging of Single Nuclear Pore Complexes by Scanning Electrochemical Microscopy","authors":"Ran Chen, Pavithra Pathirathna, Ryan J. Balla, Jiyeon Kim and Shigeru Amemiya*, ","doi":"10.1021/acs.analchem.4c01890","DOIUrl":"10.1021/acs.analchem.4c01890","url":null,"abstract":"<p >The nuclear pore complex (NPC) is a proteinaceous nanopore that solely and selectively regulates the molecular transport between the cytoplasm and nucleus of a eukaryotic cell. The ∼50 nm-diameter pore of the NPC perforates the double-membrane nuclear envelope to mediate both passive and facilitated molecular transport, thereby playing paramount biological and biomedical roles. Herein, we visualize single NPCs by scanning electrochemical microscopy (SECM). The high spatial resolution is accomplished by employing ∼25 nm-diameter ion-selective nanopipets to monitor the passive transport of tetrabutylammonium at individual NPCs. SECM images are quantitatively analyzed by employing the finite element method to confirm that this work represents the highest-resolution nanoscale SECM imaging of biological samples. Significantly, we apply the powerful imaging technique to address the long-debated origin of the central plug of the NPC. Nanoscale SECM imaging demonstrates that unplugged NPCs are more permeable to the small probe ion than are plugged NPCs. This result supports the hypothesis that the central plug is not an intrinsic transporter, but is an impermeable macromolecule, e.g., a ribonucleoprotein, trapped in the nanopore. Moreover, this result also supports the transport mechanism where the NPC is divided into the central pathway for RNA export and the peripheral pathway for protein import to efficiently mediate the bidirectional traffic.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.analchem.4c01890","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-20DOI: 10.1021/acs.analchem.4c01900
Shengrui Xu*, Huimin Li, Li Xiao, Suling Feng, Jing Fan* and Janusz Pawliszyn,
A simple, sustainable, and sensitive monitoring approach of micro/nanoplastics (MNPs) in aqueous samples is crucial since it helps in assessing the extent of contamination and understanding the potential risks associated with their presence without causing additional stress to the environment. In this study, a novel strategy for qualitative and quantitative determination of MNPs in water by direct solid-phase microextraction (SPME) coupled with gas chromatography–mass spectrometry (GC-MS) was proposed for the first time. Spherical poly(methyl methacrylate) (PMMA) and irregularly shaped polyvinyl dichloride (PVDC) were used to evaluate the feasibility and performance of the proposed method. The results demonstrated that both PMMA and PVDC MNPs were efficiently extracted by the homemade SPME coating of nitrogen-doped porous carbons (N-SPCs) and exhibited sufficient thermal decomposition in the GC-MS injection port. Excellent extraction performances of N-SPCs coating for MNPs are attributed to hydrophobic cross-linking, electrostatic forcing, hydrogen bonding, and pore trapping. Methyl methacrylate was identified as the marker for PMMA, while 1,3-dichlorobenzene and 1,3,5-trichlorobenzene were the indicators for PVDC. Under the optimal extraction and decomposition conditions, the proposed method exhibited ultrahigh sensitivity, with a limit of detection of 0.0041 μg/L for PMMA and 0.0054 μg/L for PVDC. Notably, a programmed temperature strategy for the GC-MS injector was developed to discriminate and eliminate the potential interferences of intrinsic indicator compounds. Owing to the integration of sampling, extraction, injection, and decomposition into one step by SPME, the proposed method demonstrates exceptional sensitivity, eliminating the necessity for complex sample pretreatment procedures and the use of organic solvents. Finally, the proposed method was successfully applied in the determination of PMMA and PVDC MNPs in real aqueous samples.
{"title":"Monitoring Poly(methyl methacrylate) and Polyvinyl Dichloride Micro/Nanoplastics in Water by Direct Solid-Phase Microextraction Coupled to Gas Chromatography–Mass Spectrometry","authors":"Shengrui Xu*, Huimin Li, Li Xiao, Suling Feng, Jing Fan* and Janusz Pawliszyn, ","doi":"10.1021/acs.analchem.4c01900","DOIUrl":"10.1021/acs.analchem.4c01900","url":null,"abstract":"<p >A simple, sustainable, and sensitive monitoring approach of micro/nanoplastics (MNPs) in aqueous samples is crucial since it helps in assessing the extent of contamination and understanding the potential risks associated with their presence without causing additional stress to the environment. In this study, a novel strategy for qualitative and quantitative determination of MNPs in water by direct solid-phase microextraction (SPME) coupled with gas chromatography–mass spectrometry (GC-MS) was proposed for the first time. Spherical poly(methyl methacrylate) (PMMA) and irregularly shaped polyvinyl dichloride (PVDC) were used to evaluate the feasibility and performance of the proposed method. The results demonstrated that both PMMA and PVDC MNPs were efficiently extracted by the homemade SPME coating of nitrogen-doped porous carbons (N-SPCs) and exhibited sufficient thermal decomposition in the GC-MS injection port. Excellent extraction performances of N-SPCs coating for MNPs are attributed to hydrophobic cross-linking, electrostatic forcing, hydrogen bonding, and pore trapping. Methyl methacrylate was identified as the marker for PMMA, while 1,3-dichlorobenzene and 1,3,5-trichlorobenzene were the indicators for PVDC. Under the optimal extraction and decomposition conditions, the proposed method exhibited ultrahigh sensitivity, with a limit of detection of 0.0041 μg/L for PMMA and 0.0054 μg/L for PVDC. Notably, a programmed temperature strategy for the GC-MS injector was developed to discriminate and eliminate the potential interferences of intrinsic indicator compounds. Owing to the integration of sampling, extraction, injection, and decomposition into one step by SPME, the proposed method demonstrates exceptional sensitivity, eliminating the necessity for complex sample pretreatment procedures and the use of organic solvents. Finally, the proposed method was successfully applied in the determination of PMMA and PVDC MNPs in real aqueous samples.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430867","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}
Hydrogen peroxide (H2O2) overexpressed in mitochondria has been regarded as a key biomarker in the pathological processes of various diseases. However, there is currently a lack of suitable mitochondria-targetable near-infrared (NIR) probes for the visualization of H2O2 in multiple diseases, such as PM2.5 exposure-induced lung injury, hepatic ischemia-reperfusion injury (HIRI), nonalcoholic fatty liver (NAFL), hepatic fibrosis (HF), and malignant tumor tissues containing clinical cancer patient samples. Herein, we conceived a novel NIR fluorescent probe (HCy-H2O2) by introducing pentafluorobenzenesulfonyl as a H2O2 sensing unit into the NIR hemicyanine platform. HCy-H2O2 exhibits good sensitivity and selectivity toward H2O2, accompanied by a remarkable “turn-on” fluorescence signal at 720 nm. Meanwhile, HCy-H2O2 has stable mitochondria-targetable ability and permits monitoring of the up-generated H2O2 level during mitophagy. Furthermore, using HCy-H2O2, we have successfully observed an overproduced mitochondrial H2O2 in ambient PM2.5 exposure-induced lung injury, HIRI, NAFL, and HF models through NIR fluorescence imaging. Significantly, the visualization of H2O2 has been achieved in both tumor-bear mice as well as surgical specimens of cancer patients, making HCy-H2O2 a promising tool for cancer diagnosis and imaging-guided surgery.
{"title":"Mitochondria-Targetable Near-Infrared Fluorescent Probe for Visualization of Hydrogen Peroxide in Lung Injury, Liver Injury, and Tumor Models","authors":"Qi Zan, Kunyi Zhao, Ruijin Li, Yongming Yang, Xihua Yang*, Wenzhong Li, Gangli Zhang, Chuan Dong, Shaomin Shuang* and Li Fan*, ","doi":"10.1021/acs.analchem.3c05479","DOIUrl":"10.1021/acs.analchem.3c05479","url":null,"abstract":"<p >Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) overexpressed in mitochondria has been regarded as a key biomarker in the pathological processes of various diseases. However, there is currently a lack of suitable mitochondria-targetable near-infrared (NIR) probes for the visualization of H<sub>2</sub>O<sub>2</sub> in multiple diseases, such as PM<sub>2.5</sub> exposure-induced lung injury, hepatic ischemia-reperfusion injury (HIRI), nonalcoholic fatty liver (NAFL), hepatic fibrosis (HF), and malignant tumor tissues containing clinical cancer patient samples. Herein, we conceived a novel NIR fluorescent probe (<b>HCy-H</b><sub><b>2</b></sub><b>O</b><sub><b>2</b></sub>) by introducing pentafluorobenzenesulfonyl as a H<sub>2</sub>O<sub>2</sub> sensing unit into the NIR hemicyanine platform. <b>HCy-H</b><sub><b>2</b></sub><b>O</b><sub><b>2</b></sub> exhibits good sensitivity and selectivity toward H<sub>2</sub>O<sub>2</sub>, accompanied by a remarkable “turn-on” fluorescence signal at 720 nm. Meanwhile, <b>HCy-H</b><sub><b>2</b></sub><b>O</b><sub><b>2</b></sub> has stable mitochondria-targetable ability and permits monitoring of the up-generated H<sub>2</sub>O<sub>2</sub> level during mitophagy. Furthermore, using <b>HCy-H</b><sub><b>2</b></sub><b>O</b><sub><b>2</b></sub>, we have successfully observed an overproduced mitochondrial H<sub>2</sub>O<sub>2</sub> in ambient PM<sub>2.5</sub> exposure-induced lung injury, HIRI, NAFL, and HF models through NIR fluorescence imaging. Significantly, the visualization of H<sub>2</sub>O<sub>2</sub> has been achieved in both tumor-bear mice as well as surgical specimens of cancer patients, making <b>HCy-H</b><sub><b>2</b></sub><b>O</b><sub><b>2</b></sub> a promising tool for cancer diagnosis and imaging-guided surgery.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141430866","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 : 2024-06-19DOI: 10.1021/acs.analchem.4c01858
Javier García-Bellido, Montserrat Redondo-Velasco, Laura Freije-Carrelo, Gaëtan Burnens, Mariella Moldovan, Brice Bouyssiere, Pierre Giusti* and Jorge Ruiz Encinar*,
This work introduces a new element-selective gas chromatography detector for the accurate quantification of traces of volatile oxygen-containing compounds in complex samples without the need for specific standards. The key to this approach is the use of oxygen highly enriched in 18O as the oxidizing gas in a combustion unit (800 °C) that allows us to directly and unambiguously detect the natural oxygen present in the GC-separated compounds through its incorporation into the volatile species formed after their combustion and their subsequent degradation to 16O in the ion source. The unspecific signal due to the low 16O abundance in the oxidizing gas could be compensated by measuring the m/z 12 that comes as well from the CO2 degradation. Equimolarity was proved with several O-containing compounds with different sizes and functionalities. A detection limit of 28 pg of injected O was achieved, which is the lowest ever reported for any GC detector, which barely worsened to 55 and 214 pg of O when the oxygenate partially or completely coeluted with a very abundant matrix compound. Validation was attained by the analysis of a SRM to obtain accurate (99–103%) and precise (1–4% RSD) results. Robustness was tested after spiking a hydrotreated diesel with 10 O-compounds at the ppm level, which could be discriminated from the matrix crowd and quantified (mean recovery of 102 ± 9%) with a single generic standard. Finally, it was also successfully applied to easily spot and quantify the 33 oxygenates naturally present in a complex wood bio-oil sample.
{"title":"Sensitive Detection and Quantification of Oxygenated Compounds in Complex Samples Using GC-Combustion-MS","authors":"Javier García-Bellido, Montserrat Redondo-Velasco, Laura Freije-Carrelo, Gaëtan Burnens, Mariella Moldovan, Brice Bouyssiere, Pierre Giusti* and Jorge Ruiz Encinar*, ","doi":"10.1021/acs.analchem.4c01858","DOIUrl":"10.1021/acs.analchem.4c01858","url":null,"abstract":"<p >This work introduces a new element-selective gas chromatography detector for the accurate quantification of traces of volatile oxygen-containing compounds in complex samples without the need for specific standards. The key to this approach is the use of oxygen highly enriched in <sup>18</sup>O as the oxidizing gas in a combustion unit (800 °C) that allows us to directly and unambiguously detect the natural oxygen present in the GC-separated compounds through its incorporation into the volatile species formed after their combustion and their subsequent degradation to <sup>16</sup>O in the ion source. The unspecific signal due to the low <sup>16</sup>O abundance in the oxidizing gas could be compensated by measuring the <i>m</i>/<i>z</i> 12 that comes as well from the CO<sub>2</sub> degradation. Equimolarity was proved with several O-containing compounds with different sizes and functionalities. A detection limit of 28 pg of injected O was achieved, which is the lowest ever reported for any GC detector, which barely worsened to 55 and 214 pg of O when the oxygenate partially or completely coeluted with a very abundant matrix compound. Validation was attained by the analysis of a SRM to obtain accurate (99–103%) and precise (1–4% RSD) results. Robustness was tested after spiking a hydrotreated diesel with 10 O-compounds at the ppm level, which could be discriminated from the matrix crowd and quantified (mean recovery of 102 ± 9%) with a single generic standard. Finally, it was also successfully applied to easily spot and quantify the 33 oxygenates naturally present in a complex wood bio-oil sample.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.analchem.4c01858","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141435919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}