Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been widely applied in molecular diagnostics. Unlike the Ct value quantification method of PCR, the CRISPR system mainly relies on the rise of the rate of the fluorescence signal to indicate the concentration of the target nucleic acid, which is susceptible to system errors caused by various factors, such as reaction conditions and instrument performance. Therefore, establishing internal controls is essential to improve the accuracy, reliability, and commercial feasibility of the CRISPR system. However, the nonspecific trans-cleavage activity of Cas proteins presents a challenge in establishing internal controls. In this study, we developed unified nucleic acid detection with a single-tube, one-enzyme system (UNISON) for accurate nucleic acid detection with internal controls. By extending the crRNA and modifying it with different fluorophores and quenchers, we achieved that the specific target can only specifically cleave the corresponding folded crRNA and generate a corresponding fluorescence signal. With this design, we established an internal control, achieving accurate and reliable detection of clinical samples of the hepatitis B virus. Integrating internal controls into the CRISPR/Cas system demonstrates significant potential in medical diagnostics and virus monitoring.
{"title":"A Single-Tube, Single-Enzyme Clustered Regularly Interspaced Short Palindromic Repeats System (UNISON) with Internal Controls for Accurate Nucleic Acid Detection","authors":"Jialing Zhong, Xiaolong Wu, Chuanghao Guo, Conghui Liu, Qianling Zhang, Yong Chen, Yizhen Liu","doi":"10.1021/acs.analchem.4c03403","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03403","url":null,"abstract":"Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been widely applied in molecular diagnostics. Unlike the Ct value quantification method of PCR, the CRISPR system mainly relies on the rise of the rate of the fluorescence signal to indicate the concentration of the target nucleic acid, which is susceptible to system errors caused by various factors, such as reaction conditions and instrument performance. Therefore, establishing internal controls is essential to improve the accuracy, reliability, and commercial feasibility of the CRISPR system. However, the nonspecific <i>trans</i>-cleavage activity of Cas proteins presents a challenge in establishing internal controls. In this study, we developed unified nucleic acid detection with a single-tube, one-enzyme system (UNISON) for accurate nucleic acid detection with internal controls. By extending the crRNA and modifying it with different fluorophores and quenchers, we achieved that the specific target can only specifically cleave the corresponding folded crRNA and generate a corresponding fluorescence signal. With this design, we established an internal control, achieving accurate and reliable detection of clinical samples of the hepatitis B virus. Integrating internal controls into the CRISPR/Cas system demonstrates significant potential in medical diagnostics and virus monitoring.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"34 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142684909","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}
Hydrogen sulfide (H2S) is an important bioactive molecule that plays a significant role in various functions, particularly in the living brain, where it is closely linked to cognition, memory, and several neurological diseases. Consequently, developing effective detection methods for H2S is essential for studying brain functions and the underlying mechanisms of these diseases. This study aims to construct a novel photoelectrochemical (PEC) microelectrode Ti/TiO2@HSP for the quantitative monitoring of H2S levels in the living brain. The PEC microelectrode Ti/TiO2@HSP is formed by covalently bonding a specifically designed organic PEC probe HSP, which possesses a D-π–A structure, to the surface of TiO2 nanotubes generated via in situ anodic oxidation of titanium wire. The PEC probe HSP can effectively react with H2S and generate significant photocurrent response under long-wavelength excitation light (560 nm), thereby achieving quantitative detection of H2S. The sensor demonstrates high sensitivity and good selectivity. In vivo experiments utilizing the PEC microelectrode Ti/TiO2@HSP enable the monitoring of dynamic changes in H2S levels across various regions of the mouse brain. The findings reveal that in normal mice, the concentration of H2S in the hippocampus is significantly higher than in the striatum and cerebral cortex. Additionally, following propargylglycine drug stimulation, H2S concentrations in different brain regions were observed to decrease, with the most substantial reduction noted in the hippocampus. This suggests that cystathionine γ-lyase (CSE) is the primary enzyme responsible for H2S production in this area, while the striatum exhibits a less pronounced decrease in H2S concentration, indicating a reliance on alternative enzymatic pathways for H2S production. Therefore, this study not only successfully develops a high-performance H2S detection sensor but also provides new experimental tools and theoretical foundations for further exploring the roles of H2S in neurophysiological and pathological processes.
{"title":"Development of a Photoelectrochemical Microelectrode Using an Organic Probe for Monitoring Hydrogen Sulfide in Living Brains","authors":"Yuanqiang Hao, Yewen Yang, Wenhui Wang, Hui Gu, Wansong Chen, Chunlan Li, Peisheng Zhang, Rongjin Zeng, Maotian Xu, Shu Chen","doi":"10.1021/acs.analchem.4c05336","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05336","url":null,"abstract":"Hydrogen sulfide (H<sub>2</sub>S) is an important bioactive molecule that plays a significant role in various functions, particularly in the living brain, where it is closely linked to cognition, memory, and several neurological diseases. Consequently, developing effective detection methods for H<sub>2</sub>S is essential for studying brain functions and the underlying mechanisms of these diseases. This study aims to construct a novel photoelectrochemical (PEC) microelectrode Ti/TiO<sub>2</sub>@HSP for the quantitative monitoring of H<sub>2</sub>S levels in the living brain. The PEC microelectrode Ti/TiO<sub>2</sub>@HSP is formed by covalently bonding a specifically designed organic PEC probe HSP, which possesses a D-π–<i>A</i> structure, to the surface of TiO<sub>2</sub> nanotubes generated via in situ anodic oxidation of titanium wire. The PEC probe HSP can effectively react with H<sub>2</sub>S and generate significant photocurrent response under long-wavelength excitation light (560 nm), thereby achieving quantitative detection of H<sub>2</sub>S. The sensor demonstrates high sensitivity and good selectivity. In vivo experiments utilizing the PEC microelectrode Ti/TiO<sub>2</sub>@HSP enable the monitoring of dynamic changes in H<sub>2</sub>S levels across various regions of the mouse brain. The findings reveal that in normal mice, the concentration of H<sub>2</sub>S in the hippocampus is significantly higher than in the striatum and cerebral cortex. Additionally, following propargylglycine drug stimulation, H<sub>2</sub>S concentrations in different brain regions were observed to decrease, with the most substantial reduction noted in the hippocampus. This suggests that cystathionine γ-lyase (CSE) is the primary enzyme responsible for H<sub>2</sub>S production in this area, while the striatum exhibits a less pronounced decrease in H<sub>2</sub>S concentration, indicating a reliance on alternative enzymatic pathways for H<sub>2</sub>S production. Therefore, this study not only successfully develops a high-performance H<sub>2</sub>S detection sensor but also provides new experimental tools and theoretical foundations for further exploring the roles of H<sub>2</sub>S in neurophysiological and pathological processes.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"18 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142690757","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 : 2024-11-22DOI: 10.1021/acs.analchem.4c02011
Amanda Helms, Vincent Chang, Stacy A. Malaker, Jennifer S. Brodbelt
Deciphering the pattern and abundance of O-glycosylation of mucin domain proteins, glycoproteins heavily implicated in cancer and other diseases, remains an ongoing challenge. Both the macro- and microheterogeneity of glycosylation complicates the analysis, motivating the development of new strategies for structural characterization of this diverse class of glycoproteins. Here we combine digestion of mucin domain proteins using a targeted protease, Enhancin from Serratia marcescens (SmE), with ultraviolet photodissociation (UVPD) mass spectrometry to advance glycan mapping and elucidation of O-glycosylation trends of densely glycosylated mucin proteins. UVPD facilitates identification of O-glycoforms of mucin domain proteins TIM-1, MUC-1 and MUC-16. Additionally, UVPD elucidates several glycoforms of MUC-16 and contributes to the discovery of O-glycosylation across tandem repeats of MUC-1.
{"title":"Unraveling O-Glycan Diversity of Mucins: Insights from SmE Mucinase and Ultraviolet Photodissociation Mass Spectrometry","authors":"Amanda Helms, Vincent Chang, Stacy A. Malaker, Jennifer S. Brodbelt","doi":"10.1021/acs.analchem.4c02011","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c02011","url":null,"abstract":"Deciphering the pattern and abundance of <i>O</i>-glycosylation of mucin domain proteins, glycoproteins heavily implicated in cancer and other diseases, remains an ongoing challenge. Both the macro- and microheterogeneity of glycosylation complicates the analysis, motivating the development of new strategies for structural characterization of this diverse class of glycoproteins. Here we combine digestion of mucin domain proteins using a targeted protease, Enhancin from <i>Serratia marcescens</i> (SmE), with ultraviolet photodissociation (UVPD) mass spectrometry to advance glycan mapping and elucidation of <i>O</i>-glycosylation trends of densely glycosylated mucin proteins. UVPD facilitates identification of <i>O</i>-glycoforms of mucin domain proteins TIM-1, MUC-1 and MUC-16. Additionally, UVPD elucidates several glycoforms of MUC-16 and contributes to the discovery of <i>O</i>-glycosylation across tandem repeats of MUC-1.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"198 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142684908","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 : 2024-11-22DOI: 10.1021/acs.analchem.4c03914
Bin Guo, Xiaofei Sun, Shan Tao, Tian Tian, Haozhi Lei
As medical advancements turn most cancers into manageable chronic diseases, new challenges arise in cancer recurrence monitoring. Detecting circulating tumor cells (CTCs) is crucial for monitoring cancer recurrence, but the current methods are cumbersome and costly. This study developed a new CTC detection system combining DNA aptamer recognition, hybridization chain reaction (HCR) technology, and DNA logic devices, enabling the one-step recognition of CTCs by identifying multiple membrane proteins. After catalytically active Au nanoparticles were attached through reduction synthesis in situ onto the DNA hybridization strands of the CTCs surface, a 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric reaction was used to detect CTCs concentration via peroxidase-like catalysis. With this CTCs detection reporting system, we achieved an LOD of 4 cells/mL using an ultraviolet–visible (UV–vis) spectrophotometer. At certain concentrations, CTCs could even be detected visually without the need for an instrument. The development of this CTCs detection reporting system provided a convenient, reliable, and cost-effective detection strategy for widespread CTCs-based cancer recurrence monitoring.
随着医疗技术的进步，大多数癌症都变成了可以控制的慢性病，癌症复发监测也面临着新的挑战。检测循环肿瘤细胞（CTC）对监测癌症复发至关重要，但目前的方法繁琐且成本高昂。这项研究开发了一种新的 CTC 检测系统，该系统结合了 DNA 类似物识别、杂交链式反应（HCR）技术和 DNA 逻辑器件，通过识别多种膜蛋白，实现了对 CTC 的一步识别。通过还原合成将催化活性金纳米粒子原位附着在 CTCs 表面的 DNA 杂交链上后，利用 3,3′,5,5′-四甲基联苯胺（TMB）比色反应，通过过氧化物酶样催化作用检测 CTCs 的浓度。利用这种 CTCs 检测报告系统，我们使用紫外-可见（UV-vis）分光光度计检测到的最低检测限为 4 个细胞/毫升。在某些浓度下，甚至不需要仪器就能通过肉眼检测到 CTCs。这种 CTCs 检测报告系统的开发为广泛开展基于 CTCs 的癌症复发监测提供了一种方便、可靠和经济有效的检测策略。
{"title":"Utilizing DNA Logic Device for Precise Detection of Circulating Tumor Cells via High Catalytic Activity Au Nanoparticle Anchoring","authors":"Bin Guo, Xiaofei Sun, Shan Tao, Tian Tian, Haozhi Lei","doi":"10.1021/acs.analchem.4c03914","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03914","url":null,"abstract":"As medical advancements turn most cancers into manageable chronic diseases, new challenges arise in cancer recurrence monitoring. Detecting circulating tumor cells (CTCs) is crucial for monitoring cancer recurrence, but the current methods are cumbersome and costly. This study developed a new CTC detection system combining DNA aptamer recognition, hybridization chain reaction (HCR) technology, and DNA logic devices, enabling the one-step recognition of CTCs by identifying multiple membrane proteins. After catalytically active Au nanoparticles were attached through reduction synthesis in situ onto the DNA hybridization strands of the CTCs surface, a 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric reaction was used to detect CTCs concentration via peroxidase-like catalysis. With this CTCs detection reporting system, we achieved an LOD of 4 cells/mL using an ultraviolet–visible (UV–vis) spectrophotometer. At certain concentrations, CTCs could even be detected visually without the need for an instrument. The development of this CTCs detection reporting system provided a convenient, reliable, and cost-effective detection strategy for widespread CTCs-based cancer recurrence monitoring.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"2 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142684911","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 : 2024-11-22DOI: 10.1021/acs.analchem.4c03498
Daniel-Ralph Hermann, Georg Ramer, Bernhard Lendl
Proteins are characterized by their complex levels of structures, which in turn define their function. Understanding and evaluating these structures is therefore crucial to illuminating biological processes. One of the possible analytical methods is vibrational circular dichroism (VCD), which expands the structural sensitivity of classical infrared (IR) absorbance spectroscopy by the chiral sensitivity of circular dichroism. While this technique is powerful, it is plagued by low signal intensities and long measurement times. Here we present an optical setup leveraging the high brilliance of a quantum cascade laser to measure proteins in D2O at a path length of 204 μm. It was compared to classical Fourier-transform infrared spectroscopy (FT-IR) in terms of noise levels and in its applicability to secondary structure elucidation of proteins. Protein concentrations as low as 2 mg/mL were accessible by the laser-based system at a measurement time of 1 h. Further increase of the time resolution was possible by adapting the emission to cover only the amide I’ band. This allowed for the collection of spectral data at a measurement time of 5 min without a loss of performance. With this high time resolution, we are confident that dynamic processes of protein can now be monitored by VCD, increasing our understanding of these reactions.
{"title":"External Cavity Quantum Cascade Laser Vibrational Circular Dichroism Spectroscopy for Fast and Sensitive Analysis of Proteins at Low Concentrations","authors":"Daniel-Ralph Hermann, Georg Ramer, Bernhard Lendl","doi":"10.1021/acs.analchem.4c03498","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03498","url":null,"abstract":"Proteins are characterized by their complex levels of structures, which in turn define their function. Understanding and evaluating these structures is therefore crucial to illuminating biological processes. One of the possible analytical methods is vibrational circular dichroism (VCD), which expands the structural sensitivity of classical infrared (IR) absorbance spectroscopy by the chiral sensitivity of circular dichroism. While this technique is powerful, it is plagued by low signal intensities and long measurement times. Here we present an optical setup leveraging the high brilliance of a quantum cascade laser to measure proteins in D<sub>2</sub>O at a path length of 204 μm. It was compared to classical Fourier-transform infrared spectroscopy (FT-IR) in terms of noise levels and in its applicability to secondary structure elucidation of proteins. Protein concentrations as low as 2 mg/mL were accessible by the laser-based system at a measurement time of 1 h. Further increase of the time resolution was possible by adapting the emission to cover only the amide I’ band. This allowed for the collection of spectral data at a measurement time of 5 min without a loss of performance. With this high time resolution, we are confident that dynamic processes of protein can now be monitored by VCD, increasing our understanding of these reactions.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"55 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142684910","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 : 2024-11-22DOI: 10.1021/acs.analchem.4c05022
Santiago García, Gustavo Carmona-Santiago, Arturo Jiménez-Sánchez
The development of small-molecule fluorescent probes has revolutionized the monitoring of in vivo physicochemical parameters, offering unprecedented insights into biological processes. In this perspective, we critically examine recent advances and trends in the design and application of fluorescent probes for real-time in vivo monitoring of subcellular environments. Traditional concepts such as membrane potential, microviscosity, and micropolarity have been superseded by more biologically relevant parameters like membrane voltage, tension, and hydration, enhancing the accuracy of physiological assessments. This redefinition not only presents an evolved concept with broader applications in monitoring subcellular dynamics but also addresses the unmet needs of subcellular biology more effectively. We also highlight the limitations of commonly used probes in providing specific information about the redox environment, noting their nonspecificity to oxidants and the influence of various chemical interactions. These probes typically rely on free radical mechanisms and require metal catalysts to react with hydrogen peroxide. They include naphthalimide, fluorescein, BODIPY, rhodamine, cyanine cores to cover the UV–vis–near-infrared window. The motif of this perspective is to provide critical insights into trending fluorescent-based systems employed in real-time or in vivo physicochemical-responsive monitoring, thus aiming to inform and inspire further research in creating robust and efficient fluorescent probes for comprehensive in vivo monitoring applications.
{"title":"Redefining Molecular Probes for Monitoring Subcellular Environment: A Perspective","authors":"Santiago García, Gustavo Carmona-Santiago, Arturo Jiménez-Sánchez","doi":"10.1021/acs.analchem.4c05022","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c05022","url":null,"abstract":"The development of small-molecule fluorescent probes has revolutionized the monitoring of <i>in vivo</i> physicochemical parameters, offering unprecedented insights into biological processes. In this perspective, we critically examine recent advances and trends in the design and application of fluorescent probes for real-time <i>in vivo</i> monitoring of subcellular environments. Traditional concepts such as membrane potential, microviscosity, and micropolarity have been superseded by more biologically relevant parameters like membrane voltage, tension, and hydration, enhancing the accuracy of physiological assessments. This redefinition not only presents an evolved concept with broader applications in monitoring subcellular dynamics but also addresses the unmet needs of subcellular biology more effectively. We also highlight the limitations of commonly used probes in providing specific information about the redox environment, noting their nonspecificity to oxidants and the influence of various chemical interactions. These probes typically rely on free radical mechanisms and require metal catalysts to react with hydrogen peroxide. They include naphthalimide, fluorescein, BODIPY, rhodamine, cyanine cores to cover the UV–vis–near-infrared window. The motif of this perspective is to provide critical insights into trending fluorescent-based systems employed in real-time or <i>in vivo</i> physicochemical-responsive monitoring, thus aiming to inform and inspire further research in creating robust and efficient fluorescent probes for comprehensive <i>in vivo</i> monitoring applications.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"24 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142690755","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 : 2024-11-22DOI: 10.1021/acs.analchem.4c03107
Adam R. Bernicky, Boyd Davis, Milen Kadiyski, Hans-Peter Loock
Solid preconcentrated ore samples used in pyrometallurgical copper smelters are analyzed by flame emission spectroscopy using a specialized flame optical emission spectroscopy (OES), system. Over 8500 complex spectra are categorized using an artificial neural network (ANN) that was optimized to have 10 hidden layers with 40 nodes per layer. The ANN was able to quantify the elemental content of all samples to within better than 1.5 mass% and was able to identify the prevalent minerals to within better than 2.5 mass%. The flame temperature was obtained with an uncertainty of σ < 3 K and the particle size to within 2 μm. The results are found to be superior to those obtained to a nonlinear partial least-squares fit model, which is equivalent to an ANN having no hidden layers.
{"title":"Mineralogical Analysis of Solid-Sample Flame Emission Spectra by Machine Learning","authors":"Adam R. Bernicky, Boyd Davis, Milen Kadiyski, Hans-Peter Loock","doi":"10.1021/acs.analchem.4c03107","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03107","url":null,"abstract":"Solid preconcentrated ore samples used in pyrometallurgical copper smelters are analyzed by flame emission spectroscopy using a specialized flame optical emission spectroscopy (OES), system. Over 8500 complex spectra are categorized using an artificial neural network (ANN) that was optimized to have 10 hidden layers with 40 nodes per layer. The ANN was able to quantify the elemental content of all samples to within better than 1.5 mass% and was able to identify the prevalent minerals to within better than 2.5 mass%. The flame temperature was obtained with an uncertainty of σ < 3 K and the particle size to within 2 μm. The results are found to be superior to those obtained to a nonlinear partial least-squares fit model, which is equivalent to an ANN having no hidden layers.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"254 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142684907","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 : 2024-11-22DOI: 10.1021/acs.analchem.4c03973
Songeun Kim, Jisoo Im, Shan X. Wang, Jung-Rok Lee
Planar DNA biosensors employ surface-tethered oligonucleotide probes to capture target molecules for diagnostic applications. To improve the sensitivity and specificity of biosensing, hybridization affinities should be enhanced, and cross-hybridization with off-targets must be minimized. To this end, assays can be designed using the thermodynamic properties of hybridization between probes and on-targets or off-targets based on Gibbs free energies and melting temperatures. However, the nature of heterogeneous hybridization between the probes on the surface and the targets in a solution imposes challenges in predicting precise hybridization affinities and the degree of cross-hybridization due to indeterminable thermodynamic penalties induced by the solid surface and its status. Herein, we suggest practical and convenient guidelines for designing oligonucleotide probes based on data obtained from planar magnetic biosensors and thermodynamic properties calculated by using easily accessible solution-phase prediction. The suggested requirements comprised Gibbs free energy ≥ −7.5 kcal mol–1 and melting temperature ≤10 °C below the hybridization temperature, and we validated for the absence of cross-hybridization. Additionally, the effects of secondary structures such as hairpins and homodimers were investigated for better oligonucleotide probe designs. We believe that these practical guidelines will assist researchers in developing planar magnetic biosensors with high sensitivity and specificity for the detection of new targets.
平面 DNA 生物传感器采用表面系留寡核苷酸探针捕获目标分子，用于诊断应用。为提高生物传感的灵敏度和特异性，应增强杂交亲和力，并尽量减少与非目标的交叉杂交。为此，可以根据吉布斯自由能和熔化温度，利用探针与靶上或靶下杂交的热力学特性来设计检测方法。然而，表面探针与溶液中靶标之间的异质杂交性质给预测精确的杂交亲和力和交叉杂交程度带来了挑战，因为固体表面及其状态引起的热力学惩罚是不确定的。在此，我们根据从平面磁性生物传感器获得的数据和利用易于获得的溶液相预测计算出的热力学性质，提出了设计寡核苷酸探针的实用便捷指南。建议的要求包括吉布斯自由能≥ -7.5 kcal mol-1，熔化温度低于杂交温度≤10 °C，我们还验证了无交叉杂交。此外，我们还研究了二级结构（如发夹和同源二聚体）对更好的寡核苷酸探针设计的影响。我们相信，这些实用指南将有助于研究人员开发具有高灵敏度和特异性的平面磁性生物传感器，用于检测新的目标。
{"title":"Design and Validation of Specific Oligonucleotide Probes on Planar Magnetic Biosensors","authors":"Songeun Kim, Jisoo Im, Shan X. Wang, Jung-Rok Lee","doi":"10.1021/acs.analchem.4c03973","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03973","url":null,"abstract":"Planar DNA biosensors employ surface-tethered oligonucleotide probes to capture target molecules for diagnostic applications. To improve the sensitivity and specificity of biosensing, hybridization affinities should be enhanced, and cross-hybridization with off-targets must be minimized. To this end, assays can be designed using the thermodynamic properties of hybridization between probes and on-targets or off-targets based on Gibbs free energies and melting temperatures. However, the nature of heterogeneous hybridization between the probes on the surface and the targets in a solution imposes challenges in predicting precise hybridization affinities and the degree of cross-hybridization due to indeterminable thermodynamic penalties induced by the solid surface and its status. Herein, we suggest practical and convenient guidelines for designing oligonucleotide probes based on data obtained from planar magnetic biosensors and thermodynamic properties calculated by using easily accessible solution-phase prediction. The suggested requirements comprised Gibbs free energy ≥ −7.5 kcal mol<sup>–1</sup> and melting temperature ≤10 °C below the hybridization temperature, and we validated for the absence of cross-hybridization. Additionally, the effects of secondary structures such as hairpins and homodimers were investigated for better oligonucleotide probe designs. We believe that these practical guidelines will assist researchers in developing planar magnetic biosensors with high sensitivity and specificity for the detection of new targets.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"7 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142685041","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 : 2024-11-21DOI: 10.1021/acs.analchem.4c02038
Songyuan Yao, Tra D. Nguyen, Yunpeng Lan, Wen Yang, Dan Chen, Yihan Shao, Zhibo Yang
Single-cell mass spectrometry (SCMS) is an emerging tool for studying cell heterogeneity according to variation of molecular species in single cells. Although it has become increasingly common to employ machine learning models in SCMS data analysis, such as the classification of cell phenotypes, the existing machine learning models often suffer from low adaptability and transferability. In addition, SCMS studies of rare cells can be restricted by limited number of cell samples. To overcome these limitations, we performed SCMS analyses of melanoma cancer cell lines with two phenotypes (i.e., primary and metastatic cells). We then developed a meta-learning-based model, MetaPhenotype, that can be trained using a small amount of SCMS data to accurately classify cells into primary or metastatic phenotypes. Our results show that compared with standard transfer learning models, MetaPhenotype can rapidly predict and achieve a high accuracy of over 90% with fewer new training samples. Overall, our work opens the possibility of accurate cell phenotype classification based on fewer SCMS samples, thus lowering the demand for sample acquisition.
{"title":"MetaPhenotype: A Transferable Meta-Learning Model for Single-Cell Mass Spectrometry-Based Cell Phenotype Prediction Using Limited Number of Cells","authors":"Songyuan Yao, Tra D. Nguyen, Yunpeng Lan, Wen Yang, Dan Chen, Yihan Shao, Zhibo Yang","doi":"10.1021/acs.analchem.4c02038","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c02038","url":null,"abstract":"Single-cell mass spectrometry (SCMS) is an emerging tool for studying cell heterogeneity according to variation of molecular species in single cells. Although it has become increasingly common to employ machine learning models in SCMS data analysis, such as the classification of cell phenotypes, the existing machine learning models often suffer from low adaptability and transferability. In addition, SCMS studies of rare cells can be restricted by limited number of cell samples. To overcome these limitations, we performed SCMS analyses of melanoma cancer cell lines with two phenotypes (i.e., primary and metastatic cells). We then developed a meta-learning-based model, MetaPhenotype, that can be trained using a small amount of SCMS data to accurately classify cells into primary or metastatic phenotypes. Our results show that compared with standard transfer learning models, MetaPhenotype can rapidly predict and achieve a high accuracy of over 90% with fewer new training samples. Overall, our work opens the possibility of accurate cell phenotype classification based on fewer SCMS samples, thus lowering the demand for sample acquisition.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"11 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142678842","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}
Biomarker discovery and application are paramount for the early diagnosis, treatment, and prognosis assessment of diseases. Novel proteomic strategies have been developed for high-efficiency biomarker screening. However, evaluating various strategies and applying them for the in-depth mining of biomarkers from blood need to be elucidated. Herein, we systematically evaluated the technical characteristics of three representative biomarker discovery strategies, including the most popular DIA proteomics, and two promising strategies targeting the cancer-secreted proteome or extracellular vesicle proteome, and integrated them into one multidimensional serum proteomic strategy. The results showed that the three strategies each have unique characteristics in terms of sensitivity, reproducibility, and protein coverage and are highly complementary in biomarker discovery. The integrated multidimensional serum proteomic strategy achieves deep and comprehensive coverage of the serum proteome, discovers more cancer markers, and helps achieve a more accurate multicancer (breast, lung, stomach, liver, and colorectum) diagnosis with 87.5% localization accuracy.
{"title":"Efficient Cancer Biomarker Screening and Multicancer Detection Enabled by a Multidimensional Serum Proteomic Strategy","authors":"Anqi Hu, Jiayi Zhang, Lei Zhang, Zhenxin Wang, Jiawei Dai, Ling Lin, Guoquan Yan, Fenglin Shen, Huali Shen","doi":"10.1021/acs.analchem.4c03006","DOIUrl":"https://doi.org/10.1021/acs.analchem.4c03006","url":null,"abstract":"Biomarker discovery and application are paramount for the early diagnosis, treatment, and prognosis assessment of diseases. Novel proteomic strategies have been developed for high-efficiency biomarker screening. However, evaluating various strategies and applying them for the in-depth mining of biomarkers from blood need to be elucidated. Herein, we systematically evaluated the technical characteristics of three representative biomarker discovery strategies, including the most popular DIA proteomics, and two promising strategies targeting the cancer-secreted proteome or extracellular vesicle proteome, and integrated them into one multidimensional serum proteomic strategy. The results showed that the three strategies each have unique characteristics in terms of sensitivity, reproducibility, and protein coverage and are highly complementary in biomarker discovery. The integrated multidimensional serum proteomic strategy achieves deep and comprehensive coverage of the serum proteome, discovers more cancer markers, and helps achieve a more accurate multicancer (breast, lung, stomach, liver, and colorectum) diagnosis with 87.5% localization accuracy.","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":"23 1","pages":""},"PeriodicalIF":7.4,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142678845","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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