Pub Date : 2025-04-09DOI: 10.1021/acs.analchem.5c00452
Xiangdong Yu, Sohyun Park, Younju Joung, Mengdan Lu, Ji Qi, Jaebum Choo
This study presents the development of a dual-function microdroplet sensor utilizing surface-enhanced Raman scattering (SERS) technology to identify and quantify Influenza A and COVID-19 viruses. The proposed microfluidic device incorporates compartments for two-phase segmented droplet generation, merging, splitting, and detection. Both viral strains were identified by isolating magnetic antibody-antigen complexes from the liquid medium using a magnetized bar embedded in the microfluidic channel. Concurrent Raman spectroscopic readings were obtained as suspended droplets containing residual SERS-active nanoparticles traversed the interrogation zone of the focused laser beam. Precise quantitative analysis was accomplished by correcting characteristic Raman peak intensities for both viruses with internal standards, while ensemble averaging Raman signals from multiple droplets ensured high reproducibility. This dual-function SERS microdroplet sensor represents a novel in vitro diagnostic approach capable of rapidly distinguishing between COVID-19 and Influenza A with high sensitivity and reproducibility. When coupled with a portable Raman spectrophotometer, the device shows significant potential as a diagnostic tool for swift and in situ detection of both viral pathogens.
{"title":"Dual-Function SERS Microdroplet Sensor for Rapid Differentiation of Influenza a and SARS-CoV-2.","authors":"Xiangdong Yu, Sohyun Park, Younju Joung, Mengdan Lu, Ji Qi, Jaebum Choo","doi":"10.1021/acs.analchem.5c00452","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00452","url":null,"abstract":"<p><p>This study presents the development of a dual-function microdroplet sensor utilizing surface-enhanced Raman scattering (SERS) technology to identify and quantify Influenza A and COVID-19 viruses. The proposed microfluidic device incorporates compartments for two-phase segmented droplet generation, merging, splitting, and detection. Both viral strains were identified by isolating magnetic antibody-antigen complexes from the liquid medium using a magnetized bar embedded in the microfluidic channel. Concurrent Raman spectroscopic readings were obtained as suspended droplets containing residual SERS-active nanoparticles traversed the interrogation zone of the focused laser beam. Precise quantitative analysis was accomplished by correcting characteristic Raman peak intensities for both viruses with internal standards, while ensemble averaging Raman signals from multiple droplets ensured high reproducibility. This dual-function SERS microdroplet sensor represents a novel in vitro diagnostic approach capable of rapidly distinguishing between COVID-19 and Influenza A with high sensitivity and reproducibility. When coupled with a portable Raman spectrophotometer, the device shows significant potential as a diagnostic tool for swift and in situ detection of both viral pathogens.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":""},"PeriodicalIF":6.7,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143810194","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 : 2025-04-09DOI: 10.1021/acs.analchem.5c01237
Ximeng Liu, Xuan Liu, Boping Li, Xinglei Zhang, Bin Hu
Onsite, safe, and reliable mass spectrometry (MS) analysis of hazardous and radioactive samples plays a crucial role in timely chemical emergency management and response in real environments. The current study reports the development of a smart MS robot by integrating miniature MS, quadruped robot, switchable robotic arm sampler, and direct ionization for remote-controlled chemical analysis of complex samples in inaccessible hazardous and radioactive environments. High automation and excellent analytical performance have been achieved in the real-time analysis of volatile toxic substances in air and onsite detection of explosive particles in air aerosols. Successful detection of hazardous compounds has been performed from raw wastewater. The chemical analysis of radioactive ore samples has also been demonstrated. Low limits of detection at ng/g or ng/mL (signal-to-noise ratio, S/N = 3) and good relative standard deviation (RSD < 12.0%, n = 6) were obtained by the MS robot for analyzing different gaseous, aerosol, liquid, and solid samples. The remote detection results of the MS robot were further validated. The reported study encourages the future development of a smart lab-on-robot, which functions with smart operation to replace the traditional laboratory procedures for MS analysis of dangerous chemical and environmental samples.
{"title":"Lab-on-Robot: Unmanned Mass Spectrometry Robot for Direct Sample Analysis in Hazardous and Radioactive Environments","authors":"Ximeng Liu, Xuan Liu, Boping Li, Xinglei Zhang, Bin Hu","doi":"10.1021/acs.analchem.5c01237","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c01237","url":null,"abstract":"Onsite, safe, and reliable mass spectrometry (MS) analysis of hazardous and radioactive samples plays a crucial role in timely chemical emergency management and response in real environments. The current study reports the development of a smart MS robot by integrating miniature MS, quadruped robot, switchable robotic arm sampler, and direct ionization for remote-controlled chemical analysis of complex samples in inaccessible hazardous and radioactive environments. High automation and excellent analytical performance have been achieved in the real-time analysis of volatile toxic substances in air and onsite detection of explosive particles in air aerosols. Successful detection of hazardous compounds has been performed from raw wastewater. The chemical analysis of radioactive ore samples has also been demonstrated. Low limits of detection at ng/g or ng/mL (signal-to-noise ratio, S/N = 3) and good relative standard deviation (RSD < 12.0%, <i>n</i> = 6) were obtained by the MS robot for analyzing different gaseous, aerosol, liquid, and solid samples. The remote detection results of the MS robot were further validated. The reported study encourages the future development of a smart lab-on-robot, which functions with smart operation to replace the traditional laboratory procedures for MS analysis of dangerous chemical and environmental samples.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"8 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806365","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 : 2025-04-08DOI: 10.1021/acs.analchem.4c07110
Qi Wu, Siying Li, Xinqi Long, Lei Liu, Qiyang Zhao, Yongliang Cui, Yaohai Zhang, Yue He
The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of p-aminophenyl β-D-galactopyranoside to generate p-aminophenol. This compound subsequently facilitates the decomposition of MnO2 nanosheets, leading to the generation of Mn2+ ions. The Mn2+ ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn2+-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.
{"title":"β-Galactosidase-Mediated, Mn2+-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim","authors":"Qi Wu, Siying Li, Xinqi Long, Lei Liu, Qiyang Zhao, Yongliang Cui, Yaohai Zhang, Yue He","doi":"10.1021/acs.analchem.4c07110","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c07110","url":null,"abstract":"The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of <i>p</i>-aminophenyl β-<span>D</span>-galactopyranoside to generate <i>p</i>-aminophenol. This compound subsequently facilitates the decomposition of MnO<sub>2</sub> nanosheets, leading to the generation of Mn<sup>2+</sup> ions. The Mn<sup>2+</sup> ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn<sup>2+</sup>-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"25 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798269","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}
Sirtuin 2 (SIRT2) is involved in the pathological processes of many diseases and is especially regarded as a potential therapeutic target for diabetes and neurodegenerative diseases. Imaging SIRT2 protein in live cells has important value for rapidly detecting SIRT2 and high-throughput screening SIRT2 modulators. As far as we know, there has been no method for imaging SIRT2 in live cells up to now. Here, we present a novel aptamer (Apt)-based “turn-on” fluorescent biosensor for imaging SIRT2 in live cells. To develop the recognition element of the biosensor, our work first discovered 12 aptamers (Apt) with high affinity to SIRT2 (Kd = 123.3–154.5 nM) using the magnetic beads-based systemic evolution of ligands by exponential enrichment (MB-SELEX) and selected Apt 45 (Kd = 123.3 nM) to fabricate the “turn-on” fluorescent biosensor, FAM-Apt 45/Black Hole Quencher1 (BHQ1)-cDNA/Au nanospheres, which had excellent specificity and low cytotoxicity. The experiment results demonstrated that the biosensor could image SIRT2 in three different cell lines, including Schwann, H9c2, and HUVECs cells. Further, we established a platform for screening SIRT2 modulators with the biosensor and discovered three SIRT2 modulators (astragaloside II, chlorogenic acid, and tanshinone IIA) that could increase SIRT2 levels in Schwann cells damaged by high glucose and lipid. This work provides an aptamer-based fluorescent biosensor for high-throughput screening of protein modulators at the cellular level, which could be a universal approach to screening aimed protein modulators by replacing the corresponding aptamer.
{"title":"A Novel Aptamer-Based Fluorescent Biosensor for Imaging SIRT2 in Live Cells and Screening Its Modulators","authors":"Shufang Wang, Haoran Li, Yugang Lin, Ningtao Cheng","doi":"10.1021/acs.analchem.5c00066","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00066","url":null,"abstract":"Sirtuin 2 (SIRT2) is involved in the pathological processes of many diseases and is especially regarded as a potential therapeutic target for diabetes and neurodegenerative diseases. Imaging SIRT2 protein in live cells has important value for rapidly detecting SIRT2 and high-throughput screening SIRT2 modulators. As far as we know, there has been no method for imaging SIRT2 in live cells up to now. Here, we present a novel aptamer (Apt)-based “turn-on” fluorescent biosensor for imaging SIRT2 in live cells. To develop the recognition element of the biosensor, our work first discovered 12 aptamers (Apt) with high affinity to SIRT2 (<i>K</i><sub>d</sub> = 123.3–154.5 nM) using the magnetic beads-based systemic evolution of ligands by exponential enrichment (MB-SELEX) and selected Apt 45 (<i>K</i><sub>d</sub> = 123.3 nM) to fabricate the “turn-on” fluorescent biosensor, FAM-Apt 45/Black Hole Quencher1 (BHQ1)-cDNA/Au nanospheres, which had excellent specificity and low cytotoxicity. The experiment results demonstrated that the biosensor could image SIRT2 in three different cell lines, including Schwann, H9c2, and HUVECs cells. Further, we established a platform for screening SIRT2 modulators with the biosensor and discovered three SIRT2 modulators (astragaloside II, chlorogenic acid, and tanshinone IIA) that could increase SIRT2 levels in Schwann cells damaged by high glucose and lipid. This work provides an aptamer-based fluorescent biosensor for high-throughput screening of protein modulators at the cellular level, which could be a universal approach to screening aimed protein modulators by replacing the corresponding aptamer.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"93 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806133","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 : 2025-04-08DOI: 10.1021/acs.analchem.4c06549
Ryo Imai, Hideaki Kano, Toshiaki Hattori
We propose a method to measure enzymatic reactions using the C–D stretching vibrational spectra of CD3 groups that exist next to the site where a chemical bond changes. In the proposed method, temporal changes in the concentrations of the substrate and product are measured from changes in the C–D stretching spectra of the CD3 groups. Vibrational spectra are obtained using multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy, which can obtain vibrational spectra much faster than spontaneous Raman spectroscopy. We tested the proposed method by examining the dehydrogenation reaction of isopropyl alcohol-1,1,1,3,3,3-D6 (IPA-D6) catalyzed by alcohol dehydrogenase and found that the change in concentrations of the substrate and product, IPA-D6 and acetone-D6, respectively, was successfully measured from the C–D stretching spectra.
{"title":"Monitoring Enzymatic Reactions through Probing Chemical Bond Changes by the CD3 Group Using Multiplex Coherent Anti-Stokes Raman Scattering","authors":"Ryo Imai, Hideaki Kano, Toshiaki Hattori","doi":"10.1021/acs.analchem.4c06549","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06549","url":null,"abstract":"We propose a method to measure enzymatic reactions using the C–D stretching vibrational spectra of CD<sub>3</sub> groups that exist next to the site where a chemical bond changes. In the proposed method, temporal changes in the concentrations of the substrate and product are measured from changes in the C–D stretching spectra of the CD<sub>3</sub> groups. Vibrational spectra are obtained using multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy, which can obtain vibrational spectra much faster than spontaneous Raman spectroscopy. We tested the proposed method by examining the dehydrogenation reaction of isopropyl alcohol-1,1,1,3,3,3-D6 (IPA-D6) catalyzed by alcohol dehydrogenase and found that the change in concentrations of the substrate and product, IPA-D6 and acetone-D6, respectively, was successfully measured from the C–D stretching spectra.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"300 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798265","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 : 2025-04-08DOI: 10.1021/acs.analchem.5c00672
Tong Xu, Yingrui Zhang, Shiyu Chen, Zengnan Wu, Xian-Li Meng, Yi Zhang, Jin-Ming Lin
Hydrogel microparticles are versatile tools for organ modeling due to their simplicity, uniformity, and customizability, yet their limited physiological relevance constrains practical applications. In this study, a heart microparticle model that incorporates endocardial and myocardial structures and functions was developed. Hydrogel microparticles with rough surfaces, embedded with cardiomyocytes, were created using a custom-designed microfluidic device. Surface modification with matrigel enhanced the adhesion and connectivity of endothelial cells, enabling the formation of a densely packed endothelial layer. Real-time analysis, combining microparticle culture with a microfluidic chip-mass spectrometry system, demonstrated the utility of these particles in detecting the cardiotoxicity of heart-related drugs. For example, the analysis revealed that the cardiotoxicity of aconitine and Tie-bang-chui (TBC) was associated with elevated lactate and succinate levels, while processed TBC mitigated this toxicity of TBC by reducing these metabolites. These biomimetic microparticle models provide a novel platform for real-time metabolite analysis and cardiotoxicity research.
{"title":"Biomimetic Microparticles with Myocardial and Endocardial Integration for Drug Toxicity Studies","authors":"Tong Xu, Yingrui Zhang, Shiyu Chen, Zengnan Wu, Xian-Li Meng, Yi Zhang, Jin-Ming Lin","doi":"10.1021/acs.analchem.5c00672","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00672","url":null,"abstract":"Hydrogel microparticles are versatile tools for organ modeling due to their simplicity, uniformity, and customizability, yet their limited physiological relevance constrains practical applications. In this study, a heart microparticle model that incorporates endocardial and myocardial structures and functions was developed. Hydrogel microparticles with rough surfaces, embedded with cardiomyocytes, were created using a custom-designed microfluidic device. Surface modification with matrigel enhanced the adhesion and connectivity of endothelial cells, enabling the formation of a densely packed endothelial layer. Real-time analysis, combining microparticle culture with a microfluidic chip-mass spectrometry system, demonstrated the utility of these particles in detecting the cardiotoxicity of heart-related drugs. For example, the analysis revealed that the cardiotoxicity of aconitine and Tie-bang-chui (TBC) was associated with elevated lactate and succinate levels, while processed TBC mitigated this toxicity of TBC by reducing these metabolites. These biomimetic microparticle models provide a novel platform for real-time metabolite analysis and cardiotoxicity research.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"40 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806139","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 : 2025-04-08DOI: 10.1021/acs.analchem.5c01239
Denise Di Lena, Edoardo Sisti, Erik Brass, Erica Belforte, Bruna Marini, Alessandro Porchetta, Laura Squarcia, Eleonora Da Pozzo, Alessandro Bertucci, Rudy Ippodrino
Therapeutic drug monitoring (TDM) is increasingly valuable for tailoring personalized therapy, particularly in managing chronic inflammatory diseases where overtreatment can cause significant side effects. Monoclonal antibodies (mAbs), a primary therapeutic approach for these conditions, face challenges from antidrug antibodies (ADAs), which can reduce mAb bioavailability and efficacy. To address these issues, we utilized Tumor Necrosis Factor α (TNF-α) as a binding moiety in a fluorescence-based programmable nanosensor within the NanoHYBRID (NH) platform developed by Ulisse Biomed S.p.A. By directly conjugating TNF-α to DNA probes, we developed a rapid, homogeneous, affinity-based assay capable of detecting multiple mAbs targeting distinct epitopes on the same protein. This NH platform effectively detected therapeutic concentrations of clinically relevant mAbs, such as Infliximab, Adalimumab, and Golimumab, in blood serum samples in a one-step process, bypassing the need for time-intensive washing steps. Moreover, the NH sensor exhibited heightened sensitivity to the presence of ADA, which impacted drug quantification, indicating its utility for monitoring bioavailable mAb levels. Compared to ELISA, the NH platform demonstrated superior sensitivity to ADAs, suggesting its potential as a highly specific, modular solution for TDM. This modular design allows the NH platform to create multiepitope nanosensors capable of measuring bioavailable mAbs in a single step.
治疗药物监测（TDM）在定制个性化疗法方面的价值与日俱增，尤其是在治疗慢性炎症性疾病方面，过度治疗可能会导致严重的副作用。单克隆抗体（mAbs）是治疗这些疾病的主要方法，但它面临着抗药抗体（ADAs）的挑战，因为抗药抗体会降低 mAb 的生物利用度和疗效。为了解决这些问题，我们利用肿瘤坏死因子α（TNF-α）作为Ulisse Biomed S.p.A.公司开发的NanoHYBRID（NH）平台中基于荧光的可编程纳米传感器的结合分子。通过直接将TNF-α与DNA探针结合，我们开发出了一种快速、均质、基于亲和力的检测方法，能够检测针对同一蛋白质上不同表位的多种mAb。这种 NH 平台可有效检测血清样本中临床相关 mAbs（如英夫利昔单抗、阿达木单抗和戈利木单抗）的治疗浓度，一步到位，无需耗时的洗涤步骤。此外，NH 传感器对影响药物定量的 ADA 的存在表现出更高的灵敏度，这表明它可用于监测生物可利用的 mAb 水平。与酶联免疫吸附法相比，NH 平台对 ADA 的灵敏度更高，这表明它有潜力成为 TDM 的高特异性模块化解决方案。这种模块化设计使 NH 平台能够创建多位点纳米传感器，只需一个步骤就能测量生物可利用的 mAb。
{"title":"Rapid, Single-Step Monitoring of Monoclonal Antibody Bioavailability by Using a TNF-α-Based Multiepitope DNA Nanoswitch","authors":"Denise Di Lena, Edoardo Sisti, Erik Brass, Erica Belforte, Bruna Marini, Alessandro Porchetta, Laura Squarcia, Eleonora Da Pozzo, Alessandro Bertucci, Rudy Ippodrino","doi":"10.1021/acs.analchem.5c01239","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c01239","url":null,"abstract":"Therapeutic drug monitoring (TDM) is increasingly valuable for tailoring personalized therapy, particularly in managing chronic inflammatory diseases where overtreatment can cause significant side effects. Monoclonal antibodies (mAbs), a primary therapeutic approach for these conditions, face challenges from antidrug antibodies (ADAs), which can reduce mAb bioavailability and efficacy. To address these issues, we utilized Tumor Necrosis Factor α (TNF-α) as a binding moiety in a fluorescence-based programmable nanosensor within the NanoHYBRID (NH) platform developed by Ulisse Biomed S.p.A. By directly conjugating TNF-α to DNA probes, we developed a rapid, homogeneous, affinity-based assay capable of detecting multiple mAbs targeting distinct epitopes on the same protein. This NH platform effectively detected therapeutic concentrations of clinically relevant mAbs, such as Infliximab, Adalimumab, and Golimumab, in blood serum samples in a one-step process, bypassing the need for time-intensive washing steps. Moreover, the NH sensor exhibited heightened sensitivity to the presence of ADA, which impacted drug quantification, indicating its utility for monitoring bioavailable mAb levels. Compared to ELISA, the NH platform demonstrated superior sensitivity to ADAs, suggesting its potential as a highly specific, modular solution for TDM. This modular design allows the NH platform to create multiepitope nanosensors capable of measuring bioavailable mAbs in a single step.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"37 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798268","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 : 2025-04-08DOI: 10.1021/acs.analchem.4c06950
Yaxin Zheng, Xinyue Ma, Shuyao Zhou, Wenwen Lei, Xuan Luo, Lei Zhou, Keming Xu, Wenying Zhong
Tracking autophagy in cancer cells is crucial for enhancing cancer therapies. Existing methods are often inefficient and cannot distinguish cancer from normal cells during autophagy. Herein, a sequentially activated peptide probe, NBD-1p-Dabcyl, was developed for achieving cancer cell-specific imaging of autophagy. The probe self-assembled and fluoresced brightly upon sequential processing by alkaline phosphatase (ALP) and autophagy-related protease (ATG4B), where NBD-1p-Dabcyl was dephosphorylated by ALP to give NBD-1-Dabcyl, which was then processed by ATG4B into nanofibers emitting strong fluorescence. Notably, the bright fluorescence of NBD was observed in cancer cells MDA-MB-231 and HeLa, while normal cells NIH3T3 exhibited weaker fluorescence, allowing differentiation between cancer and normal cells using a rapamycin (Rap)-induced autophagy cell model. The enhanced fluorescence in cancer cells was attributed to the higher activities of intracellular ALP and ATG4B. Next, NBD-1p-Dabcyl was used to assess the inhibition efficiency of an autophagy inhibitor NSC 185058 in MDA-MB-231 cells, where a strong correlation between fluorescence intensity and inhibitor concentration suggested that NBD-1p-Dabcyl could predict the activity of autophagy inhibitors. Finally, animal experiments revealed that NBD-1p-Dabcyl effectively facilitated in situ fluorescence imaging of autophagy in tumor tissues. The design of this sequentially activated peptide probe offers a practical approach for monitoring autophagy in cancer cells, enabling high-throughput screening of autophagy inhibitors for cancer therapy.
{"title":"Alkaline Phosphatase and ATG4B Sequentially Activated Fluorescent Probe for Cancer Cell-Specific Live Imaging of Autophagy","authors":"Yaxin Zheng, Xinyue Ma, Shuyao Zhou, Wenwen Lei, Xuan Luo, Lei Zhou, Keming Xu, Wenying Zhong","doi":"10.1021/acs.analchem.4c06950","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06950","url":null,"abstract":"Tracking autophagy in cancer cells is crucial for enhancing cancer therapies. Existing methods are often inefficient and cannot distinguish cancer from normal cells during autophagy. Herein, a sequentially activated peptide probe, NBD-1p-Dabcyl, was developed for achieving cancer cell-specific imaging of autophagy. The probe self-assembled and fluoresced brightly upon sequential processing by alkaline phosphatase (ALP) and autophagy-related protease (ATG4B), where NBD-1p-Dabcyl was dephosphorylated by ALP to give NBD-1-Dabcyl, which was then processed by ATG4B into nanofibers emitting strong fluorescence. Notably, the bright fluorescence of NBD was observed in cancer cells MDA-MB-231 and HeLa, while normal cells NIH3T3 exhibited weaker fluorescence, allowing differentiation between cancer and normal cells using a rapamycin (Rap)-induced autophagy cell model. The enhanced fluorescence in cancer cells was attributed to the higher activities of intracellular ALP and ATG4B. Next, NBD-1p-Dabcyl was used to assess the inhibition efficiency of an autophagy inhibitor NSC 185058 in MDA-MB-231 cells, where a strong correlation between fluorescence intensity and inhibitor concentration suggested that NBD-1p-Dabcyl could predict the activity of autophagy inhibitors. Finally, animal experiments revealed that NBD-1p-Dabcyl effectively facilitated in situ fluorescence imaging of autophagy in tumor tissues. The design of this sequentially activated peptide probe offers a practical approach for monitoring autophagy in cancer cells, enabling high-throughput screening of autophagy inhibitors for cancer therapy.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"29 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798266","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}
Cathodic photoelectrochemical (PEC) assay with superior anti-interference capacity and high photocorrosion resistance demonstrates great advantages in real sample analysis. However, it suffers from unsatisfactory cathodic PEC response, resulting in inferior detection performance. Herein, a hot-hole injection effect (HIE)-enabled PEC signal modulation strategy with exceptional target responsiveness is first proposed based on atom-shared plasmonic Bi-Bi2O3 (AS-BBO) hetero-nanostructure with enriched oxygen vacancy. The coshared Bi atoms at the AS-BBO heterointerface foster an atomic-level intimate-contact interface between Bi2O3 and plasmonic Bi. This elaborately designed configuration effectively shortens the carrier diffusion distance and reduces the interfacial energy barrier, thereby enabling efficient injection of short-lived hot holes from plasmonic Bi into Bi2O3 photocathode, leading to a greatly increased charge carrier density. Meanwhile, the oxygen vacancies efficiently prolong the lifetime of the charge carriers in AS-BBO, further improving photo-to-current conversion efficiency. Consequently, the AS-BBO generated a significantly enhanced cathodic PEC signal due to the HIE facilitated by oxygen vacancies. To realize sensitive photocathodic analysis, target-level controlled HIE efficacy was designed by exploiting CoFe2O4 as a hole sink to harvest photoinduced holes from AS-BBO, which resulted in a sharp quenching of the robust initial photocurrent signal owing to diminished HIE. Leveraging this substantial photocurrent variation, a highly sensitive paper-based photocathodic sensing platform was developed for microRNA-221 detection, achieving a low detection limit (80 aM) and a wide linear range (0.25 fM to 2 nM). This work laid the foundation for exploiting HIE as an efficient signal modulation strategy for high-performance photocathodic analysis.
{"title":"Hot-Hole Injection-Enabled Efficient Signal Modulation for Boosting Sensitive Paper-Based Photocathodic Analysis through an Atom-Shared Plasmonic Hetero-Nanostructure","authors":"Haihan Yu, Chuanyi Tu, Hongshuo Liu, Lina Zhang, Chaomin Gao, Yan Zhang, Peihua Zhu, Shenguang Ge, Hongmei Yang, Jinghua Yu","doi":"10.1021/acs.analchem.5c00536","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00536","url":null,"abstract":"Cathodic photoelectrochemical (PEC) assay with superior anti-interference capacity and high photocorrosion resistance demonstrates great advantages in real sample analysis. However, it suffers from unsatisfactory cathodic PEC response, resulting in inferior detection performance. Herein, a hot-hole injection effect (HIE)-enabled PEC signal modulation strategy with exceptional target responsiveness is first proposed based on atom-shared plasmonic Bi-Bi<sub>2</sub>O<sub>3</sub> (AS-BBO) hetero-nanostructure with enriched oxygen vacancy. The coshared Bi atoms at the AS-BBO heterointerface foster an atomic-level intimate-contact interface between Bi<sub>2</sub>O<sub>3</sub> and plasmonic Bi. This elaborately designed configuration effectively shortens the carrier diffusion distance and reduces the interfacial energy barrier, thereby enabling efficient injection of short-lived hot holes from plasmonic Bi into Bi<sub>2</sub>O<sub>3</sub> photocathode, leading to a greatly increased charge carrier density. Meanwhile, the oxygen vacancies efficiently prolong the lifetime of the charge carriers in AS-BBO, further improving photo-to-current conversion efficiency. Consequently, the AS-BBO generated a significantly enhanced cathodic PEC signal due to the HIE facilitated by oxygen vacancies. To realize sensitive photocathodic analysis, target-level controlled HIE efficacy was designed by exploiting CoFe<sub>2</sub>O<sub>4</sub> as a hole sink to harvest photoinduced holes from AS-BBO, which resulted in a sharp quenching of the robust initial photocurrent signal owing to diminished HIE. Leveraging this substantial photocurrent variation, a highly sensitive paper-based photocathodic sensing platform was developed for microRNA-221 detection, achieving a low detection limit (80 aM) and a wide linear range (0.25 fM to 2 nM). This work laid the foundation for exploiting HIE as an efficient signal modulation strategy for high-performance photocathodic analysis.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"43 3 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806138","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}
Chemiluminescence (CL) as a powerful analytical tool has garnered increasing interest. However, traditional molecular-based CL luminophores suffer from low emission efficiency due to limited total CL photons emitted per luminophore, driving efforts to explore amplified strategies or novel probes to boost the emission. Although metal nanoclusters (NCs) as luminescent nanoprobes have been extensively studied for electrochemiluminescence and photoluminescence (PL) owing to their intriguing luminescent properties, the CL performance using metal NCs as emitters is often ignored. Herein, based on the synergistic effect within the bimetallic NCs, a series of glutathione-coated Au–Ag bimetallic NCs (GSH-AuAg NCs) were optimized by adjusting precursor ratios and achieved the maximum CL response at a Au:Ag molar ratio of 5:1. To our surprise, CL emission with GSH-AuAg NCs as emitters was triggered with oxidant reagents such as KMnO4, and bimetallic NCs display boosted CL emission (ca. 6.2-fold) compared to monometallic NCs owing to the synergistic effect on enhancing the emission efficiency. Surface-defect-involved CL was revealed by collecting the CL spectra with a maximum emission wavelength of around 750 nm and an obvious red shift of 140 nm compared to PL spectra. The mechanism reveals the KMnO4-injected hole into the valence band through redox reactions with GSH ligands, leading to CL emission by efficient radiative charge recombination with pre-existing electron. A sensing platform based on the GSH-AuAg NCs/oxidant system was constructed for sensing H2O2 and glucose, demonstrating the potential of GSH-AuAg NCs as CL emitters in analytical applications.
{"title":"Surface-Defect-Involved Chemiluminescence Boosted by Gold–Silver Bimetallic Nanoclusters for Bioanalysis","authors":"Tongtong Zhai, Luyao Zhang, Sipeng Tian, Zhangpeng Xu, Xiushuang Fan, Jing Li, Erkang Wang","doi":"10.1021/acs.analchem.5c00219","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00219","url":null,"abstract":"Chemiluminescence (CL) as a powerful analytical tool has garnered increasing interest. However, traditional molecular-based CL luminophores suffer from low emission efficiency due to limited total CL photons emitted per luminophore, driving efforts to explore amplified strategies or novel probes to boost the emission. Although metal nanoclusters (NCs) as luminescent nanoprobes have been extensively studied for electrochemiluminescence and photoluminescence (PL) owing to their intriguing luminescent properties, the CL performance using metal NCs as emitters is often ignored. Herein, based on the synergistic effect within the bimetallic NCs, a series of glutathione-coated Au–Ag bimetallic NCs (GSH-AuAg NCs) were optimized by adjusting precursor ratios and achieved the maximum CL response at a Au:Ag molar ratio of 5:1. To our surprise, CL emission with GSH-AuAg NCs as emitters was triggered with oxidant reagents such as KMnO<sub>4</sub>, and bimetallic NCs display boosted CL emission (ca. 6.2-fold) compared to monometallic NCs owing to the synergistic effect on enhancing the emission efficiency. Surface-defect-involved CL was revealed by collecting the CL spectra with a maximum emission wavelength of around 750 nm and an obvious red shift of 140 nm compared to PL spectra. The mechanism reveals the KMnO<sub>4</sub>-injected hole into the valence band through redox reactions with GSH ligands, leading to CL emission by efficient radiative charge recombination with pre-existing electron. A sensing platform based on the GSH-AuAg NCs/oxidant system was constructed for sensing H<sub>2</sub>O<sub>2</sub> and glucose, demonstrating the potential of GSH-AuAg NCs as CL emitters in analytical applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"18 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143806135","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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