Pub Date : 2026-02-04DOI: 10.1021/acs.analchem.5c03375
Tomos E. Morgan, Alina Theisen, Sean Ellacott, Anisha Haris, Christopher A. Wootton, Julia Y. Rho, Mark P. Barrow, Anthony W. T. Bristow, Sébastien Perrier, Peter B. O’Connor
Cyclic peptide–polymer conjugates offer a unique biocompatible system with many advantages but come at the cost of being analytically challenging. Developing further analytical techniques of complex polymer-conjugate systems is key to understanding synthetic and medicinal properties. In this contribution, a synthetic cyclic peptide–polymer conjugate is analyzed using electron capture dissociation (ECD), infrared multiphoton absorption dissociation (IRMPD), and 193 nm ultraviolet photodissociation (UVPD) on the same mass spectrometry system. IRMPD and UVPD were shown to effectively characterize unconjugated cyclic peptide species. ECD was less informative during cyclic peptide analysis due to the production of multiple sequence scrambling fragments and radical side chain losses. ECD was shown to produce extensive fragmentation and enable the characterization of conjugated side chains of cyclic species. ECD and IRMPD thus provided complementary data, enabling the target analysis of conjugated systems. UVPD effectively characterized both the cyclic peptide and the conjugating polymer in one experiment, being able to produce complete cyclic peptide fragmentation via b/y fragment pathways and polymer fragmentation via a/x poly(2-ethyl-2-oxazoline) fragment pathways.
{"title":"Cyclic Peptide–Polymer Conjugate Characterization Using 193 nm Ultraviolet Photodissociation Tandem Mass Spectrometry","authors":"Tomos E. Morgan, Alina Theisen, Sean Ellacott, Anisha Haris, Christopher A. Wootton, Julia Y. Rho, Mark P. Barrow, Anthony W. T. Bristow, Sébastien Perrier, Peter B. O’Connor","doi":"10.1021/acs.analchem.5c03375","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c03375","url":null,"abstract":"Cyclic peptide–polymer conjugates offer a unique biocompatible system with many advantages but come at the cost of being analytically challenging. Developing further analytical techniques of complex polymer-conjugate systems is key to understanding synthetic and medicinal properties. In this contribution, a synthetic cyclic peptide–polymer conjugate is analyzed using electron capture dissociation (ECD), infrared multiphoton absorption dissociation (IRMPD), and 193 nm ultraviolet photodissociation (UVPD) on the same mass spectrometry system. IRMPD and UVPD were shown to effectively characterize unconjugated cyclic peptide species. ECD was less informative during cyclic peptide analysis due to the production of multiple sequence scrambling fragments and radical side chain losses. ECD was shown to produce extensive fragmentation and enable the characterization of conjugated side chains of cyclic species. ECD and IRMPD thus provided complementary data, enabling the target analysis of conjugated systems. UVPD effectively characterized both the cyclic peptide and the conjugating polymer in one experiment, being able to produce complete cyclic peptide fragmentation via <i>b</i>/<i>y</i> fragment pathways and polymer fragmentation via <i>a</i>/<i>x</i> poly(2-ethyl-2-oxazoline) fragment pathways.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"42 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146116155","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 : 2026-02-04DOI: 10.1021/acs.analchem.5c04295
Alexis N. Edwards,Madeline G. Bannon,Michael S. Cordes,Elyssia S. Gallagher
Native ion mobility spectrometry-mass spectrometry (IMS-MS) is routinely used for analysis of folded proteins and protein complexes. For many proteins, the three-dimensional structure is maintained during electrospray ionization (ESI) as the protein transitions to the gas phase, allowing for detailed investigation of the gaseous, ionic protein’s structure and stability. Much of the native IMS-MS research has been conducted in positive-ion mode (+ESI), even when the protein of interest has a net-negative charge in solution at physiological pH. We hypothesize that analyzing a protein in the polarity that is opposite to its solution-phase charge, such as analyzing net-negative proteins by +ESI-MS, disrupts the network of noncovalent-bonding interactions within the protein to a greater extent than using the polarity that matches the protein’s solution-phase charge, resulting in differences in protein stability. Herein, we show that while most protein ions have similar initial, folded structures in +ESI and negative-ion mode (−ESI), positive and negative ions exhibit significant differences in gas-phase stability. Furthermore, the energy required to cause this unfolding is often greater in the polarity corresponding to the solution-phase charge of the protein, indicating that the protein is more stable in that polarity. Thus, this work highlights the necessity of considering polarity when conducting native IMS-MS experiments.
{"title":"Investigating the Effect of Isoelectric Points on the Gas-Phase Stability of Native-like Proteins Analyzed in Positive- versus Negative-Ion Mode by IMS-MS","authors":"Alexis N. Edwards,Madeline G. Bannon,Michael S. Cordes,Elyssia S. Gallagher","doi":"10.1021/acs.analchem.5c04295","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c04295","url":null,"abstract":"Native ion mobility spectrometry-mass spectrometry (IMS-MS) is routinely used for analysis of folded proteins and protein complexes. For many proteins, the three-dimensional structure is maintained during electrospray ionization (ESI) as the protein transitions to the gas phase, allowing for detailed investigation of the gaseous, ionic protein’s structure and stability. Much of the native IMS-MS research has been conducted in positive-ion mode (+ESI), even when the protein of interest has a net-negative charge in solution at physiological pH. We hypothesize that analyzing a protein in the polarity that is opposite to its solution-phase charge, such as analyzing net-negative proteins by +ESI-MS, disrupts the network of noncovalent-bonding interactions within the protein to a greater extent than using the polarity that matches the protein’s solution-phase charge, resulting in differences in protein stability. Herein, we show that while most protein ions have similar initial, folded structures in +ESI and negative-ion mode (−ESI), positive and negative ions exhibit significant differences in gas-phase stability. Furthermore, the energy required to cause this unfolding is often greater in the polarity corresponding to the solution-phase charge of the protein, indicating that the protein is more stable in that polarity. Thus, this work highlights the necessity of considering polarity when conducting native IMS-MS experiments.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"91 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111275","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 : 2026-02-04DOI: 10.1021/acs.analchem.5c06805
Yi Yang,Licheng Yu,Haiyang Wang,Liang He,Pengli Bai,Xiwen He,Langxing Chen,Yukui Zhang
In this work, porphyrinic metal–organic frameworks (MOFs) with longitudinal Pt deposition were developed for the inspired establishment of opto/catalytic adjuvants referenced for a dual-path glycoprotein assay. In the compositional cooperation, porphyrinic MOFs were synthesized through Zr oxoclusters and polytopic porphyrin ligands into a benchmark template (PCN-222), followed by catalytic Pt nanowire deposition (PtNW@PCN-222). As for signal reflection, the fluorogenic species (i.e., resazurin and amplex red) were integrated into zeolitic imidazolate framework-8 (ZIF-8) via epitaxial shell growth to guide lectin integration. The specific performance toward glycoprotein recognition was guaranteed by both antibody–antigen interaction as well as glycan epitope binding through lectin affinity, and the signal response was initiated by textural decomposition of the ZIF-8 skeleton in a controllable manner. On one hand, PtNW@PCN-222 can function as the catalytic adjuvant to exert the fluorogenic reactions: a reductive N-deoxygenation of resazurin and an oxidative N-deacetylation of amplex red, in which both the redox reactions generated the fluorescent species of resorufin products. On the other hand, PtNW@PCN-222 with endogenous periodical arrangement of porphyrin ligands can be tailored as an optical adjuvant for reporting signal reference. For benefits, the comprehensive functions leaned on the catalytic performance and optical reference property from PtNW@PCN-222 can be harvested for a ratiometric glycoprotein assay. Besides, due to the variant catalytic converter of PtNW@PCN-222 involved in the two fluorogenic reactions, the proposed glycoprotein assay exhibited a range of 0.03–3 nM with a detection limit of 7.91 pM in the reductive pathway, and showed a linear range from 0.06 to 10 nM with a detection limit of 21.64 pM in the oxidative pathway. Collectively, our proposed glycoprotein assay may provide a new thought in opto/catalytic MOF adjuvant-empowered ratiometric sensing in the dual-path reflection manner, which may also reinforce the accurate detection capability for the glycoprotein assay.
{"title":"Porphyrinic Metal–Organic Frameworks with Longitudinal Pt Deposition Referenced as Opto/Catalytic Adjuvants for Dual-Path Glycoprotein Assay","authors":"Yi Yang,Licheng Yu,Haiyang Wang,Liang He,Pengli Bai,Xiwen He,Langxing Chen,Yukui Zhang","doi":"10.1021/acs.analchem.5c06805","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c06805","url":null,"abstract":"In this work, porphyrinic metal–organic frameworks (MOFs) with longitudinal Pt deposition were developed for the inspired establishment of opto/catalytic adjuvants referenced for a dual-path glycoprotein assay. In the compositional cooperation, porphyrinic MOFs were synthesized through Zr oxoclusters and polytopic porphyrin ligands into a benchmark template (PCN-222), followed by catalytic Pt nanowire deposition (PtNW@PCN-222). As for signal reflection, the fluorogenic species (i.e., resazurin and amplex red) were integrated into zeolitic imidazolate framework-8 (ZIF-8) via epitaxial shell growth to guide lectin integration. The specific performance toward glycoprotein recognition was guaranteed by both antibody–antigen interaction as well as glycan epitope binding through lectin affinity, and the signal response was initiated by textural decomposition of the ZIF-8 skeleton in a controllable manner. On one hand, PtNW@PCN-222 can function as the catalytic adjuvant to exert the fluorogenic reactions: a reductive N-deoxygenation of resazurin and an oxidative N-deacetylation of amplex red, in which both the redox reactions generated the fluorescent species of resorufin products. On the other hand, PtNW@PCN-222 with endogenous periodical arrangement of porphyrin ligands can be tailored as an optical adjuvant for reporting signal reference. For benefits, the comprehensive functions leaned on the catalytic performance and optical reference property from PtNW@PCN-222 can be harvested for a ratiometric glycoprotein assay. Besides, due to the variant catalytic converter of PtNW@PCN-222 involved in the two fluorogenic reactions, the proposed glycoprotein assay exhibited a range of 0.03–3 nM with a detection limit of 7.91 pM in the reductive pathway, and showed a linear range from 0.06 to 10 nM with a detection limit of 21.64 pM in the oxidative pathway. Collectively, our proposed glycoprotein assay may provide a new thought in opto/catalytic MOF adjuvant-empowered ratiometric sensing in the dual-path reflection manner, which may also reinforce the accurate detection capability for the glycoprotein assay.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"295 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111269","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 : 2026-02-04DOI: 10.1021/acs.analchem.5c06616
Yue Zhang,Xianjie Zheng,Zelin Jiang,Xu Wang
As the most prevalent form of genetic variation, single nucleotide polymorphisms (SNPs) are strongly associated with disease pathogenesis, microbial resistance, and pathogenicity. The rapid mutation rates of viruses such as influenza A and SARS-CoV-2 highlight the urgent need for multifunctional diagnostic tools capable of effectively distinguishing emerging variants. However, current clinical SNPs detection methods often require complex primer/probe designs and sophisticated instruments, which limits their wide application in resource-constrained settings. Here, we developed the LIVE-SNP method, a rapid ligation-mediated isothermal assay for the visual evaluation of SNPs with high sensitivity and specificity. This method employs multicolor fluorescent probes to establish multiplex assays for the simultaneous identification of different SNPs, allowing for direct discrimination of target types through visual observation of fluorescent signals. The established method exhibits high specificity in identifying SNPs in both RNA and DNA. It can accurately detect drug-resistant single-nucleotide mutations in influenza A virus and efficiently identify antimicrobial resistance mutations in bacteria. Furthermore, we have developed a compact hand-held device integrating heating and fluorescence detection modules, which facilitates rapid on-site detection of pathogenic microorganisms and their variants, providing a powerful technical platform for applications in food safety, genotyping, and infectious disease control.
{"title":"Rapid and Sensitive Visual Detection of Gene Mutations via LIVE-SNP","authors":"Yue Zhang,Xianjie Zheng,Zelin Jiang,Xu Wang","doi":"10.1021/acs.analchem.5c06616","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c06616","url":null,"abstract":"As the most prevalent form of genetic variation, single nucleotide polymorphisms (SNPs) are strongly associated with disease pathogenesis, microbial resistance, and pathogenicity. The rapid mutation rates of viruses such as influenza A and SARS-CoV-2 highlight the urgent need for multifunctional diagnostic tools capable of effectively distinguishing emerging variants. However, current clinical SNPs detection methods often require complex primer/probe designs and sophisticated instruments, which limits their wide application in resource-constrained settings. Here, we developed the LIVE-SNP method, a rapid ligation-mediated isothermal assay for the visual evaluation of SNPs with high sensitivity and specificity. This method employs multicolor fluorescent probes to establish multiplex assays for the simultaneous identification of different SNPs, allowing for direct discrimination of target types through visual observation of fluorescent signals. The established method exhibits high specificity in identifying SNPs in both RNA and DNA. It can accurately detect drug-resistant single-nucleotide mutations in influenza A virus and efficiently identify antimicrobial resistance mutations in bacteria. Furthermore, we have developed a compact hand-held device integrating heating and fluorescence detection modules, which facilitates rapid on-site detection of pathogenic microorganisms and their variants, providing a powerful technical platform for applications in food safety, genotyping, and infectious disease control.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"7 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111272","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 development of conventional orthosteric drugs targeting G protein-coupled receptors (GPCRs) is often hindered by off-target effects and low subtype selectivity. Allosteric ligands enable precise control of receptor signaling through selective conformational changes, highlighting their critical importance as a promising therapeutic strategy and as a key frontier in modern drug discovery. To enhance GPCR ligand screening efficacy, surface-enhanced Raman spectroscopy (SERS), with sensitivity and narrow spectral peaks, was employed to outline a methodology based on allosteric aptamer binding and stabilizing conformations of the β2 adrenergic receptor (β2AR) for identifying GPCR ligands by fabricating a sandwich-type SERS sensor. The method involved immobilization of β2AR on magnetic plasmonic nanoparticles by label-free click chemistry, probing and stabilization of the allosteric conformational changes by RNA aptamer, and amplification and detection of the signal by SERS-active nanoparticles. The success of the allosteric β2AR assay was confirmed through multiple analytical techniques, including SERS, SEM, UV–vis spectroscopy, and particle size analysis. This methodology demonstrates broad applicability, characterized by rapid response, high sensitivity, and excellent selectivity. BI167107 could be detected within a range of 10–9–10–6 M, with a detection limit of 49 pM. Such a sandwich biosensor successfully identified the agonist, antagonist, and allosteric inhibitor of β2AR from the ligand complex in both aqueous solution and serum. This method is expected to become an alternative for high-throughput and multiplexed drug screening when it comes to a compound library.
{"title":"An Allosteric Assay for Identifying Ligands Binding to β2 Adrenergic Receptor by Surface-Enhanced Raman Scattering (SERS)-Active Nanoparticles","authors":"Yunshan Wang,Nan Lu,Peiyao Wu,Yue Chu,Yonghao Xing,Jing Wang,Xinfeng Zhao","doi":"10.1021/acs.analchem.5c07045","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c07045","url":null,"abstract":"The development of conventional orthosteric drugs targeting G protein-coupled receptors (GPCRs) is often hindered by off-target effects and low subtype selectivity. Allosteric ligands enable precise control of receptor signaling through selective conformational changes, highlighting their critical importance as a promising therapeutic strategy and as a key frontier in modern drug discovery. To enhance GPCR ligand screening efficacy, surface-enhanced Raman spectroscopy (SERS), with sensitivity and narrow spectral peaks, was employed to outline a methodology based on allosteric aptamer binding and stabilizing conformations of the β2 adrenergic receptor (β2AR) for identifying GPCR ligands by fabricating a sandwich-type SERS sensor. The method involved immobilization of β2AR on magnetic plasmonic nanoparticles by label-free click chemistry, probing and stabilization of the allosteric conformational changes by RNA aptamer, and amplification and detection of the signal by SERS-active nanoparticles. The success of the allosteric β2AR assay was confirmed through multiple analytical techniques, including SERS, SEM, UV–vis spectroscopy, and particle size analysis. This methodology demonstrates broad applicability, characterized by rapid response, high sensitivity, and excellent selectivity. BI167107 could be detected within a range of 10–9–10–6 M, with a detection limit of 49 pM. Such a sandwich biosensor successfully identified the agonist, antagonist, and allosteric inhibitor of β2AR from the ligand complex in both aqueous solution and serum. This method is expected to become an alternative for high-throughput and multiplexed drug screening when it comes to a compound library.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"108 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111267","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 : 2026-02-04DOI: 10.1021/acs.analchem.5c06945
Zhuandi Zhu,Mingshuai Fan,Yubao Lan,Zhengang Han,Yaqi Zhao,Yanjun Feng,Xiaowan Li,Xiaoquan Lu
Carbazole derivatives have great potential in the field of electrochemiluminescence (ECL) due to their excellent photoelectric properties. The ECL properties of carbazole derivatives have been studied by modifying different substituents on carbazolyl benzene. Among them, DPA-BFCz shows obvious aggregated enhanced luminescence (AEE) and excellent ECL performance. Notably, scanning photoelectrochemical microscopy (SPECM) and intensity-modulated photocurrent spectroscopy (IMPS) are used to analyze the charge transfer kinetics of the emitters. In addition, a sensor of Cyt c with an ECL-resonance energy transfer (RET) mechanism is developed by taking advantage of the superior ECL performance of DPA-BFCz, with a concentration range of 1.0 to 500 nM and a detection limit of 0.20 nM. This work presents a novel strategy for rapid and sensitive detection of Cyt c.
{"title":"Excellent Electrochemiluminescence Performance of Carbazolyl Benzene via Substituent Optimization: A Structure–Performance Relationship Study","authors":"Zhuandi Zhu,Mingshuai Fan,Yubao Lan,Zhengang Han,Yaqi Zhao,Yanjun Feng,Xiaowan Li,Xiaoquan Lu","doi":"10.1021/acs.analchem.5c06945","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c06945","url":null,"abstract":"Carbazole derivatives have great potential in the field of electrochemiluminescence (ECL) due to their excellent photoelectric properties. The ECL properties of carbazole derivatives have been studied by modifying different substituents on carbazolyl benzene. Among them, DPA-BFCz shows obvious aggregated enhanced luminescence (AEE) and excellent ECL performance. Notably, scanning photoelectrochemical microscopy (SPECM) and intensity-modulated photocurrent spectroscopy (IMPS) are used to analyze the charge transfer kinetics of the emitters. In addition, a sensor of Cyt c with an ECL-resonance energy transfer (RET) mechanism is developed by taking advantage of the superior ECL performance of DPA-BFCz, with a concentration range of 1.0 to 500 nM and a detection limit of 0.20 nM. This work presents a novel strategy for rapid and sensitive detection of Cyt c.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"22 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111268","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}
Colorectal cancer (CRC) continues to represent a serious threat to global health, and its diagnosis faces significant challenges, especially in the early phases of malignant tumors. Herein, we demonstrate that BRD2 RNA can serve as a potent noninvasive CRC biomarker and construct a CRISPR/Cas13a-engineered RNA-based fluorogenic biosensor for label-free detection of BRD2 RNA in colorectal tissues. In this assay, the specific recognition of BRD2 RNA by the substrate probe activates Cas13a/crRNA, leading to the trans-cleavage of the substrate probe and the generation of the T7 promoter sequence. The resulting T7 promoter subsequently induces efficient transcription amplification to synthesize abundant Pepper RNA aptamers that can light up HBC620. Leveraging the synergistic advantages of Cas13a precision, efficient transcription amplification, and superior signal-to-noise ratio of RNA aptamer-fluorophore complex, this fluorogenic biosensor enables sensitive detection of BRD2 RNA down to 0.39 fM and accurate quantification of its expression at the single-cell level. In addition, this fluorogenic biosensor can successfully distinguish CRC patient tissues from adjacent normal tissues based on distinct BRD2 RNA expression profiles. Importantly, the programmability of crRNA makes this fluorogenic biosensor readily adapted for detecting a broad range of RNA targets (e.g., noncoding RNAs and viral RNAs) by simply modifying the spacer sequence of crRNA, providing a new paradigm for early clinical diagnostics.
{"title":"CRISPR/Cas13a-Engineered RNA-Based Fluorogenic Biosensor for Label-Free Quantification of RNA in Colorectal Tissues.","authors":"Yu-Chen Xu, Wen-Jing Liu, Chen-Chen Li, Dandan Zhang, Fei Ma, Chun-Yang Zhang","doi":"10.1021/acs.analchem.5c07694","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c07694","url":null,"abstract":"<p><p>Colorectal cancer (CRC) continues to represent a serious threat to global health, and its diagnosis faces significant challenges, especially in the early phases of malignant tumors. Herein, we demonstrate that BRD2 RNA can serve as a potent noninvasive CRC biomarker and construct a CRISPR/Cas13a-engineered RNA-based fluorogenic biosensor for label-free detection of BRD2 RNA in colorectal tissues. In this assay, the specific recognition of BRD2 RNA by the substrate probe activates Cas13a/crRNA, leading to the <i>trans</i>-cleavage of the substrate probe and the generation of the T7 promoter sequence. The resulting T7 promoter subsequently induces efficient transcription amplification to synthesize abundant Pepper RNA aptamers that can light up HBC620. Leveraging the synergistic advantages of Cas13a precision, efficient transcription amplification, and superior signal-to-noise ratio of RNA aptamer-fluorophore complex, this fluorogenic biosensor enables sensitive detection of BRD2 RNA down to 0.39 fM and accurate quantification of its expression at the single-cell level. In addition, this fluorogenic biosensor can successfully distinguish CRC patient tissues from adjacent normal tissues based on distinct BRD2 RNA expression profiles. Importantly, the programmability of crRNA makes this fluorogenic biosensor readily adapted for detecting a broad range of RNA targets (e.g., noncoding RNAs and viral RNAs) by simply modifying the spacer sequence of crRNA, providing a new paradigm for early clinical diagnostics.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":" ","pages":""},"PeriodicalIF":6.7,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146117136","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}
Real-time monitoring of intracellular oxygen (O2) is crucial for understanding cell proliferation, differentiation, and metabolic processes, as well as for observing cellular behavior within hypoxic tissues associated with conditions such as cancer, ischemia, and chronic kidney disease. We developed small-molecule intracellular O2 probes based on the cationic Ir(III) complexes C6-BA and C545T-BA (C6 = coumarin 6, C545T = coumarin 545T, and BA = N,N′-dibutylethylenediamine) and investigated intracellular O2 levels using a commercially available microplate reader and phosphorescence lifetime imaging microscope (PLIM). The synthesized Ir(III) complexes exhibited a strong visible absorption band with a molar absorption coefficient of approximately 100,000 dm–3 mol–1 cm–1 due to singlet ligand-centered π,π* transitions originating from the C6 and C545T ligands. C6-BA and C545T-BA exhibited bright emission with high quantum yield (0.65 for C6-BA, 0.44 for C545T-BA) and remarkably long emission lifetimes (34.8 μs for C6-BA, 53.7 μs for C545T-BA) in N2-saturated acetonitrile. A comprehensive study of cellular analyses of these complexes revealed that C6-BA and C545T-BA are efficiently taken up by cultured cells and exhibit high O2 sensitivity. The phosphorescence lifetime of living cells stained with each Ir(III) complex determined by measuring time-resolved emission using a microplate reader enabled tracking of real-time changes in cellular O2 tension after treatment with metabolic stimulants. Furthermore, we acquired PLIM images of living cells stained with C6-BA followed by treatment with metabolic stimulants. Time-lapse PLIM analysis showed a heterogeneous O2 distribution within the field of view and variations in O2 partial pressure between individual cells.
{"title":"Intracellular Oxygen Sensing Using Long-Phosphorescence-Lifetime Cationic Ir(III) Complexes With Coumarin 6 and 545T Ligands","authors":"Aoi Horikoshi,Tatsuya Hirose,Shuichi Shiozaki,Toshitada Yoshihara","doi":"10.1021/acs.analchem.5c06799","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c06799","url":null,"abstract":"Real-time monitoring of intracellular oxygen (O2) is crucial for understanding cell proliferation, differentiation, and metabolic processes, as well as for observing cellular behavior within hypoxic tissues associated with conditions such as cancer, ischemia, and chronic kidney disease. We developed small-molecule intracellular O2 probes based on the cationic Ir(III) complexes C6-BA and C545T-BA (C6 = coumarin 6, C545T = coumarin 545T, and BA = N,N′-dibutylethylenediamine) and investigated intracellular O2 levels using a commercially available microplate reader and phosphorescence lifetime imaging microscope (PLIM). The synthesized Ir(III) complexes exhibited a strong visible absorption band with a molar absorption coefficient of approximately 100,000 dm–3 mol–1 cm–1 due to singlet ligand-centered π,π* transitions originating from the C6 and C545T ligands. C6-BA and C545T-BA exhibited bright emission with high quantum yield (0.65 for C6-BA, 0.44 for C545T-BA) and remarkably long emission lifetimes (34.8 μs for C6-BA, 53.7 μs for C545T-BA) in N2-saturated acetonitrile. A comprehensive study of cellular analyses of these complexes revealed that C6-BA and C545T-BA are efficiently taken up by cultured cells and exhibit high O2 sensitivity. The phosphorescence lifetime of living cells stained with each Ir(III) complex determined by measuring time-resolved emission using a microplate reader enabled tracking of real-time changes in cellular O2 tension after treatment with metabolic stimulants. Furthermore, we acquired PLIM images of living cells stained with C6-BA followed by treatment with metabolic stimulants. Time-lapse PLIM analysis showed a heterogeneous O2 distribution within the field of view and variations in O2 partial pressure between individual cells.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"1 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111270","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}
Cyclic organochlorine chemicals (COCs), including dieldrin, endrin, endosulfan, aldrin, heptachlor, chlordane, and toxaphene, persist globally as carcinogenic and neurotoxic persistent organic pollutants, posing bioaccumulation risks. In this study, we designed two haptens based on the common hexachlorocyclopentadiene and norbornane structural scaffolds shared by these seven COCs. The design rationale was evaluated using computational chemistry assistance and validated through animal immunization, followed by immunological analysis data. Based on the predicted hapten H1, a broad-spectrum monoclonal antibody (2A11) was prepared with a sensitivity as low as 14.91 ng/mL. Through the molecular recognition mechanism, the key amino acid residues, HIS-31 and TYR-33, responsible for the broad-spectrum binding and sensitivity to COCs were elucidated. Subsequently, a rapid and broad-spectrum colloidal gold immunochromatographic assay (GICA) was developed. The visual detection limits for the seven COCs were determined to be 10–100 ng/mL in water and 50–500 ng/g in both fish and soil, respectively. Furthermore, results from the analysis of unknown samples showed a good agreement between GICA and gas chromatography-tandem mass spectrometry. The computer-aided chemistry-based hapten prediction strategy effectively guided the preparation of antibodies for broad-spectrum recognition of COCs, enabling their rapid screening and detection.
{"title":"Molecular Recognition-Based Detection: Antibody Dipsticks for Cyclic Organochlorine Chemicals","authors":"Jinyan Li,Lingling Guo,Aihua Qu,Chuanlai Xu,Hua Kuang,Xinxin Xu","doi":"10.1021/acs.analchem.5c06442","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c06442","url":null,"abstract":"Cyclic organochlorine chemicals (COCs), including dieldrin, endrin, endosulfan, aldrin, heptachlor, chlordane, and toxaphene, persist globally as carcinogenic and neurotoxic persistent organic pollutants, posing bioaccumulation risks. In this study, we designed two haptens based on the common hexachlorocyclopentadiene and norbornane structural scaffolds shared by these seven COCs. The design rationale was evaluated using computational chemistry assistance and validated through animal immunization, followed by immunological analysis data. Based on the predicted hapten H1, a broad-spectrum monoclonal antibody (2A11) was prepared with a sensitivity as low as 14.91 ng/mL. Through the molecular recognition mechanism, the key amino acid residues, HIS-31 and TYR-33, responsible for the broad-spectrum binding and sensitivity to COCs were elucidated. Subsequently, a rapid and broad-spectrum colloidal gold immunochromatographic assay (GICA) was developed. The visual detection limits for the seven COCs were determined to be 10–100 ng/mL in water and 50–500 ng/g in both fish and soil, respectively. Furthermore, results from the analysis of unknown samples showed a good agreement between GICA and gas chromatography-tandem mass spectrometry. The computer-aided chemistry-based hapten prediction strategy effectively guided the preparation of antibodies for broad-spectrum recognition of COCs, enabling their rapid screening and detection.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"17 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111273","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 : 2026-02-04DOI: 10.1021/acs.analchem.5c05489
Willmann Antonio Jiménez Morales,María del Pilar Cañizares-Macías
An innovative method, termed FRAP/ABTS-SIA, was developed to simultaneously integrate the FRAP and ABTS antioxidant assays within a single sequential injection analysis (SIA) system with spectrophotometric detection. Leveraging the kinetic differences between the assays and controlling the dispersion, a compact aspiration sequence (antioxidant–FRAP–ABTS–antioxidant-water) was optimized using a central composite design, defining a flow rate of 40 μL s–1 and aspiration volumes of 43, 38, 38, 43, and 100 μL, respectively. The system incorporated a helical reaction coil positioned before the detector, allowing the antioxidant–FRAP bolus to react while the ABTS–antioxidant–water sequence was aspirated into the holding coil. This configuration enhanced the FRAP signal and enabled clear separation of both analytical responses. Compared to conventional batch protocols, this strategy reduced FRAP reagent concentrations by 70% and ABTS•+ radical concentrations by 50%. The method delivers responses within a 2 min run, achieving a throughput of ∼30 samples h–1. Linearity was confirmed for both assays over the range 10–120 μmol L–1 Trolox, with detection limits of 0.031 μmol L–1 (FRAP) and 0.0047 μmol L–1 (ABTS). Intralaboratory precision was below 2% RSD, and recoveries ranged from 97.3 to 106.2% (FRAP) and 92.8 to 105.4% (ABTS). The method was successfully applied to complex food matrices─including coffees, wines, juices, and spices─showing correlations ≥0.99 with microplate reference assays. High-throughput, reagent savings, metrological robustness, and simplified data processing position FRAP/ABTS-SIA as an efficient and reliable tool for routine antioxidant capacity evaluation in food and biomedical applications.
{"title":"A Breakthrough SIA-Based Dual Assay for Simultaneous Evaluation of Antioxidant Capacity via ABTS and FRAP Mechanisms","authors":"Willmann Antonio Jiménez\u0000Morales,María del Pilar Cañizares-Macías","doi":"10.1021/acs.analchem.5c05489","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c05489","url":null,"abstract":"An innovative method, termed FRAP/ABTS-SIA, was developed to simultaneously integrate the FRAP and ABTS antioxidant assays within a single sequential injection analysis (SIA) system with spectrophotometric detection. Leveraging the kinetic differences between the assays and controlling the dispersion, a compact aspiration sequence (antioxidant–FRAP–ABTS–antioxidant-water) was optimized using a central composite design, defining a flow rate of 40 μL s–1 and aspiration volumes of 43, 38, 38, 43, and 100 μL, respectively. The system incorporated a helical reaction coil positioned before the detector, allowing the antioxidant–FRAP bolus to react while the ABTS–antioxidant–water sequence was aspirated into the holding coil. This configuration enhanced the FRAP signal and enabled clear separation of both analytical responses. Compared to conventional batch protocols, this strategy reduced FRAP reagent concentrations by 70% and ABTS•+ radical concentrations by 50%. The method delivers responses within a 2 min run, achieving a throughput of ∼30 samples h–1. Linearity was confirmed for both assays over the range 10–120 μmol L–1 Trolox, with detection limits of 0.031 μmol L–1 (FRAP) and 0.0047 μmol L–1 (ABTS). Intralaboratory precision was below 2% RSD, and recoveries ranged from 97.3 to 106.2% (FRAP) and 92.8 to 105.4% (ABTS). The method was successfully applied to complex food matrices─including coffees, wines, juices, and spices─showing correlations ≥0.99 with microplate reference assays. High-throughput, reagent savings, metrological robustness, and simplified data processing position FRAP/ABTS-SIA as an efficient and reliable tool for routine antioxidant capacity evaluation in food and biomedical applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"8 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2026-02-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146111274","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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