Pub Date : 2025-04-23DOI: 10.1021/acs.analchem.5c01013
Yanwen Liu, Su Li, Xinmeng Wang, Xiya Liu, Juan Wang, Zhihong Liu
The development of long-term and in situ in vivo monitoring techniques is critical for environmental biology, life sciences, and analytical chemistry. However, existing in vivo analysis methods are limited by the complex and large instruments or adverse impacts of rigid implanted substrates on living organisms, making it difficult to achieve continuous in situ detection. Herein, taking advantage of the flexibility and biocompatibility of the hydrogel fiber and solving its instability or opacity problems caused by ionic or polymer conduction for hydrogel fibers, a photoelectrochemical (PEC) hydrogel fiber free of conventional rigid substrate support is successfully prepared and achieves long-term tracking of persistent organic pollutants in free-behaving fish, timely identifying their environmental ecological risks. This support-free PEC fiber exhibits fascinating properties of electrical and light conductivity, flexibility, antifouling ability, and biocompatibility, allowing it to be implanted in vivo for 70 days without experiencing significant loss of sensing performance and causing apparent inflammation and immune responses. Moreover, the fabricated fiber not only achieves in vitro pentachlorophenol detection with high selectivity, low detection limit, good reproducibility, and dual-mode sensing but also realizes in vivo monitoring of pentachlorophenol enriched in fish brain for up to 70 days with satisfactory reliability, unraveling its tempting potential for various in vivo application.
{"title":"Support-Free Implantable Photoelectrochemical Hydrogel Fiber Enables Long-Term Monitoring in Free-Behaving Organisms","authors":"Yanwen Liu, Su Li, Xinmeng Wang, Xiya Liu, Juan Wang, Zhihong Liu","doi":"10.1021/acs.analchem.5c01013","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c01013","url":null,"abstract":"The development of long-term and in situ in vivo monitoring techniques is critical for environmental biology, life sciences, and analytical chemistry. However, existing in vivo analysis methods are limited by the complex and large instruments or adverse impacts of rigid implanted substrates on living organisms, making it difficult to achieve continuous in situ detection. Herein, taking advantage of the flexibility and biocompatibility of the hydrogel fiber and solving its instability or opacity problems caused by ionic or polymer conduction for hydrogel fibers, a photoelectrochemical (PEC) hydrogel fiber free of conventional rigid substrate support is successfully prepared and achieves long-term tracking of persistent organic pollutants in free-behaving fish, timely identifying their environmental ecological risks. This support-free PEC fiber exhibits fascinating properties of electrical and light conductivity, flexibility, antifouling ability, and biocompatibility, allowing it to be implanted in vivo for 70 days without experiencing significant loss of sensing performance and causing apparent inflammation and immune responses. Moreover, the fabricated fiber not only achieves in vitro pentachlorophenol detection with high selectivity, low detection limit, good reproducibility, and dual-mode sensing but also realizes in vivo monitoring of pentachlorophenol enriched in fish brain for up to 70 days with satisfactory reliability, unraveling its tempting potential for various in vivo application.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"108 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143862485","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 : 2025-04-23DOI: 10.1021/acs.analchem.5c00035
Salman Khan, Sneha Asok, Veda V. Dasari, Sharayu Magar, Bhagyasree Paila, Anil K. Suresh
Nanomedicine is increasingly being utilized in addressing various eye ailments and holds immense potential in rectifying ocular diseases; however, the interactions between nanomedicines and their route of administration via tear fluid remain poorly understood. When nanoparticles are introduced into the tear fluid, a layer of protein corona is formed on their surface that not only influences the properties and biological fate of nanoparticles but also potentially interferes with the function of endogenous proteins. To investigate the interactions between gold nanoaprticles (AuNPs) and tear fluid, focusing on the physicochemical changes of the particles, and to quantitatively and qualitatively identify the key proteins involved in the corona formation, we employed label-free techniques for material and biophysical characterizations along with proteomic analyses and mass spectrometry. The AuNPs remained stable without forming aggregates, showing only an ∼31 nm increase in hydrodynamic diameter after interacting with tear fluid. Notably, their overall zeta potential increased significantly from −12 to −23 eV due to the supplemented charge by the adsorbed proteins. Proteomic analysis and liquid chromatography/mass spectrometry (LC-MS/MS) identified 31 proteins that were bound with the nanoparticles from a total of 174 proteins that were detected in the tear fluid. Bioinformatic classification revealed an enrichment of specific proteins essential for ocular health; proteins such as clusterin, lactotransferrin, adenosine triphosphate (ATP) synthase, lysozyme, alpha enolase, keratin, apolipoprotein, and epidermal growth factor receptor (EGFR) with pivotal roles in anti-inflammatory, immune response, cell adhesion, cellular organization, plasminogen activation, cell signaling, stress response, and corneal epithelial homeostasis. Overall, our study provides an unresolved comprehensive map of the tear protein corona landscape and its impact on nanoparticle behavior in the tear fluid. These insights must be considered and are valuable for designing safer and more effective nanomedicines for the treatment of various eye diseases.
{"title":"Feeling of an Eye When It Meets the Unseen “Nano”","authors":"Salman Khan, Sneha Asok, Veda V. Dasari, Sharayu Magar, Bhagyasree Paila, Anil K. Suresh","doi":"10.1021/acs.analchem.5c00035","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00035","url":null,"abstract":"Nanomedicine is increasingly being utilized in addressing various eye ailments and holds immense potential in rectifying ocular diseases; however, the interactions between nanomedicines and their route of administration via tear fluid remain poorly understood. When nanoparticles are introduced into the tear fluid, a layer of protein corona is formed on their surface that not only influences the properties and biological fate of nanoparticles but also potentially interferes with the function of endogenous proteins. To investigate the interactions between gold nanoaprticles (AuNPs) and tear fluid, focusing on the physicochemical changes of the particles, and to quantitatively and qualitatively identify the key proteins involved in the corona formation, we employed label-free techniques for material and biophysical characterizations along with proteomic analyses and mass spectrometry. The AuNPs remained stable without forming aggregates, showing only an ∼31 nm increase in hydrodynamic diameter after interacting with tear fluid. Notably, their overall zeta potential increased significantly from −12 to −23 eV due to the supplemented charge by the adsorbed proteins. Proteomic analysis and liquid chromatography/mass spectrometry (LC-MS/MS) identified 31 proteins that were bound with the nanoparticles from a total of 174 proteins that were detected in the tear fluid. Bioinformatic classification revealed an enrichment of specific proteins essential for ocular health; proteins such as clusterin, lactotransferrin, adenosine triphosphate (ATP) synthase, lysozyme, alpha enolase, keratin, apolipoprotein, and epidermal growth factor receptor (EGFR) with pivotal roles in anti-inflammatory, immune response, cell adhesion, cellular organization, plasminogen activation, cell signaling, stress response, and corneal epithelial homeostasis. Overall, our study provides an unresolved comprehensive map of the tear protein corona landscape and its impact on nanoparticle behavior in the tear fluid. These insights must be considered and are valuable for designing safer and more effective nanomedicines for the treatment of various eye diseases.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"42 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866236","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 : 2025-04-23DOI: 10.1021/acs.analchem.5c00714
Jiří Kohoutek, Juan I. Sánchez-Avila, Marie Smutná, Petr Janků, Jana Klánová, Klára Hilscherová
Many analytical methods for thyroid hormone (TH) determination lack sensitivity and/or specificity. The thyroid hormone metabolites (THMs) are usually not measured at all. This study describes the development of sensitive high-throughput analytical methods for determining the total concentration and free fraction of TH and THM in the human serum. For the analysis of the TOTAL fraction, we employed protein precipitation and anionic exchanger solid-phase extraction. For the FREE fraction, ultrafiltration and salt-out liquid partitioning were used. Derivatization using dansyl chloride was employed to enhance the sensitivity of HPLC-ESI-MS/MS analysis. Both protocols were validated according to the European Analytical Guidelines (2002/657/EC). We obtained very good recoveries (73–115%) and precision. Interday coefficients of variation (CVs) for most of the analytes ranged from 1.2 to 16.4%. The sensitivity was excellent with detection limits in the sub ppt range for the majority of TH and THM. A significant enhancement in sensitivity (>10 fold) was achieved through derivatization. The applicability was proved on a set of samples from pregnant women enrolled in the CELSPAC cohort (n = 120). Our TH reference ranges are in good agreement with those reported in the literature. The methods also allowed us to quantify the levels of 11 THM, including some previously undetected THM in total and free fractions, and proved to be suitable for high-throughput routine TH and THM analyses. Our approach offers an important advancement in thyroid hormone analysis. To the best of our knowledge, it is for the first time that data for T1A and T2A as well as for free THM levels in the human serum are published in the literature. Moreover, our study also brings the first information about the levels of most of the THM in pregnant women.
{"title":"Determination of Thyroid Hormones and 11 Metabolites in the Human Serum Using a Simple Derivatization Strategy and Analysis by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry","authors":"Jiří Kohoutek, Juan I. Sánchez-Avila, Marie Smutná, Petr Janků, Jana Klánová, Klára Hilscherová","doi":"10.1021/acs.analchem.5c00714","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00714","url":null,"abstract":"Many analytical methods for thyroid hormone (TH) determination lack sensitivity and/or specificity. The thyroid hormone metabolites (THMs) are usually not measured at all. This study describes the development of sensitive high-throughput analytical methods for determining the total concentration and free fraction of TH and THM in the human serum. For the analysis of the TOTAL fraction, we employed protein precipitation and anionic exchanger solid-phase extraction. For the FREE fraction, ultrafiltration and salt-out liquid partitioning were used. Derivatization using dansyl chloride was employed to enhance the sensitivity of HPLC-ESI-MS/MS analysis. Both protocols were validated according to the European Analytical Guidelines (2002/657/EC). We obtained very good recoveries (73–115%) and precision. Interday coefficients of variation (CVs) for most of the analytes ranged from 1.2 to 16.4%. The sensitivity was excellent with detection limits in the sub ppt range for the majority of TH and THM. A significant enhancement in sensitivity (>10 fold) was achieved through derivatization. The applicability was proved on a set of samples from pregnant women enrolled in the CELSPAC cohort (<i>n</i> = 120). Our TH reference ranges are in good agreement with those reported in the literature. The methods also allowed us to quantify the levels of 11 THM, including some previously undetected THM in total and free fractions, and proved to be suitable for high-throughput routine TH and THM analyses. Our approach offers an important advancement in thyroid hormone analysis. To the best of our knowledge, it is for the first time that data for T1A and T2A as well as for free THM levels in the human serum are published in the literature. Moreover, our study also brings the first information about the levels of most of the THM in pregnant women.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"5 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866238","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 : 2025-04-23DOI: 10.1021/acs.analchem.4c05889
Jiyao Wang, Ruina Zhang, Shihao Li, Binghang Du, Xiaohao Wang, Kai Ni
In Ion Mobility Spectrometry (IMS), ion gates are essential for controlling ion flow, significantly impacting detection sensitivity and resolution. Despite various optimization methods, a clear approach is needed to define the performance limits of ion gates. This study proposes a linear relationship between peak intensity (Ip) and resolving power (Rp) at low ion quantities, introducing it as a reference for evaluating the optimization level. The Tri-State gate (TSG) enables the accurate study of this Ip–Rp relationship, but ion leakage of TSG disrupts control over narrow ion swarms, thereby limiting Rp. After the causes of ion leakage were examined, a preshaping method that removes the gate-opening phase was proposed to precisely control ion injection. This method leverages ion leakage to improve Rp while ensuring the linearity between Ip and Rp. The preserved linearity means avoiding excessive ion loss and shape distortion, ultimately leading to optimal resolving power. Simulations and experiments demonstrate that precise voltage adjustments effectively minimize ion leakage, enhancing resolving power by 50% (reaching a maximum of 106), while the corresponding decrease in signal intensity follows the Ip–Rp linear relationship. This approach expands the accessible range of Ip–Rp combinations without altering their fundamental relationship, reduces discrimination, and establishes a new evaluation method for optimizing ion gate performance in IMS.
{"title":"Achieving Improved Ion Swarm Shaping Based on Ion Leakage Control in Ion Mobility Spectrometry","authors":"Jiyao Wang, Ruina Zhang, Shihao Li, Binghang Du, Xiaohao Wang, Kai Ni","doi":"10.1021/acs.analchem.4c05889","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05889","url":null,"abstract":"In Ion Mobility Spectrometry (IMS), ion gates are essential for controlling ion flow, significantly impacting detection sensitivity and resolution. Despite various optimization methods, a clear approach is needed to define the performance limits of ion gates. This study proposes a linear relationship between peak intensity (<i>I</i><sub>p</sub>) and resolving power (<i>R</i><sub>p</sub>) at low ion quantities, introducing it as a reference for evaluating the optimization level. The Tri-State gate (TSG) enables the accurate study of this <i>I</i><sub>p</sub>–<i>R</i><sub>p</sub> relationship, but ion leakage of TSG disrupts control over narrow ion swarms, thereby limiting <i>R</i><sub>p</sub>. After the causes of ion leakage were examined, a preshaping method that removes the gate-opening phase was proposed to precisely control ion injection. This method leverages ion leakage to improve <i>R</i><sub>p</sub> while ensuring the linearity between <i>I</i><sub>p</sub> and <i>R</i><sub>p</sub>. The preserved linearity means avoiding excessive ion loss and shape distortion, ultimately leading to optimal resolving power. Simulations and experiments demonstrate that precise voltage adjustments effectively minimize ion leakage, enhancing resolving power by 50% (reaching a maximum of 106), while the corresponding decrease in signal intensity follows the <i>I</i><sub>p</sub>–<i>R</i><sub>p</sub> linear relationship. This approach expands the accessible range of <i>I</i><sub>p</sub>–<i>R</i><sub>p</sub> combinations without altering their fundamental relationship, reduces discrimination, and establishes a new evaluation method for optimizing ion gate performance in IMS.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"42 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866786","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 : 2025-04-22DOI: 10.1021/acs.analchem.4c07081
Yue Liu, Yunping Wu, Yi Liu, Qiang Zhang, Hong Yuan, Shen Li, Zhi Li, Bo Wang, Yangyang Chang, Meng Liu
CRISPR-Cas technologies have emerged as powerful biosensing tools for the sensitive and specific detection of non-nucleic acid targets. However, existing biosensing strategies suffer from poor compatibility across diverse targets due to the complicated engineering of crRNA and DNA activator required for the CRISPR-Cas activity regulation. Herein, we report a novel and straightforward strategy for designing CRISPR-Cas12a-based biosensors that function by switching structures from single-stranded (ss)DNA/CRISPR-Cas12a assembly to DNA activator/CRISPR-Cas12a complex in the presence of target bacterium. The strategy begins with a ssDNA assembly made of a trans-acting RNA-cleaving DNAzyme (tRCD) and an RNA/DNA chimeric substrate (RCS). The ssDNA assembly has the ability to bind Cas12a nonspecifically, thus indeed blocking the CRISPR-Cas12a activity. By exploiting the specific recognition and cleavage capacities of tRCD for RCS in the presence of a target, the target-bound tRCD and the cleaved RCS are released from Cas12a, thus restoring the CRISPR-Cas12a activity. This method has been successfully applied for the sensitive (detection limit: 102 CFU/mL) detection of Escherichia coli (E. coli, EC) and Burkholderia gladioli (B. gladioli, BG). For the blind testing of 30 clinical urine samples, it exhibited 100% sensitivity and 100% specificity in identifying E. coli-associated urinary tract infections (UTIs).
{"title":"Arrest of CRISPR-Cas12a by Nonspecific Single-Stranded DNA for Biosensing","authors":"Yue Liu, Yunping Wu, Yi Liu, Qiang Zhang, Hong Yuan, Shen Li, Zhi Li, Bo Wang, Yangyang Chang, Meng Liu","doi":"10.1021/acs.analchem.4c07081","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c07081","url":null,"abstract":"CRISPR-Cas technologies have emerged as powerful biosensing tools for the sensitive and specific detection of non-nucleic acid targets. However, existing biosensing strategies suffer from poor compatibility across diverse targets due to the complicated engineering of crRNA and DNA activator required for the CRISPR-Cas activity regulation. Herein, we report a novel and straightforward strategy for designing CRISPR-Cas12a-based biosensors that function by switching structures from single-stranded (ss)DNA/CRISPR-Cas12a assembly to DNA activator/CRISPR-Cas12a complex in the presence of target bacterium. The strategy begins with a ssDNA assembly made of a trans-acting RNA-cleaving DNAzyme (tRCD) and an RNA/DNA chimeric substrate (RCS). The ssDNA assembly has the ability to bind Cas12a nonspecifically, thus indeed blocking the CRISPR-Cas12a activity. By exploiting the specific recognition and cleavage capacities of tRCD for RCS in the presence of a target, the target-bound tRCD and the cleaved RCS are released from Cas12a, thus restoring the CRISPR-Cas12a activity. This method has been successfully applied for the sensitive (detection limit: 10<sup>2</sup> CFU/mL) detection of <i>Escherichia coli</i> (<i>E. coli</i>, EC) and <i>Burkholderia gladioli</i> (<i>B. gladioli</i>, BG). For the blind testing of 30 clinical urine samples, it exhibited 100% sensitivity and 100% specificity in identifying <i>E. coli</i>-associated urinary tract infections (UTIs).","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"17 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857455","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 : 2025-04-22DOI: 10.1021/acs.analchem.5c00783
Jin Wang, Xiangmei Li, Hongtao Lei, Juewen Liu
Being a neurotoxin and carcinogen, acrylamide has been an important target for developing biosensors. DNA aptamers are attractive for making biosensors due to their programmable structure, low cost, and ease of modification. However, DNA aptamers have poor affinities to low-binding epitope target molecules such as acrylamide. In this work, an aptamer for acrylamide was isolated with an apparent Kd of 10.5 mM using a thioflavin T fluorescence assay and 4.7 mM using the fluorescence strand-displacement assay. To improve binding affinity, acrylamide was reacted with xanthydrol to form a covalent adduct, and a new aptamer selected for this adduct achieved a Kd of 20 nM using the strand-displacement assay, representing an improvement of 235,000-fold. Using the strand-displacement biosensor, a limit of detection of 4.2 nM was achieved for the adduct. This work demonstrates a practical route to convert low epitope targets to high-affinity targets for aptamer binding and bioanalytical applications.
{"title":"Increasing Aptamer Affinity from Millimolar to Nanomolar by Forming a Covalent Adduct for Detecting Acrylamide","authors":"Jin Wang, Xiangmei Li, Hongtao Lei, Juewen Liu","doi":"10.1021/acs.analchem.5c00783","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00783","url":null,"abstract":"Being a neurotoxin and carcinogen, acrylamide has been an important target for developing biosensors. DNA aptamers are attractive for making biosensors due to their programmable structure, low cost, and ease of modification. However, DNA aptamers have poor affinities to low-binding epitope target molecules such as acrylamide. In this work, an aptamer for acrylamide was isolated with an apparent <i>K</i><sub>d</sub> of 10.5 mM using a thioflavin T fluorescence assay and 4.7 mM using the fluorescence strand-displacement assay. To improve binding affinity, acrylamide was reacted with xanthydrol to form a covalent adduct, and a new aptamer selected for this adduct achieved a <i>K</i><sub>d</sub> of 20 nM using the strand-displacement assay, representing an improvement of 235,000-fold. Using the strand-displacement biosensor, a limit of detection of 4.2 nM was achieved for the adduct. This work demonstrates a practical route to convert low epitope targets to high-affinity targets for aptamer binding and bioanalytical applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"20 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857457","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 : 2025-04-22DOI: 10.1021/acs.analchem.4c05730
Anatolii Spesyvyi, Marek Cebecauer, Ján Žabka, Agnieszka Olżyńska, Michaela Malečková, Zuzana Johanovská, Miroslav Polášek, Ales Charvat, Bernd Abel
Extracellular vesicles (EVs) are membranous particles released by cells and are considered to be promising sources of biomarkers for various diseases. Mass spectrometry (MS) analysis of EVs requires a sample of purified and detergent-lysed EVs. Purification of EVs is laborious, based on size, density, or surface nature, and requires large amounts of the source material (e.g., blood, spinal fluid). We have employed synthetically produced large unilamellar lipid vesicles (LUVs) as analogs of EVs to demonstrate an alternative approach to vesicle separation for subsequent mass spectrometry analysis of their composition. Mass-to-charge ratio m/z separation by frequency-controlled quadrupole was employed to filter narrow-size distributions of LUVs from a water sample. Lipid vesicles were positively charged with nanoelectrospray and transferred into a vacuum using two wide m/z-range frequency-controlled quadrupoles. The m/z, charges, and masses of individual vesicles were obtained by the nondestructive single-pass charge detector. The resolving mode of the second quadrupole with m/z RSD < 10% allowed to separate size selected distributions of vesicles with modal diameters of 88, 112, 130, 162, and 190 nm at corresponding quadrupole m/z settings of 2.5 × 105, 5 × 105, 8 × 105, 1.5 × 106, and 2.5 × 106, respectively with a rate of 20–100 counts per minute. The distributions of bioparticles with masses between 108 and 1010 Da were separated from human blood serum in the pilot experiment. The presented approach for lipid vesicle separation encourages the development of new techniques for the direct mass-spectrometric analysis of biomarkers in MS-separated EVs in a vacuum.
{"title":"Separation and Detection of Charged Unilamellar Vesicles in Vacuum by a Frequency-Controlled Quadrupole Mass Sensor","authors":"Anatolii Spesyvyi, Marek Cebecauer, Ján Žabka, Agnieszka Olżyńska, Michaela Malečková, Zuzana Johanovská, Miroslav Polášek, Ales Charvat, Bernd Abel","doi":"10.1021/acs.analchem.4c05730","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05730","url":null,"abstract":"Extracellular vesicles (EVs) are membranous particles released by cells and are considered to be promising sources of biomarkers for various diseases. Mass spectrometry (MS) analysis of EVs requires a sample of purified and detergent-lysed EVs. Purification of EVs is laborious, based on size, density, or surface nature, and requires large amounts of the source material (e.g., blood, spinal fluid). We have employed synthetically produced large unilamellar lipid vesicles (LUVs) as analogs of EVs to demonstrate an alternative approach to vesicle separation for subsequent mass spectrometry analysis of their composition. Mass-to-charge ratio <i>m</i>/<i>z</i> separation by frequency-controlled quadrupole was employed to filter narrow-size distributions of LUVs from a water sample. Lipid vesicles were positively charged with nanoelectrospray and transferred into a vacuum using two wide <i>m</i>/<i>z</i>-range frequency-controlled quadrupoles. The <i>m</i>/<i>z</i>, charges, and masses of individual vesicles were obtained by the nondestructive single-pass charge detector. The resolving mode of the second quadrupole with <i>m</i>/<i>z</i> RSD < 10% allowed to separate size selected distributions of vesicles with modal diameters of 88, 112, 130, 162, and 190 nm at corresponding quadrupole <i>m</i>/<i>z</i> settings of 2.5 × 10<sup>5</sup>, 5 × 10<sup>5</sup>, 8 × 10<sup>5</sup>, 1.5 × 10<sup>6</sup>, and 2.5 × 10<sup>6</sup>, respectively with a rate of 20–100 counts per minute. The distributions of bioparticles with masses between 10<sup>8</sup> and 10<sup>10</sup> Da were separated from human blood serum in the pilot experiment. The presented approach for lipid vesicle separation encourages the development of new techniques for the direct mass-spectrometric analysis of biomarkers in MS-separated EVs in a vacuum.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"6 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857454","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 : 2025-04-22DOI: 10.1021/acs.analchem.5c00089
Xinli Yang, Hongwu Li, Dexing Li, Maoguo Luo, Renxiao Liu, Yinglu Ji, Chunhui Wang, Xiaochun Wu, Guanglu Ge
Single-particle inductively coupled plasma mass spectrometry (spICP-MS) is a sensitive and convenient technique for characterizing nanoparticles in suspension, enabling the determination of particle size, particle size distribution (PSD) and particle number concentration (PNC) from time-resolved signals of particle events. Accurate acquisition and modeling of event intensity distributions (EIDs) are critical steps in expanding functionality and improving measurement accuracy. In this work, we explored the broadening factors of EID, establishing and validating a robust instrument response function (IRF) in the form of a mixed Poisson distribution that reliably correlates PSD with EID across varying operating conditions. The EID tailing caused by particle coincidence is quantified and eliminated through Monte Carlo simulations grounded in the homogeneous Poisson process, and then the recovered EID is deconvoluted by IRF to yield high-fidelity PSD, improving the accuracy of PSD and PNC obtained by spICP-MS. For monodisperse gold nanoparticles (AuNPs) and AuNP mixtures, stable PSDs can be recovered from the broadened EIDs by IRF deconvolution, yielding results closely aligned with those obtained by transmission electron microscopy, thus increasing the size resolution to about 7 nm in both simulated and actual samples. The application of IRF to the measurement of nanoparticle agglomerates was also demonstrated, and the probability mass function of agglomeration numbers was successfully resolved. This technique is expected to leverage the high-throughput advantages of spICP-MS in the quantification of nanoparticle mixtures or agglomerates.
{"title":"Modeling, Validation, and Application of Instrument Response Function in the Form of Mixed Poisson Distribution for Single-Particle ICP-MS","authors":"Xinli Yang, Hongwu Li, Dexing Li, Maoguo Luo, Renxiao Liu, Yinglu Ji, Chunhui Wang, Xiaochun Wu, Guanglu Ge","doi":"10.1021/acs.analchem.5c00089","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00089","url":null,"abstract":"Single-particle inductively coupled plasma mass spectrometry (spICP-MS) is a sensitive and convenient technique for characterizing nanoparticles in suspension, enabling the determination of particle size, particle size distribution (PSD) and particle number concentration (PNC) from time-resolved signals of particle events. Accurate acquisition and modeling of event intensity distributions (EIDs) are critical steps in expanding functionality and improving measurement accuracy. In this work, we explored the broadening factors of EID, establishing and validating a robust instrument response function (IRF) in the form of a mixed Poisson distribution that reliably correlates PSD with EID across varying operating conditions. The EID tailing caused by particle coincidence is quantified and eliminated through Monte Carlo simulations grounded in the homogeneous Poisson process, and then the recovered EID is deconvoluted by IRF to yield high-fidelity PSD, improving the accuracy of PSD and PNC obtained by spICP-MS. For monodisperse gold nanoparticles (AuNPs) and AuNP mixtures, stable PSDs can be recovered from the broadened EIDs by IRF deconvolution, yielding results closely aligned with those obtained by transmission electron microscopy, thus increasing the size resolution to about 7 nm in both simulated and actual samples. The application of IRF to the measurement of nanoparticle agglomerates was also demonstrated, and the probability mass function of agglomeration numbers was successfully resolved. This technique is expected to leverage the high-throughput advantages of spICP-MS in the quantification of nanoparticle mixtures or agglomerates.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"41 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143857456","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 : 2025-04-22DOI: 10.1021/acs.analchem.4c06606
Hong Yan, Mingpan Qi, Hong Li
CRISPR/Cas9 is widely recognized as the most effective, efficient, and precise genome editing tool, inspiring numerous applications in basic science, medicine, and biotechnology. In the CRISPR/Cas9 system, single guide RNA (sgRNA) and Cas9 enzyme form a ribonucleoprotein complex that specifically and effectively cleaves target DNA. Accurate sequencing of sgRNA, particularly identifying the target sequence within the first 20 nucleotides (nt) at the 5′-end, is crucial for quality assurance and regulatory compliance. In this study, we used site-directed cleavage using ribonuclease H (RNase H) and DNAzyme for the first time to digest 100 nt sgRNA, achieving full sequencing with 100% coverage by analyzing the two cleaved fragments separately via LC MS/MS. We evaluated four different DNA-RNA chimeras as capture probes for the RNase H site-directed cleavage approach, finding that the chimera with four deoxynucleotides provided the most specific cleavage. Compared to RNase H, the DNAzyme demonstrated higher specificity and stability for 100 nt sgRNA digestion, successfully identifying up to 200 nucleotides of large RNA with 100% sequence coverage by fully sequencing the four short cleaved fragments. Due to the high specificity of DNAzyme cleavage, we used this method to study the designed 5′-end N-X truncated impurities of 100 nt sgRNA, demonstrating accurate identification and relative quantification. For 100 nt sgRNA, the limited available cleavage site was set on the scaffold sequence for both site-directed cleavage approaches, and the captured probes designed for RNase H and DNAzyme can be universally applied to sequence all 100 nt sgRNAs because of the conserved scaffold sequence.
CRISPR/Cas9 被公认为最有效、高效和精确的基因组编辑工具，在基础科学、医学和生物技术领域得到了广泛应用。在 CRISPR/Cas9 系统中，单导 RNA（sgRNA）和 Cas9 酶形成核糖核蛋白复合物，能特异、有效地切割目标 DNA。对 sgRNA 进行精确测序，特别是识别 5′端前 20 个核苷酸（nt）内的靶序列，对于保证质量和符合法规要求至关重要。在这项研究中，我们首次使用核糖核酸酶 H（RNase H）和 DNA 酶进行定点定向裂解来消化 100 nt sgRNA，通过 LC MS/MS 分别分析两个裂解片段，实现了 100%覆盖率的全测序。我们评估了四种不同的 DNA-RNA 嵌合体作为 RNase H 定点裂解方法的捕获探针，发现含有四个脱氧核苷酸的嵌合体能提供最特异的裂解。与RNase H相比，DNA酶在消化100 nt sgRNA时表现出更高的特异性和稳定性，通过对四个短裂解片段进行完全测序，成功鉴定了多达200个核苷酸的大RNA，序列覆盖率达100%。由于 DNA 酶裂解的特异性很高，我们用这种方法研究了设计的 100 nt sgRNA 的 5′ 端 N-X 截短杂质，结果表明能准确识别和相对定量。对于 100 nt sgRNA，有限的裂解位点被设置在两种定点裂解方法的支架序列上，由于支架序列的保守性，为 RNase H 和 DNAzyme 设计的捕获探针可普遍应用于所有 100 nt sgRNA 的测序。
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Pub Date : 2025-04-22DOI: 10.1021/acs.analchem.4c06853
Yue Zhu, Lei Zhang, Sha Li, Yingying Luo, Long Xiao, Qiao Yu, Zhong-Xing Jiang, Xin Zhou, Shizhen Chen
Caspase-3 (Cas-3) is a crucial cysteine protease involved in executing cell apoptosis, a process that is a hallmark of tumor cell apoptosis response to chemotherapy. Developing molecular imaging probes that specifically detect apoptotic signals during tumor treatment is considered to be one of the most important and effective strategies for reducing cancer-associated death rates and improving treatment outcomes. However, achieving a dual-enhanced bimodal probe in a single molecule remains a significant challenge. In this study, we developed a 1H/19F dual-enhanced magnetic resonance imaging (MRI) probe, CF3DEVDFFFK(Fmoc)-Gd, responsive to caspase-3 for in vivo imaging of apoptotic cells. Upon interaction with caspase-3, CF3DEVDFFFK(Fmoc)-Gd efficiently splits into two components CF3DEVD and FFFK(Fmoc)-C2-Gd, where FFFK(Fmoc)-C2-Gd subsequently self-assembles into nanofibers. This process activates both 19F MRI and 1H MRI, with longitudinal relaxivity (r1) increasing from 9.38 ± 0.22 to 23.24 ± 2.33 mM–1 s–1 at 0.5 T and turning on the 19F MRI signal due to the absence of the paramagnetic relaxation enhancement (PRE) effect. In vivo imaging results demonstrated that, after systemic administration, CF3DEVDFFFK(Fmoc)-Gd effectively accumulated in apoptotic 4T1 tumor tissues, resulting in significantly enhanced 1H MRI signals for visualization of caspase-3 activity in doxorubicin-treated apoptotic 4T1 tumor tissues, with signal intensity three times greater than that of Gd-DTPA. 19F MRI further complemented 1H MRI, with a notable recovery of the 19F MRI signal after intratumoral injection. These results confirm that CF3DEVDFFFK(Fmoc)-Gd effectively reports tumor apoptosis through combined 1H and 19F MRI, offering a promising approach for the preliminary assessment of antitumor efficacy in vivo.
{"title":"Caspase-3-Responsive Dual-Enhanced 1H/19F MRI Bimodal Probe for In Vivo Tumor Apoptosis Imaging","authors":"Yue Zhu, Lei Zhang, Sha Li, Yingying Luo, Long Xiao, Qiao Yu, Zhong-Xing Jiang, Xin Zhou, Shizhen Chen","doi":"10.1021/acs.analchem.4c06853","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06853","url":null,"abstract":"Caspase-3 (Cas-3) is a crucial cysteine protease involved in executing cell apoptosis, a process that is a hallmark of tumor cell apoptosis response to chemotherapy. Developing molecular imaging probes that specifically detect apoptotic signals during tumor treatment is considered to be one of the most important and effective strategies for reducing cancer-associated death rates and improving treatment outcomes. However, achieving a dual-enhanced bimodal probe in a single molecule remains a significant challenge. In this study, we developed a <sup>1</sup>H/<sup>19</sup>F dual-enhanced magnetic resonance imaging (MRI) probe, <b>CF</b><sub><b>3</b></sub><b>DEVDFFFK(Fmoc)-Gd</b>, responsive to caspase-3 for <i>in vivo</i> imaging of apoptotic cells. Upon interaction with caspase-3, <b>CF</b><sub><b>3</b></sub><b>DEVDFFFK(Fmoc)-Gd</b> efficiently splits into two components <b>CF</b><sub><b>3</b></sub><b>DEVD</b> and <b>FFFK(Fmoc)-C</b><sub><b>2</b></sub><b>-Gd</b>, where <b>FFFK(Fmoc)-C</b><sub><b>2</b></sub><b>-Gd</b> subsequently self-assembles into nanofibers. This process activates both <sup>19</sup>F MRI and <sup>1</sup>H MRI, with longitudinal relaxivity (<i>r</i><sub>1</sub>) increasing from 9.38 ± 0.22 to 23.24 ± 2.33 mM<sup>–1</sup> s<sup>–1</sup> at 0.5 T and turning on the <sup>19</sup>F MRI signal due to the absence of the paramagnetic relaxation enhancement (PRE) effect. <i>In vivo</i> imaging results demonstrated that, after systemic administration, <b>CF</b><sub><b>3</b></sub><b>DEVDFFFK(Fmoc)-Gd</b> effectively accumulated in apoptotic 4T1 tumor tissues, resulting in significantly enhanced <sup>1</sup>H MRI signals for visualization of caspase-3 activity in doxorubicin-treated apoptotic 4T1 tumor tissues, with signal intensity three times greater than that of Gd-DTPA. <sup>19</sup>F MRI further complemented <sup>1</sup>H MRI, with a notable recovery of the <sup>19</sup>F MRI signal after intratumoral injection. These results confirm that <b>CF</b><sub><b>3</b></sub><b>DEVDFFFK(Fmoc)-Gd</b> effectively reports tumor apoptosis through combined <sup>1</sup>H and <sup>19</sup>F MRI, offering a promising approach for the preliminary assessment of antitumor efficacy <i>in vivo</i>.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"47 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143862487","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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