Pub Date : 2025-03-31DOI: 10.1021/acs.analchem.5c00547
Tianyi Zhang, Yingying Zhao, Cong Zhu, Xi Zhu, Xiaohua Zhu, Ye Qiu, Zhou Nie, Chunyang Lei
Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor’s mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.
{"title":"CRISPR/Cas12a Protein Switch Powered Label-Free Electrochemical Biosensor for Sensitive Viral Protease Detection","authors":"Tianyi Zhang, Yingying Zhao, Cong Zhu, Xi Zhu, Xiaohua Zhu, Ye Qiu, Zhou Nie, Chunyang Lei","doi":"10.1021/acs.analchem.5c00547","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00547","url":null,"abstract":"Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor’s mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"103 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143745558","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 : 2025-03-31DOI: 10.1021/acs.analchem.4c05502
Yingbo Pan, Dawei Wang, Ruyue Wei, Shuqi Wang, Yufan Li, Wei Pan, Ping Zhou, Na Li, Bo Tang
Early cancer screening is essential for reducing cancer-related mortality and improving survival rates. Simultaneous detection of multiple tumor markers can enhance the accuracy and specificity of cancer diagnosis, helping us to mitigate false-positive results associated with single-marker analysis. Here, we have developed a lateral flow detection platform that combines recombinase polymerase amplification (RPA), CRISPR Cas9, and catalyzed hairpin assembly (CHA) for the simultaneous detection of KRAS ctDNA and miRNA-223 in lung cancer. The CRISPR Cas9 system acts as a linking element, enabling specific recognition and binding to RPA amplicons of KRAS ctDNA while facilitating the capture of Au-DNA-Bio nanoparticles (NPs), thereby producing a stronger detection signal through Au NPs aggregation. The CHA system enhances this platform by providing sensitive detection of miRNA-223. Our platform was tested on a limited number of clinical saliva samples, demonstrating feasibility but requiring further validation with larger cohorts.
{"title":"Lateral Flow Platform for Lung Cancer Diagnosis through Simultaneous Detection of ctDNA and MicroRNA","authors":"Yingbo Pan, Dawei Wang, Ruyue Wei, Shuqi Wang, Yufan Li, Wei Pan, Ping Zhou, Na Li, Bo Tang","doi":"10.1021/acs.analchem.4c05502","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05502","url":null,"abstract":"Early cancer screening is essential for reducing cancer-related mortality and improving survival rates. Simultaneous detection of multiple tumor markers can enhance the accuracy and specificity of cancer diagnosis, helping us to mitigate false-positive results associated with single-marker analysis. Here, we have developed a lateral flow detection platform that combines recombinase polymerase amplification (RPA), CRISPR Cas9, and catalyzed hairpin assembly (CHA) for the simultaneous detection of KRAS ctDNA and miRNA-223 in lung cancer. The CRISPR Cas9 system acts as a linking element, enabling specific recognition and binding to RPA amplicons of KRAS ctDNA while facilitating the capture of Au-DNA-Bio nanoparticles (NPs), thereby producing a stronger detection signal through Au NPs aggregation. The CHA system enhances this platform by providing sensitive detection of miRNA-223. Our platform was tested on a limited number of clinical saliva samples, demonstrating feasibility but requiring further validation with larger cohorts.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"13 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737045","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 formation of Au–S bonding is commonly used for the fabrication of biosensors through self-assembly, but the stability of the Au–S bonding is not always satisfying in complex biological systems, as they contain biothiols like glutathione that may displace the self-assembled thiolated molecules. To address this issue, we explored the utilization of iridium–thiol interaction to form highly stable Ir–S bonding through self-assembly, and an electrochemical biosensor was developed by immobilizing antifouling thiol-peptides onto an electrode modified with Ir nanoparticles. The Ir–S bond was verified to be more robust than the Au–S bond, which ensured effective peptide immobilization and reduced displacement by biothiols. Additionally, we integrated functionalized peptides specifically designed for murine double minute 2 (MDM2) biological assays, resulting in a highly stable and sensitive platform for quantifying MDM2 in biological matrices. The explored Ir–S binding offers a new avenue for the self-assembly of thiolated molecules to develop ultrarobust biosensors and bioelectronics with enhanced reliability in complex biological environments.
{"title":"Ir–S Bonding Is Superior to Au–S Bonding for the Construction of Robust Antifouling Biosensors through Self-Assembly","authors":"Wenqing Wang, Yuanyuan Zhang, Baoping Zhu, Mingjun Shi, Rui Han, Xiliang Luo","doi":"10.1021/acs.analchem.4c06742","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c06742","url":null,"abstract":"The formation of Au–S bonding is commonly used for the fabrication of biosensors through self-assembly, but the stability of the Au–S bonding is not always satisfying in complex biological systems, as they contain biothiols like glutathione that may displace the self-assembled thiolated molecules. To address this issue, we explored the utilization of iridium–thiol interaction to form highly stable Ir–S bonding through self-assembly, and an electrochemical biosensor was developed by immobilizing antifouling thiol-peptides onto an electrode modified with Ir nanoparticles. The Ir–S bond was verified to be more robust than the Au–S bond, which ensured effective peptide immobilization and reduced displacement by biothiols. Additionally, we integrated functionalized peptides specifically designed for murine double minute 2 (MDM2) biological assays, resulting in a highly stable and sensitive platform for quantifying MDM2 in biological matrices. The explored Ir–S binding offers a new avenue for the self-assembly of thiolated molecules to develop ultrarobust biosensors and bioelectronics with enhanced reliability in complex biological environments.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"40 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737046","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 : 2025-03-31DOI: 10.1021/acs.analchem.4c03543
Hongtu Cheng, Jie Feng, Yang Shen, Zhi Fang
Photoacoustic spectroscopy (PAS) gas sensing systems are generally classified into resonant and nonresonant types. However, it remains unclear whether signals at both resonant and nonresonant frequencies can be demodulated by one demodulation channel and one pass cell to achieve the simultaneous detection of two target molecules. In this study, a cantilever-enhanced photoacoustic (PA) gas detection system was utilized to measure H2S and CO2, both of which have been identified as typical fault gases in SF6 gas-insulated electrical equipment. The vibration modes of the cantilever beam at its first resonant and nonresonant frequencies were simulated and analyzed. Utilizing a laser interferometer, the vibration signals of a cantilever beam were measured. The results confirm that the resonant and nonresonant components of the vibration signal can be demodulated simultaneously. Gas detection experiments were conducted using a cantilever beam-based PAS gas sensing unit. Measurements in the ternary gas mixtures demonstrated the feasibility of simultaneously detecting H2S and CO2 by extracting components at 34 and 349 Hz. The PA signals exhibited a strong linear relationship with the gas concentrations, and the detection limits of H2S and CO2 are 2.55 and 84.69 ppm, respectively.
{"title":"Cantilever-Enhanced Photoacoustic Spectroscopy: Simultaneous Demodulation of Resonant and Nonresonant Signals for the Measurement of Dual SF6 Decomposition Components","authors":"Hongtu Cheng, Jie Feng, Yang Shen, Zhi Fang","doi":"10.1021/acs.analchem.4c03543","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03543","url":null,"abstract":"Photoacoustic spectroscopy (PAS) gas sensing systems are generally classified into resonant and nonresonant types. However, it remains unclear whether signals at both resonant and nonresonant frequencies can be demodulated by one demodulation channel and one pass cell to achieve the simultaneous detection of two target molecules. In this study, a cantilever-enhanced photoacoustic (PA) gas detection system was utilized to measure H<sub>2</sub>S and CO<sub>2</sub>, both of which have been identified as typical fault gases in SF<sub>6</sub> gas-insulated electrical equipment. The vibration modes of the cantilever beam at its first resonant and nonresonant frequencies were simulated and analyzed. Utilizing a laser interferometer, the vibration signals of a cantilever beam were measured. The results confirm that the resonant and nonresonant components of the vibration signal can be demodulated simultaneously. Gas detection experiments were conducted using a cantilever beam-based PAS gas sensing unit. Measurements in the ternary gas mixtures demonstrated the feasibility of simultaneously detecting H<sub>2</sub>S and CO<sub>2</sub> by extracting components at 34 and 349 Hz. The PA signals exhibited a strong linear relationship with the gas concentrations, and the detection limits of H<sub>2</sub>S and CO<sub>2</sub> are 2.55 and 84.69 ppm, respectively.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"58 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737273","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 : 2025-03-31DOI: 10.1021/acs.analchem.5c00062
Xinyu Zhao, Hongchao Qi, Yajie Zhang, Yufu Xu, Chenxi Li, Min Guo, Fengxiang Ma, Wei Peng, Ke Chen
The effects of H2O and SF6 on the relaxation rate of CO molecules are studied using a fiber-optic photoacoustic sensor (FOPAS) that integrates a resonant multipass photoacoustic cell with a fiber cantilever. The CO photoacoustic signal is enhanced by approximately 1 order of magnitude under the induction of H2O. SF6 has a high density compared to N2, which causes the photoacoustic resonance frequency to shift to low frequency. The photoacoustic responses of CO/SF6 and CO/N2 at low frequency are tested with and without H2O to verify that SF6 gas has a promoting effect on the relaxation process. The impact of the increasing H2O concentration on the CO/N2 photoacoustic signal manifests in three distinct stages, which are reflected in gradual increase, sharp increase, and eventual saturation. The corresponding H2O concentration ranges of the three different stages are 2000–12,000, 12,000–18,000, and above 18,000 ppm, respectively. The SF6 ratio in CO/SF6 and CO/N2 gas is precisely controlled, and the influence mechanism of the background gas mixture changes on the resonance frequency, photoacoustic response, and Q-factor is studied. Furthermore, the responsivity and noise of the fiber-optic photoacoustic CO sensor are evaluated under the induction of H2O and SF6. The detection limits of CO/N2 and CO/SF6 are 10 ppb with averaging times of 100 and 400 s, which makes this study applicable to the detection environment with N2, SF6, and N2–SF6 mixtures as background gas.
{"title":"Effects of H2O and SF6 on CO Molecular Relaxation in a Cantilever-Enhanced Fiber-Optic Photoacoustic Sensor","authors":"Xinyu Zhao, Hongchao Qi, Yajie Zhang, Yufu Xu, Chenxi Li, Min Guo, Fengxiang Ma, Wei Peng, Ke Chen","doi":"10.1021/acs.analchem.5c00062","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00062","url":null,"abstract":"The effects of H<sub>2</sub>O and SF<sub>6</sub> on the relaxation rate of CO molecules are studied using a fiber-optic photoacoustic sensor (FOPAS) that integrates a resonant multipass photoacoustic cell with a fiber cantilever. The CO photoacoustic signal is enhanced by approximately 1 order of magnitude under the induction of H<sub>2</sub>O. SF<sub>6</sub> has a high density compared to N<sub>2</sub>, which causes the photoacoustic resonance frequency to shift to low frequency. The photoacoustic responses of CO/SF<sub>6</sub> and CO/N<sub>2</sub> at low frequency are tested with and without H<sub>2</sub>O to verify that SF<sub>6</sub> gas has a promoting effect on the relaxation process. The impact of the increasing H<sub>2</sub>O concentration on the CO/N<sub>2</sub> photoacoustic signal manifests in three distinct stages, which are reflected in gradual increase, sharp increase, and eventual saturation. The corresponding H<sub>2</sub>O concentration ranges of the three different stages are 2000–12,000, 12,000–18,000, and above 18,000 ppm, respectively. The SF<sub>6</sub> ratio in CO/SF<sub>6</sub> and CO/N<sub>2</sub> gas is precisely controlled, and the influence mechanism of the background gas mixture changes on the resonance frequency, photoacoustic response, and <i>Q</i>-factor is studied. Furthermore, the responsivity and noise of the fiber-optic photoacoustic CO sensor are evaluated under the induction of H<sub>2</sub>O and SF<sub>6</sub>. The detection limits of CO/N<sub>2</sub> and CO/SF<sub>6</sub> are 10 ppb with averaging times of 100 and 400 s, which makes this study applicable to the detection environment with N<sub>2</sub>, SF<sub>6,</sub> and N<sub>2</sub>–SF<sub>6</sub> mixtures as background gas.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"72 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737047","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 : 2025-03-31DOI: 10.1021/acs.analchem.5c00284
Josiah J. Rensner, Hyojin Kim, Kiyoul Park, Edgar B. Cahoon, Young Jin Lee
Lipids make up an important class of biomolecules with diverse structures and varied chemical functions. This diversity is a major challenge in chemical analysis and limits our understanding of biological functions and regulation. A major way lipid isomers differ is by double-bond (db) position, and analyzing db-isomers is especially challenging for mass spectrometry imaging (MSI). Ozonolysis can be used to determine the db-position and has been paired with MSI before. However, previous techniques require increased analysis time to allow for gas-phase reactions within an ion trap or ion mobility cell or additional sample preparation time to allow for offline ozonation. Here, we introduce a new ozonolysis method inside the matrix-assisted laser desorption-ionization (MALDI) source, termed OzMALDI, that simultaneously produces ozonides from all unsaturated lipids. This allows us to determine db-positions without adding additional reaction time while maintaining the high mass resolution provided by Orbitrap MS. This new technique is especially effective at determining multiple db-positions in lipids containing polyunsaturated fatty acids, which is a limitation of many previous techniques. OzMALDI-MSI was applied to the analysis of rat brain and genetically engineered Camelina and soybean seed samples, demonstrating the utility of this method and uncovering novel biological information.
{"title":"OzMALDI: A Gas-Phase, In-Source Ozonolysis Reaction for Efficient Double-Bond Assignment in Mass Spectrometry Imaging with Matrix-Assisted Laser Desorption/Ionization","authors":"Josiah J. Rensner, Hyojin Kim, Kiyoul Park, Edgar B. Cahoon, Young Jin Lee","doi":"10.1021/acs.analchem.5c00284","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00284","url":null,"abstract":"Lipids make up an important class of biomolecules with diverse structures and varied chemical functions. This diversity is a major challenge in chemical analysis and limits our understanding of biological functions and regulation. A major way lipid isomers differ is by double-bond (db) position, and analyzing db-isomers is especially challenging for mass spectrometry imaging (MSI). Ozonolysis can be used to determine the db-position and has been paired with MSI before. However, previous techniques require increased analysis time to allow for gas-phase reactions within an ion trap or ion mobility cell or additional sample preparation time to allow for offline ozonation. Here, we introduce a new ozonolysis method inside the matrix-assisted laser desorption-ionization (MALDI) source, termed OzMALDI, that simultaneously produces ozonides from all unsaturated lipids. This allows us to determine db-positions without adding additional reaction time while maintaining the high mass resolution provided by Orbitrap MS. This new technique is especially effective at determining multiple db-positions in lipids containing polyunsaturated fatty acids, which is a limitation of many previous techniques. OzMALDI-MSI was applied to the analysis of rat brain and genetically engineered <i>Camelina</i> and soybean seed samples, demonstrating the utility of this method and uncovering novel biological information.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"34 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737048","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}
Aptamers are versatile sensing elements for the construction of biosensors. A common approach for signal generation in “aptasensors” involves the displacement of short complementary “probes” resulting from conformational changes upon aptamer-target binding. However, designing strands that rapidly and completely displace when the target binds is nontrivial. Typically, probes are discovered through a lengthy process of screening several potential sequences. Here, we explored properties governing probe displacement efficiency using a well-characterized aptamer for the agricultural contaminant ochratoxin A (OTA). Surprisingly, the length, probe affinity, and melting temperature did not correlate with probe displacement efficiency. We therefore developed a novel surface plasmon resonance (SPR) assay to rapidly measure target-induced displacement of probes from aptamers. Fitted displacement results from the SPR assay were correlated with fast proportional fluorescence recovery from quencher-labeled probe displacement. This new method allows for the rapid distinction of efficient probes, resulting in sensitive biosensing of OTA. Finally, we demonstrated our new method is adaptable to diverse aptamers, offering a generally applicable method to improve probe design and accelerate aptasensor development.
{"title":"A Generalizable Screening Platform for Developing Functional Aptasensors","authors":"Micaela Belleperche, Jiawen Liu, Yuhao Chen, Chuyang Zhang, Kimiya Karimi, Sabrina Leslie, Maureen McKeague","doi":"10.1021/acs.analchem.4c04120","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c04120","url":null,"abstract":"Aptamers are versatile sensing elements for the construction of biosensors. A common approach for signal generation in “aptasensors” involves the displacement of short complementary “probes” resulting from conformational changes upon aptamer-target binding. However, designing strands that rapidly and completely displace when the target binds is nontrivial. Typically, probes are discovered through a lengthy process of screening several potential sequences. Here, we explored properties governing probe displacement efficiency using a well-characterized aptamer for the agricultural contaminant ochratoxin A (OTA). Surprisingly, the length, probe affinity, and melting temperature did not correlate with probe displacement efficiency. We therefore developed a novel surface plasmon resonance (SPR) assay to rapidly measure target-induced displacement of probes from aptamers. Fitted displacement results from the SPR assay were correlated with fast proportional fluorescence recovery from quencher-labeled probe displacement. This new method allows for the rapid distinction of efficient probes, resulting in sensitive biosensing of OTA. Finally, we demonstrated our new method is adaptable to diverse aptamers, offering a generally applicable method to improve probe design and accelerate aptasensor development.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"3 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143745555","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 : 2025-03-31DOI: 10.1021/acs.analchem.4c05885
Fengfei Ma, A. Carl Sanchez, James Song, Esther Kofman, Daniela Tomazela, Laurence Fayadat-Dilman, Kanaka Hettiarachchi, Mohammad Ahmed Al-Sayah
Characterization of drug–antibody ratio (DAR) species in antibody–drug conjugates (ADCs) is crucial for assessing the developability/manufacturability and downstream development of drug candidates. Although hydrophobic interaction chromatography (HIC) is the gold standard for DAR analysis, elucidating DAR species within each HIC peak has historically been challenging. This is due to the nonvolatility and high ionic strength of conventional buffer systems, which necessitate labor-intensive offline fractionation, followed by MS analysis. To address these challenges, an innovative alternative strategy has been developed that directly couples native reversed-phase liquid chromatography (nRPLC) to high-resolution Orbitrap MS for online native MS analysis (nRPLC-MS). In collaboration with Phenomenex, two types of columns, each with a different hydrophobicity, were developed, allowing for elution with low concentration of MS-friendly salt and organic buffer. LC and MS parameters were optimized to enhance the detection of intact DAR species under high flow rate conditions. To demonstrate the feasibility of the platform for characterizing different types of ADCs, both interchain-linked (heterogeneous DAR of 0 to 8) and site-specific ADCs were evaluated. The method enables the nondenatured separation and simultaneous characterization of different DAR species, and strong correlation was observed between this approach and analysis by HIC. This integrated strategy allows unbiased characterization of DAR species without postcolumn flow splitting or peak fractionation. Furthermore, comparisons with two commonly used methods (native SEC-MS and RPLC-MS) have shown that superior separation in terms of selectivity and resolution is achieved with the nRPLC method. Notably, unconjugated antibody (DAR0) was successfully retained with a low-ionic-strength salt using this method. Moreover, the method facilitated the chromatographic separation of positional isomers of DAR4 species with different conjugation linkages, which was not achievable with traditional HIC. As a result, this method holds great promise for high-throughput screening and characterization of ADCs across conjugation methods and payload classes.
{"title":"Novel Native Reversed-Phase Liquid Chromatography (nRPLC)/MS for Antibody–Drug Conjugates (ADCs) Characterization and Drug–Antibody Ratio (DAR) Assessment","authors":"Fengfei Ma, A. Carl Sanchez, James Song, Esther Kofman, Daniela Tomazela, Laurence Fayadat-Dilman, Kanaka Hettiarachchi, Mohammad Ahmed Al-Sayah","doi":"10.1021/acs.analchem.4c05885","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05885","url":null,"abstract":"Characterization of drug–antibody ratio (DAR) species in antibody–drug conjugates (ADCs) is crucial for assessing the developability/manufacturability and downstream development of drug candidates. Although hydrophobic interaction chromatography (HIC) is the gold standard for DAR analysis, elucidating DAR species within each HIC peak has historically been challenging. This is due to the nonvolatility and high ionic strength of conventional buffer systems, which necessitate labor-intensive offline fractionation, followed by MS analysis. To address these challenges, an innovative alternative strategy has been developed that directly couples native reversed-phase liquid chromatography (nRPLC) to high-resolution Orbitrap MS for online native MS analysis (nRPLC-MS). In collaboration with Phenomenex, two types of columns, each with a different hydrophobicity, were developed, allowing for elution with low concentration of MS-friendly salt and organic buffer. LC and MS parameters were optimized to enhance the detection of intact DAR species under high flow rate conditions. To demonstrate the feasibility of the platform for characterizing different types of ADCs, both interchain-linked (heterogeneous DAR of 0 to 8) and site-specific ADCs were evaluated. The method enables the nondenatured separation and simultaneous characterization of different DAR species, and strong correlation was observed between this approach and analysis by HIC. This integrated strategy allows unbiased characterization of DAR species without postcolumn flow splitting or peak fractionation. Furthermore, comparisons with two commonly used methods (native SEC-MS and RPLC-MS) have shown that superior separation in terms of selectivity and resolution is achieved with the nRPLC method. Notably, unconjugated antibody (DAR0) was successfully retained with a low-ionic-strength salt using this method. Moreover, the method facilitated the chromatographic separation of positional isomers of DAR4 species with different conjugation linkages, which was not achievable with traditional HIC. As a result, this method holds great promise for high-throughput screening and characterization of ADCs across conjugation methods and payload classes.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"16 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143745556","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 Argonaute (Ago) protein exhibits high specificity in nucleic acid recognition and cleavage, making it highly promising for biosensing applications. Its potential is further enhanced by its independence from protospacer adjacent motif (PAM) requirements and the cost-effectiveness of using short DNA guides. Both Ago and CRISPR/Cas systems face challenges in signal amplification, which limit their ability to detect targets at ultralow concentrations. To overcome this limitation, a thermostable quadratic amplification system (T-QAS) was constructed by integrating a thermostable nicking-enzyme-mediated amplification (NEMA) strategy with TtAgo. The system leverages the high stability of T-QAS at elevated temperatures to enhance guide–target interactions and decrease false positives caused by nonspecific amplification. Additionally, nanozyme is integrated with T-QAS to construct the AIESTA platform (all-in-one isothermal enzymatic signal transduction amplifier), which is a single-tube visual sensing platform. Within the AIESTA system, T-QAS improves specificity through high operational temperatures and offers programmable functions, enabling the sensitive detection of miRNA and foodborne toxins. The combination of T-QAS and nanozyme makes AIESTA a candidate of point-of-care testing (POCT) field, showcasing the potential for biosensing in resource-limited and complex environments.
{"title":"Programmable AIESTA: All-in-One Isothermal Enzymatic Signal Transduction Amplifier for Portable Profiling","authors":"Haoran Shen, Yanling Li, Kangling Tang, Hongzhi Liang, Zhen-Lin Xu, Yingju Liu, Weipeng Liu","doi":"10.1021/acs.analchem.5c00934","DOIUrl":"https://doi.org/10.1021/acs.analchem.5c00934","url":null,"abstract":"The Argonaute (Ago) protein exhibits high specificity in nucleic acid recognition and cleavage, making it highly promising for biosensing applications. Its potential is further enhanced by its independence from protospacer adjacent motif (PAM) requirements and the cost-effectiveness of using short DNA guides. Both Ago and CRISPR/Cas systems face challenges in signal amplification, which limit their ability to detect targets at ultralow concentrations. To overcome this limitation, a thermostable quadratic amplification system (T-QAS) was constructed by integrating a thermostable nicking-enzyme-mediated amplification (NEMA) strategy with TtAgo. The system leverages the high stability of T-QAS at elevated temperatures to enhance guide–target interactions and decrease false positives caused by nonspecific amplification. Additionally, nanozyme is integrated with T-QAS to construct the AIESTA platform (all-in-one isothermal enzymatic signal transduction amplifier), which is a single-tube visual sensing platform. Within the AIESTA system, T-QAS improves specificity through high operational temperatures and offers programmable functions, enabling the sensitive detection of miRNA and foodborne toxins. The combination of T-QAS and nanozyme makes AIESTA a candidate of point-of-care testing (POCT) field, showcasing the potential for biosensing in resource-limited and complex environments.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"36 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143737049","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 : 2025-03-31DOI: 10.1021/acs.analchem.4c04750
Ling Dai, Mengjun Deng, Kena Chen, Xueping Chen, Junjie Li
The in-frame internal tandem duplication of the FLT-3 gene (FLT3-ITD), a prevalent genetic aberration, significantly contributes to treatment failure and poor prognosis in acute myeloid leukemia (AML). A robust and cost-effective assay for minimal residual disease (MRD) detection in FLT3-ITD+ AML is crucial for guiding therapeutic decisions. However, current MRD monitoring methodologies for FLT3-ITD+ patients are limited by sensitivity and adaptability, particularly for dynamically quantifying complex and heterogeneous FLT3-ITD mutations. In this study, we developed a primer competition enhanced mutation accumulation (PCEMA) technique designed to selectively enrich FLT3-ITD in the context of abundant wild-type alleles. By integrating the PCEMA with capillary electrophoresis, we significantly improved the discrimination between mutant and wild-type genes, increasing the minimum detectable sensitivity to 0.001%, comparable to next-generation sequencing. The competitive amplification between ITD-specific and universal primers facilitated the selective enrichment of mutant alleles, enabling highly sensitive and specific real-time FLT3-ITD mutation monitoring. We thoroughly evaluated the analytical performance and adoptability of the PCEMA technique in conjunction with quantitative fluorescent PCR (qPCEMA). Our results demonstrated that qPCEMA quantitatively differentiates FLT3-ITD with a mutation frequency below 0.1%, offering an effective, rapid, and reliable method for long-term FLT3-ITD monitoring in clinical AML patients. The PCEMA technique, characterized by its robustness, sensitivity, specificity, timeliness, and adoptability, presents a promising alternative for clinical FLT3-ITD mutation detection. It is anticipated to provide significant technical support for timely diagnosis, prognosis assessment, drug evaluation, and personalized treatment of AML patients, with substantial potential for clinical application.
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