Accurate identification and quantification of 5-hydroxymethylcytosine (5hmC) can help elucidate its function in gene expression and disease pathogenesis. Current 5hmC analysis methods still present challenges, especially for clinical applications, such as having a risk of false-positive results and a lack of sufficient sensitivity. Herein, a 5hmC quantification method for fragment-specific DNA sequences with extreme specificity, high sensitivity, and clinical applicability was established using a quantitative real-time PCR (qPCR)-based workflow through the combination of enzymatic digestion and biological deamination strategy (EDD-5hmC assay). The EDD-5hmC approach enriched glycosylated 5hmC via enzyme digestion and then APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-mediated deamination to efficiently differentiate between various cytosine(C) modification states, resulting in 5hmC quantification with extreme specificity such that nonspecific amplification is reduced over eight million-fold. Moreover, the nondestructive biological treatment process of the EDD-5hmC assay exhibits high sensitivity, yielding the limit of detection of 30 aM. For the first time, we measured 5hmC levels in colorectal cancer tissues and matched paracancerous tissues to evaluate the ability to differentiate colorectal cancer, with the area under the receiver operating characteristic curve of up to 82.8% for the single gene of Septin9 and 83.6% for the combinations of Septin9 and Syndecan-2 (SDC2), demonstrating the EDD-5hmC assay is a promising method with clinical applicability for accurately quantifying the 5hmC level.
{"title":"Fragment-specific Quantification of 5hmC by qPCR via a Combination of Enzymatic Digestion and Deamination: Extreme Specificity, High Sensitivity, and Clinical Applicability","authors":"XiaoHuan Peng, MengQiu Yan, Hao Yang, LinQing Zhen, LianXi Wei, Hong Xu","doi":"10.1021/acs.analchem.4c05147","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05147","url":null,"abstract":"Accurate identification and quantification of 5-hydroxymethylcytosine (5hmC) can help elucidate its function in gene expression and disease pathogenesis. Current 5hmC analysis methods still present challenges, especially for clinical applications, such as having a risk of false-positive results and a lack of sufficient sensitivity. Herein, a 5hmC quantification method for fragment-specific DNA sequences with extreme specificity, high sensitivity, and clinical applicability was established using a quantitative real-time PCR (qPCR)-based workflow through the combination of enzymatic digestion and biological deamination strategy (EDD-5hmC assay). The EDD-5hmC approach enriched glycosylated 5hmC via enzyme digestion and then APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like)-mediated deamination to efficiently differentiate between various cytosine(C) modification states, resulting in 5hmC quantification with extreme specificity such that nonspecific amplification is reduced over eight million-fold. Moreover, the nondestructive biological treatment process of the EDD-5hmC assay exhibits high sensitivity, yielding the limit of detection of 30 aM. For the first time, we measured 5hmC levels in colorectal cancer tissues and matched paracancerous tissues to evaluate the ability to differentiate colorectal cancer, with the area under the receiver operating characteristic curve of up to 82.8% for the single gene of Septin9 and 83.6% for the combinations of Septin9 and Syndecan-2 (SDC2), demonstrating the EDD-5hmC assay is a promising method with clinical applicability for accurately quantifying the 5hmC level.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"51 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142968502","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 : 2025-01-13DOI: 10.1021/acs.analchem.4c06108
Pei-Rong Bai, Na An, Yan-Zhen Wang, Yao-Yu Chen, Quan-Fei Zhu, Yu-Qi Feng
Flow injection mass spectrometry (FI-MS) is widely employed for high-throughput metabolome analysis, yet the absence of prior separation leads to significant matrix effects, thereby limiting the metabolome coverage. In this study, we introduce a novel photosensitive MS probe, iTASO-ONH2, integrated with FI-MS to establish a high-throughput strategy for submetabolome analyses. The iTASO probe features a conjugated-imino sulfonate moiety for efficient photolysis under 365 nm irradiation and a reactive group for selective metabolite labeling. The iTASO-ONH2 probe effectively and selectively labels carbonyl compounds, forming highly stable labeled products. Upon UV exposure, the labeled products rapidly release sulfonic acid-containing photolysis products, detectable with high sensitivity in ESI-negative mode and low matrix effect, offering femtomole-level detection sensitivity. The iTASO-ONH2-based FI-MS strategy was applied to fecal samples from chronic sleep-deprived and control mice, revealing 192 potential carbonyl compounds of which 37 exhibited significant alterations. Additionally, three other photosensitive probes─iTASO-NH2, iTASO-NHS, and iTASO-MAL─were synthesized to selectively label carboxyl, amino, and thiol metabolites, respectively, underscoring the versatility of the iTASO-based FI-MS strategy for submetabolomic analysis across diverse metabolite classes.
{"title":"iTASO: A Novel Photosensitive Probe for High-Throughput and Selective Submetabolomic Analysis via Flow Injection-Mass Spectrometry","authors":"Pei-Rong Bai, Na An, Yan-Zhen Wang, Yao-Yu Chen, Quan-Fei Zhu, Yu-Qi Feng","doi":"10.1021/acs.analchem.4c06108","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06108","url":null,"abstract":"Flow injection mass spectrometry (FI-MS) is widely employed for high-throughput metabolome analysis, yet the absence of prior separation leads to significant matrix effects, thereby limiting the metabolome coverage. In this study, we introduce a novel photosensitive MS probe, iTASO-ONH<sub>2</sub>, integrated with FI-MS to establish a high-throughput strategy for submetabolome analyses. The iTASO probe features a conjugated-imino sulfonate moiety for efficient photolysis under 365 nm irradiation and a reactive group for selective metabolite labeling. The iTASO-ONH<sub>2</sub> probe effectively and selectively labels carbonyl compounds, forming highly stable labeled products. Upon UV exposure, the labeled products rapidly release sulfonic acid-containing photolysis products, detectable with high sensitivity in ESI-negative mode and low matrix effect, offering femtomole-level detection sensitivity. The iTASO-ONH<sub>2</sub>-based FI-MS strategy was applied to fecal samples from chronic sleep-deprived and control mice, revealing 192 potential carbonyl compounds of which 37 exhibited significant alterations. Additionally, three other photosensitive probes─iTASO-NH<sub>2</sub>, iTASO-NHS, and iTASO-MAL─were synthesized to selectively label carboxyl, amino, and thiol metabolites, respectively, underscoring the versatility of the iTASO-based FI-MS strategy for submetabolomic analysis across diverse metabolite classes.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"74 6 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142975469","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 : 2025-01-13DOI: 10.1021/acs.analchem.4c04614
Andras Saftics, Benjamin Purnell, Balint Beres, S. Thompson, Nan Jiang, Ima Ghaeli, Carinna Lima, Brian Armstrong, Kendall Van Keuren-Jensen, Tijana Jovanovic-Talisman
Extracellular vesicles (EVs), membrane-encapsulated nanoparticles shed from all cells, are tightly involved in critical cellular functions. Moreover, EVs have recently emerged as exciting therapeutic modalities, delivery vectors, and biomarker sources. However, EVs are difficult to characterize, because they are typically small and heterogeneous in size, origin, and molecular content. Recent advances in single EV methods have addressed some of these challenges by providing sensitive tools for assessing individual vesicles; one example is our recently developed Single Extracellular VEsicle Nanoscopy (SEVEN) approach. However, these tools are typically not universally available to the general research community, as they require highly specialized equipment. Here, we show how single EV studies may be democratized via a novel method that employs super-resolution radial fluctuations (SRRF) microscopy and advanced data analysis. SRRF is compatible with a wide range of microscopes and fluorophores. We herein quantified individual EVs by combining affinity isolation (analytical protocol based on SEVEN) with SRRF microscopy and new analysis algorithms supported by machine learning-based EV assessment. Using SEVEN, we first optimized the workflow and validated the data obtained on wide-field and total internal reflection fluorescence microscopes. We further demonstrated that our approach, which we call the SEVEN-Universal Protocol (SEVEN-UP), can robustly assess the number, size, and content of plasma and recombinant EVs. Finally, we used the platform to assess RNA in EVs from conditioned cell culture media. Using SYTO RNASelect dye, we found that 18% of EVs from HEK 293T cells appear to contain RNA; these EVs were significantly larger compared with the general EV population. Altogether, we developed an economical, multiparametric, single EV characterization approach for the research community.
{"title":"Single Extracellular VEsicle Nanoscopy-Universal Protocol (SEVEN-UP): Accessible Imaging Platform for Quantitative Characterization of Single Extracellular Vesicles","authors":"Andras Saftics, Benjamin Purnell, Balint Beres, S. Thompson, Nan Jiang, Ima Ghaeli, Carinna Lima, Brian Armstrong, Kendall Van Keuren-Jensen, Tijana Jovanovic-Talisman","doi":"10.1021/acs.analchem.4c04614","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04614","url":null,"abstract":"Extracellular vesicles (EVs), membrane-encapsulated nanoparticles shed from all cells, are tightly involved in critical cellular functions. Moreover, EVs have recently emerged as exciting therapeutic modalities, delivery vectors, and biomarker sources. However, EVs are difficult to characterize, because they are typically small and heterogeneous in size, origin, and molecular content. Recent advances in single EV methods have addressed some of these challenges by providing sensitive tools for assessing individual vesicles; one example is our recently developed Single Extracellular VEsicle Nanoscopy (SEVEN) approach. However, these tools are typically not universally available to the general research community, as they require highly specialized equipment. Here, we show how single EV studies may be democratized via a novel method that employs super-resolution radial fluctuations (SRRF) microscopy and advanced data analysis. SRRF is compatible with a wide range of microscopes and fluorophores. We herein quantified individual EVs by combining affinity isolation (analytical protocol based on SEVEN) with SRRF microscopy and new analysis algorithms supported by machine learning-based EV assessment. Using SEVEN, we first optimized the workflow and validated the data obtained on wide-field and total internal reflection fluorescence microscopes. We further demonstrated that our approach, which we call the SEVEN-Universal Protocol (SEVEN-UP), can robustly assess the number, size, and content of plasma and recombinant EVs. Finally, we used the platform to assess RNA in EVs from conditioned cell culture media. Using SYTO RNASelect dye, we found that 18% of EVs from HEK 293T cells appear to contain RNA; these EVs were significantly larger compared with the general EV population. Altogether, we developed an economical, multiparametric, single EV characterization approach for the research community.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"49 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142968410","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 : 2025-01-13DOI: 10.1021/acs.analchem.4c04134
Siyu Chen, Yixuan Xie, Michael Russelle Alvarez, Ying Sheng, Yasmine Bouchibti, Vincent Chang, Carlito B. Lebrilla
Protein–protein interactions in the cell membrane are typically mediated by glycans, with terminal sialic acid often involved in these interactions. To probe the nature of the interactions, we developed quantitative cross-linking methods involving the glycans of the glycoproteins and the polypeptide moieties of proteins. We designed and synthesized biotinylated enrichable cross-linkers that were click-tagged to metabolically incorporate azido-sialic acid on cell surface glycans to allow cross-linking of the azido-glycans with lysine residues on proximal polypeptides. The glycopeptide–peptide cross-links (GPx) were enriched using biotin groups through affinity purification with streptavidin resin beads. Workflows using two linkers, one photocleavable and the other disulfide, were developed and applied to reveal the sialic acid-mediated cell-surface protein networks of PNT2 (prostate) cells. Glycopeptide–peptide pairs were identified, with 12000 GPx linked by sialylated glycoforms revealing interactions between source glycoproteins and nearly 700 target proteins. Protein–protein interactions were characterized by as many as 40 peptide pairs, and the extent of the interactions between proteins was prioritized by the number of GPx. Quantitation was performed by counting the number of GPx that identify the protein pairs. Abundant membrane proteins such as ITGB1 yielded an interactome consisting of around 400 other proteins, which were ranked from the most extensive interaction, having the largest number of GPx, to at least one. The interactome was further confirmed separately by published databases. This tool will enhance our understanding of glycosylation on protein–protein interactions and provide new potential targets for therapeutics.
{"title":"Quantitative Glycan-Protein Cross-Linking Mass Spectrometry Using Enrichable Linkers Reveals Extensive Glycan-Mediated Protein Interaction Networks","authors":"Siyu Chen, Yixuan Xie, Michael Russelle Alvarez, Ying Sheng, Yasmine Bouchibti, Vincent Chang, Carlito B. Lebrilla","doi":"10.1021/acs.analchem.4c04134","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04134","url":null,"abstract":"Protein–protein interactions in the cell membrane are typically mediated by glycans, with terminal sialic acid often involved in these interactions. To probe the nature of the interactions, we developed quantitative cross-linking methods involving the glycans of the glycoproteins and the polypeptide moieties of proteins. We designed and synthesized biotinylated enrichable cross-linkers that were click-tagged to metabolically incorporate azido-sialic acid on cell surface glycans to allow cross-linking of the azido-glycans with lysine residues on proximal polypeptides. The glycopeptide–peptide cross-links (GPx) were enriched using biotin groups through affinity purification with streptavidin resin beads. Workflows using two linkers, one photocleavable and the other disulfide, were developed and applied to reveal the sialic acid-mediated cell-surface protein networks of PNT2 (prostate) cells. Glycopeptide–peptide pairs were identified, with 12000 GPx linked by sialylated glycoforms revealing interactions between source glycoproteins and nearly 700 target proteins. Protein–protein interactions were characterized by as many as 40 peptide pairs, and the extent of the interactions between proteins was prioritized by the number of GPx. Quantitation was performed by counting the number of GPx that identify the protein pairs. Abundant membrane proteins such as ITGB1 yielded an interactome consisting of around 400 other proteins, which were ranked from the most extensive interaction, having the largest number of GPx, to at least one. The interactome was further confirmed separately by published databases. This tool will enhance our understanding of glycosylation on protein–protein interactions and provide new potential targets for therapeutics.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"118 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142975465","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}
DNAzyme-based cascade networks are effective tools to achieve ultrasensitive detection of low-abundance miRNAs. However, their designs are complicated and costly, and the operation is time-consuming. Herein, a novel simple noncascade DNAzyme network is designed and its amplification effect is comparable to or even better than many cascading ones. It is a nonenzymatic, isothermal, bicirculating amplification network consisting of two toehold-mediated strand-displacement reactions and a localized DNAzyme amplification strategy. Taking microRNA-122 as a target model, this ultrasensitive fluorescence biosensor has a detection limit of 84 zmol L–1, which is 8-orders of magnitude lower than that of the nonamplification one. The ultrasensitivity mainly benefits from the exclusive design and positive self-feedback mechanism of the ingenious bicirculating DNAzyme amplification network. In addition, the utilization of superparamagnetic Fe3O4@SiO2 particles not only helps for the localization of DNAzymes but also facilitates the rapid separation of signal probes (output DNA-CdTe QDs). This fluorescent biosensor also has the advantages of specificity, speed, thermal stability, and low cost. This novel design paves a new way to simple and effective bioamplification strategy, which may be very attractive for biosensors, DNA logic gates, and DNA computers.
{"title":"Localized Bicirculating DNAzyme Self-Feedback Amplification Strategy for Ultra-Sensitive Fluorescence Biosensing of MicroRNA","authors":"Defu Qian, Jingling Zhang, Qingqing Tan, Yuye Zhang, Qin Xu, Jing Li, Hongbo Li","doi":"10.1021/acs.analchem.4c04417","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04417","url":null,"abstract":"DNAzyme-based cascade networks are effective tools to achieve ultrasensitive detection of low-abundance miRNAs. However, their designs are complicated and costly, and the operation is time-consuming. Herein, a novel simple noncascade DNAzyme network is designed and its amplification effect is comparable to or even better than many cascading ones. It is a nonenzymatic, isothermal, bicirculating amplification network consisting of two toehold-mediated strand-displacement reactions and a localized DNAzyme amplification strategy. Taking microRNA-122 as a target model, this ultrasensitive fluorescence biosensor has a detection limit of 84 zmol L<sup>–1</sup>, which is 8-orders of magnitude lower than that of the nonamplification one. The ultrasensitivity mainly benefits from the exclusive design and positive self-feedback mechanism of the ingenious bicirculating DNAzyme amplification network. In addition, the utilization of superparamagnetic Fe<sub>3</sub>O<sub>4</sub>@SiO<sub>2</sub> particles not only helps for the localization of DNAzymes but also facilitates the rapid separation of signal probes (output DNA-CdTe QDs). This fluorescent biosensor also has the advantages of specificity, speed, thermal stability, and low cost. This novel design paves a new way to simple and effective bioamplification strategy, which may be very attractive for biosensors, DNA logic gates, and DNA computers.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"27 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142975466","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}
The outbreak of the monkeypox epidemic underscores the importance of developing a rapid and sensitive virus detection technique. Microneedles (MNs) offer minimally invasive sampling capabilities, providing a solution for the development of integrated extraction and diagnostic portable devices. Here, we report an integrated MNs and hydrogel biosensor (IMHB) platform, composed of an electronic device, an MN patch, and a hydrogel patch. The IMHB allowed for specific extraction of monkeypox virus (MPXV) directly from lesional skin and virus detection in both electrochemical and colorimetric modes. A bifunctional signal probe 3,3′,5,5′-tetramethylbenzidine (TMB) was loaded in a hydrogel patch, providing measurable signals for dual-mode sensing. Additionally, a control area was designed in this platform to collect blank samples from normal skin, enabling ratio analysis and quality control functions. This dual-mode ratiometric sensing strategy exhibited a wide range of 10–1000 ng/mL for MPXV A29 protein, with detection limits of 0.1632 and 0.3017 ng/mL for electrochemical and colorimetric assay, respectively. The developed IMHB platform provides a novel way for rapid on-site determination of MPXV, demonstrating the potential for quick intervention in the early stages of infectious diseases.
{"title":"Integrated Microneedles and Hydrogel Biosensor Platform: Toward a Diagnostic Device for Collection and Dual-Mode Sensing of Monkeypox Virus A29 Protein","authors":"Yujian Liu, Jiang Liu, Yequn Chen, Guanghui Zhang, Qiqin Wang, Yingchun Li","doi":"10.1021/acs.analchem.4c03835","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03835","url":null,"abstract":"The outbreak of the monkeypox epidemic underscores the importance of developing a rapid and sensitive virus detection technique. Microneedles (MNs) offer minimally invasive sampling capabilities, providing a solution for the development of integrated extraction and diagnostic portable devices. Here, we report an integrated MNs and hydrogel biosensor (IMHB) platform, composed of an electronic device, an MN patch, and a hydrogel patch. The IMHB allowed for specific extraction of monkeypox virus (MPXV) directly from lesional skin and virus detection in both electrochemical and colorimetric modes. A bifunctional signal probe 3,3′,5,5′-tetramethylbenzidine (TMB) was loaded in a hydrogel patch, providing measurable signals for dual-mode sensing. Additionally, a control area was designed in this platform to collect blank samples from normal skin, enabling ratio analysis and quality control functions. This dual-mode ratiometric sensing strategy exhibited a wide range of 10–1000 ng/mL for MPXV A29 protein, with detection limits of 0.1632 and 0.3017 ng/mL for electrochemical and colorimetric assay, respectively. The developed IMHB platform provides a novel way for rapid on-site determination of MPXV, demonstrating the potential for quick intervention in the early stages of infectious diseases.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"50 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142968412","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 : 2025-01-13DOI: 10.1021/acs.analchem.4c05256
Shuzhen Yue, Xuan Xu, Li-Ping Jiang, Huiqin Yao, Jun-Jie Zhu
An entropy-driven catalysis (EDC) strategy is appealing for amplified bioimaging of microRNAs in living cells; yet, complex operation procedures, lacking of cell selectivity, and insufficient accuracy hamper its further applications. Here, we introduce an ingenious all-in-one entropy-driven DNA nanomachine (termed as AIO-EDN), which can be triggered by endogenous apurinic/apyrimidinic endonuclease 1 (APE1) to achieve tumor cell-selective dual-mode imaging of microRNA. Compared with the traditional EDC strategy, the integrated design of AIO-EDN achieves autocatalytic signal amplification without extra fuel strands. Moreover, the AIO-EDN leverages an endogenous APE1 overexpressed in cancer cells to activate the EDC reaction, which, however, exerts no target sensing activity in normal cells. Combining fluorescence- and surface-enhanced Raman scattering (FL/SERS) dual-mode imaging techniques, this DNA nanomachine exhibits significantly improved accuracy and tumor cell selectivity for microRNA imaging in living cells. This study provides a new paradigm to develop an integrated EDC-based platform and shows great potential in in-depth cancer diagnosis with high precision.
{"title":"All-In-One Entropy-Driven DNA Nanomachine for Tumor Cell-Selective Fluorescence/SERS Dual-Mode Imaging of MicroRNA","authors":"Shuzhen Yue, Xuan Xu, Li-Ping Jiang, Huiqin Yao, Jun-Jie Zhu","doi":"10.1021/acs.analchem.4c05256","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05256","url":null,"abstract":"An entropy-driven catalysis (EDC) strategy is appealing for amplified bioimaging of microRNAs in living cells; yet, complex operation procedures, lacking of cell selectivity, and insufficient accuracy hamper its further applications. Here, we introduce an ingenious all-in-one entropy-driven DNA nanomachine (termed as AIO-EDN), which can be triggered by endogenous apurinic/apyrimidinic endonuclease 1 (APE1) to achieve tumor cell-selective dual-mode imaging of microRNA. Compared with the traditional EDC strategy, the integrated design of AIO-EDN achieves autocatalytic signal amplification without extra fuel strands. Moreover, the AIO-EDN leverages an endogenous APE1 overexpressed in cancer cells to activate the EDC reaction, which, however, exerts no target sensing activity in normal cells. Combining fluorescence- and surface-enhanced Raman scattering (FL/SERS) dual-mode imaging techniques, this DNA nanomachine exhibits significantly improved accuracy and tumor cell selectivity for microRNA imaging in living cells. This study provides a new paradigm to develop an integrated EDC-based platform and shows great potential in in-depth cancer diagnosis with high precision.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"16 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142975472","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 : 2025-01-13DOI: 10.1021/acs.analchem.4c05355
Lijun Hu, Lei Jiao, Chengjie Chen, Xiangkun Jia, Xiaotong Li, Dongbo Yan, Yanling Zhai, Xiaoquan Lu
The elementary mechanism and site studies of nanozyme-based inhibition reactions are ambiguous and urgently require advanced nanozymes as mediators to elucidate the inhibition effect. To this end, we develop a class of nanozymes featuring single Cu–N catalytic configurations and B–O sites as binding configurations on a porous nitrogen-doped carbon substrate (B6/CuSA) for inducing modulable inhibition transfer at the atomic level. The full redistribution of electrons across the Cu–N sites, induced by B–O sites incorporation, yields B6/CuSA with enhanced peroxidase-like activity versus CuSA. More importantly, CuSA with single Cu–N sites features in cysteine binding and expresses a competitive inhibition through coordination bonds, with an inhibition constant of 0.048 mM. Benefiting from the modulable binding way in nanozymes, B6/CuSA possesses mixed binding approaches for cysteine through noncovalent bonds and delivers a record-mixed inhibition interaction with a competitive inhibition constant of 0.054 mM and a noncompetitive inhibition constant of 0.71 mM. Based on the modulable inhibition of B6/CuSA and CuSA, a multichannel sensor array accomplishes the detection of various cancer cells, normal cells, and thiols. The design principle of this work is endowed with guidelines for the preliminary inhibition mechanism evaluation of massive potential thiols, cell discrimination, and disease prediction.
{"title":"Nanozymes with Modulable Inhibition Transfer Pathways for Thiol and Cell Identification","authors":"Lijun Hu, Lei Jiao, Chengjie Chen, Xiangkun Jia, Xiaotong Li, Dongbo Yan, Yanling Zhai, Xiaoquan Lu","doi":"10.1021/acs.analchem.4c05355","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05355","url":null,"abstract":"The elementary mechanism and site studies of nanozyme-based inhibition reactions are ambiguous and urgently require advanced nanozymes as mediators to elucidate the inhibition effect. To this end, we develop a class of nanozymes featuring single Cu–N catalytic configurations and B–O sites as binding configurations on a porous nitrogen-doped carbon substrate (B<sub>6</sub>/Cu<sub>SA</sub>) for inducing modulable inhibition transfer at the atomic level. The full redistribution of electrons across the Cu–N sites, induced by B–O sites incorporation, yields B<sub>6</sub>/Cu<sub>SA</sub> with enhanced peroxidase-like activity versus Cu<sub>SA</sub>. More importantly, Cu<sub>SA</sub> with single Cu–N sites features in cysteine binding and expresses a competitive inhibition through coordination bonds, with an inhibition constant of 0.048 mM. Benefiting from the modulable binding way in nanozymes, B<sub>6</sub>/Cu<sub>SA</sub> possesses mixed binding approaches for cysteine through noncovalent bonds and delivers a record-mixed inhibition interaction with a competitive inhibition constant of 0.054 mM and a noncompetitive inhibition constant of 0.71 mM. Based on the modulable inhibition of B<sub>6</sub>/Cu<sub>SA</sub> and Cu<sub>SA</sub>, a multichannel sensor array accomplishes the detection of various cancer cells, normal cells, and thiols. The design principle of this work is endowed with guidelines for the preliminary inhibition mechanism evaluation of massive potential thiols, cell discrimination, and disease prediction.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"36 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142975470","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}
DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites. Our correlative model, considering the cross-effects between different binding sites, showed that for ATP aptamers with two binding sites, binding of the first ATP molecule would decrease its affinity for the second at low temperatures and even completely inhibit this binding at high temperatures. Moreover, the thermodynamic parameters of protein-specific aptamers were calculated to elucidate the universality of the method. The successful analysis of cell-specific aptamers further demonstrated MDRC’s applicability in complex biological systems.
{"title":"Reducing Measurement Deviation by Metastable DNA Probes for Aptamer Thermodynamic Characterization","authors":"Yulin Du, Chunran Ma, Yuqi Zeng, Yihao Liu, Zihan Zhao, Yifan Lyu","doi":"10.1021/acs.analchem.4c05900","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05900","url":null,"abstract":"DNA reaction equilibrium-based calculations have great potential in thermodynamic characterization, but their widespread applications are hindered by significant measurement deviation of equilibrium concentration. Here, we report the advantages of metastable DNA hybridization in reducing quantification deviation of equilibrium concentration and propose a universal and standardized strategy for measuring aptamer binding energy, termed metastable DNA reference calorimetry (MDRC). We built different MDRC-based algorithms tailored to different aptamer binding models, enabling the calculation of thermodynamic parameters for aptamers with one or more binding sites. Our correlative model, considering the cross-effects between different binding sites, showed that for ATP aptamers with two binding sites, binding of the first ATP molecule would decrease its affinity for the second at low temperatures and even completely inhibit this binding at high temperatures. Moreover, the thermodynamic parameters of protein-specific aptamers were calculated to elucidate the universality of the method. The successful analysis of cell-specific aptamers further demonstrated MDRC’s applicability in complex biological systems.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"12 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142968417","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}
This study presents a novel approach that combines thermogravimetric analysis with time-of-flight mass spectrometry (TG-TOFMS), principal component analysis (PCA), and Kendrick mass defect (KMD) analysis─referred to as TG-PCA-KMD─to investigate molecular-scale structural changes and quantitatively assess the progression of thermo-oxidative degradation in glass fiber reinforced polypropylene (GF/PP). TG-TOFMS enables the simultaneous and sensitive detection of both structural changes due to thermo-oxidative degradation and compositional changes in the filler and matrix. PCA and KMD analysis are crucial for identifying specific ion series derived from the degraded PP matrix in the high-resolution mass spectra obtained through TG-TOFMS. Additionally, PCA fitting was employed to selectively extract information on the degraded components of GF/PP from differential thermogravimetric profiles. Our findings demonstrate the advantages and utility of TG-PCA-KMD in the degradation analysis of composite materials.
{"title":"High-Resolution TG-TOFMS Coupled with Principal Component Analysis and Kendrick Mass Defect Analysis: Elucidation of Molecular-Scale Degradation Behavior of Glass Fiber Reinforced Polypropylene during Thermo-Oxidative Degradation","authors":"Taiki Ozawa, Sayaka Nakamura, Hiroaki Sato, Hideyuki Shinzawa, Hideaki Hagihara, Ryota Watanabe","doi":"10.1021/acs.analchem.4c04630","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04630","url":null,"abstract":"This study presents a novel approach that combines thermogravimetric analysis with time-of-flight mass spectrometry (TG-TOFMS), principal component analysis (PCA), and Kendrick mass defect (KMD) analysis─referred to as TG-PCA-KMD─to investigate molecular-scale structural changes and quantitatively assess the progression of thermo-oxidative degradation in glass fiber reinforced polypropylene (GF/PP). TG-TOFMS enables the simultaneous and sensitive detection of both structural changes due to thermo-oxidative degradation and compositional changes in the filler and matrix. PCA and KMD analysis are crucial for identifying specific ion series derived from the degraded PP matrix in the high-resolution mass spectra obtained through TG-TOFMS. Additionally, PCA fitting was employed to selectively extract information on the degraded components of GF/PP from differential thermogravimetric profiles. Our findings demonstrate the advantages and utility of TG-PCA-KMD in the degradation analysis of composite materials.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"22 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142975467","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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