Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet-tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (Kd) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.
{"title":"Identification of Podoplanin Aptamers by SELEX for Protein Detection and Inhibition of Platelet Aggregation Stimulated by C-Type Lectin-like Receptor 2.","authors":"Hui-Ju Tsai, Kai-Wen Cheng, Jou-Chen Li, Tsai-Xiang Ruan, Ting-Hsin Chang, Jin-Ru Wang, Ching-Ping Tseng","doi":"10.3390/bios14100464","DOIUrl":"https://doi.org/10.3390/bios14100464","url":null,"abstract":"<p><p>Tumor cell-induced platelet aggregation (TCIPA) is a mechanism for the protection of tumor cells in the bloodstream and the promotion of tumor progression and metastases. The platelet C-type lectin-like receptor 2 (CLEC-2) can bind podoplanin (PDPN) on a cancer cell surface to facilitate TCIPA. Selective blockage of PDPN-mediated platelet-tumor cell interaction is a plausible strategy for inhibiting metastases. In this study, we aimed to screen for aptamers, which are the single-stranded DNA oligonucleotides that form a specific three-dimensional structure, bind to specific molecular targets with high affinity and specificity, bind to PDPN, and interfere with PDPN/CLEC-2 interactions. The systematic evolution of ligands by exponential enrichment (SELEX) was employed to enrich aptamers that recognize PDPN. The initial characterization of ssDNA pools enriched by SELEX revealed a PDPN aptamer designated as A1 displaying parallel-type G-quadruplexes and long stem-and-loop structures and binding PDPN with a material with a dissociation constant (K<sub>d</sub>) of 1.3 ± 1.2 nM. The A1 aptamer recognized both the native and denatured form of PDPN. Notably, the A1 aptamer was able to quantitatively detect PDPN proteins in Western blot analysis. The A1 aptamer could interfere with the interaction between PDPN and CLEC-2 and inhibit PDPN-induced platelet aggregation in a concentration-dependent manner. These findings indicated that the A1 aptamer is a candidate for the development of biosensors in detecting the levels of PDPN expression. The action by A1 aptamer could result in the prevention of tumor cell metastases, and if so, could become an effective pharmacological agent in treating cancer patients.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11506057/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.
CRISPR/Cas 系统以其高特异性和高灵敏度彻底改变了分子诊断领域。本综述全面概述了利用 CRISPR/Cas 系统检测 DNA 和 RNA 的原理和最新进展。本综述从探讨支持 CRISPR/Cas 系统的关键 Cas 蛋白的分子机制开始,探讨了病原体和非病原体核酸的检测,强调了 CRISPR 在识别各种遗传物质方面的关键作用。讨论延伸到 CRISPR/Cas 系统与各种信号读出技术(包括荧光、电化学和比色法)以及成像和生物传感方法的整合,强调了它们在实际应用中的优势和局限性。此外,还对目标放大、多路复用和定量检测等领域的挑战进行了批判性分析,强调了需要进一步完善的领域。最后,综述对基于 CRISPR 的核酸检测的未来发展方向进行了深入分析,强调了这些系统继续推动诊断创新的潜力,对研究、临床实践和生物技术具有广泛的影响。
{"title":"Harnessing CRISPR/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges.","authors":"Heyjin Son","doi":"10.3390/bios14100460","DOIUrl":"https://doi.org/10.3390/bios14100460","url":null,"abstract":"<p><p>The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11506544/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cheng-Xue Yu, Kuan-Hsun Huang, To-Lin Chen, Chan-Chiung Liu, Lung-Ming Fu
A novel microfluidic ractopamine (RAC) detection platform consisting of a microfluidic RAC chip and a smart analysis device is proposed for the determination of RAC concentration in meat samples. This technology utilizes gold nanoparticles (AuNPs) modified with glutamic acid (GLU) and polyethyleneimine (PEI) to measure RAC concentration in food products. When RAC is present, AuNPs aggregate through hydrogen bonding, causing noticeable changes in their optical properties, which are detected using a self-built UV-visible micro-spectrophotometer. Within the range of 5 to 80 ppb, a linear relationship exists between the absorbance ratio (A693nm/A518nm) (Y) and RAC concentration (X), expressed as Y = 0.0054X + 0.4690, with a high coefficient of determination (R2 = 0.9943). This method exhibits a detection limit of 1.0 ppb and achieves results within 3 min. The practical utility of this microfluidic assay is exemplified through the evaluation of RAC concentrations in 50 commercially available meat samples. The variance between concentrations measured using this platform and those determined via liquid chromatography-tandem mass spectrometry (LC-MS/MS) is less than 8.33%. These results underscore the viability of the microfluidic detection platform as a rapid and cost-effective solution for ensuring food safety and regulatory compliance within the livestock industry.
{"title":"Microfluidic Detection Platform for Determination of Ractopamine in Food.","authors":"Cheng-Xue Yu, Kuan-Hsun Huang, To-Lin Chen, Chan-Chiung Liu, Lung-Ming Fu","doi":"10.3390/bios14100462","DOIUrl":"https://doi.org/10.3390/bios14100462","url":null,"abstract":"<p><p>A novel microfluidic ractopamine (RAC) detection platform consisting of a microfluidic RAC chip and a smart analysis device is proposed for the determination of RAC concentration in meat samples. This technology utilizes gold nanoparticles (AuNPs) modified with glutamic acid (GLU) and polyethyleneimine (PEI) to measure RAC concentration in food products. When RAC is present, AuNPs aggregate through hydrogen bonding, causing noticeable changes in their optical properties, which are detected using a self-built UV-visible micro-spectrophotometer. Within the range of 5 to 80 ppb, a linear relationship exists between the absorbance ratio (A<sub>693nm</sub>/A<sub>518nm</sub>) (Y) and RAC concentration (X), expressed as Y = 0.0054X + 0.4690, with a high coefficient of determination (R<sup>2</sup> = 0.9943). This method exhibits a detection limit of 1.0 ppb and achieves results within 3 min. The practical utility of this microfluidic assay is exemplified through the evaluation of RAC concentrations in 50 commercially available meat samples. The variance between concentrations measured using this platform and those determined via liquid chromatography-tandem mass spectrometry (LC-MS/MS) is less than 8.33%. These results underscore the viability of the microfluidic detection platform as a rapid and cost-effective solution for ensuring food safety and regulatory compliance within the livestock industry.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11506807/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510831","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Qinggang Li, Haojie Ren, Zhenjiang Liao, Shuchang Xia, Xue Sun
The biosensors based on transcription factors (TFs) are widely used in high throughput screening of metabolic overproducers. The unsatisfactory performances (narrow detection and dynamic ranges) of biosensors limit their practical application and need more improvement. In this study, using the TF LysG (sensing lysine) as an example, a biosensor optimization method was constructed by growth-coupled screening of TF random mutant libraries. The better the performance of the biosensor, the faster the strain grows under screening pressure. A LysGE15D, A54D, and I164V-based biosensors were obtained, which were about 2-fold of the control in the detection and dynamic ranges. A lysine high-producer was screened effectively using the optimized biosensor with the production at 1.51 ± 0.30 g/L in flasks (2.22-fold of the original strain). This study provided a promising strategy for optimizing TF-based biosensors and was of high potential to be applied in the lysine high-producers screening process.
{"title":"High Throughput Screening of Transcription Factor LysG for Constructing a Better Lysine Biosensor.","authors":"Qinggang Li, Haojie Ren, Zhenjiang Liao, Shuchang Xia, Xue Sun","doi":"10.3390/bios14100455","DOIUrl":"https://doi.org/10.3390/bios14100455","url":null,"abstract":"<p><p>The biosensors based on transcription factors (TFs) are widely used in high throughput screening of metabolic overproducers. The unsatisfactory performances (narrow detection and dynamic ranges) of biosensors limit their practical application and need more improvement. In this study, using the TF LysG (sensing lysine) as an example, a biosensor optimization method was constructed by growth-coupled screening of TF random mutant libraries. The better the performance of the biosensor, the faster the strain grows under screening pressure. A LysG<sup>E15D, A54D, and I164V</sup>-based biosensors were obtained, which were about 2-fold of the control in the detection and dynamic ranges. A lysine high-producer was screened effectively using the optimized biosensor with the production at 1.51 ± 0.30 g/L in flasks (2.22-fold of the original strain). This study provided a promising strategy for optimizing TF-based biosensors and was of high potential to be applied in the lysine high-producers screening process.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11506072/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Andrzej Marcinek, Joanna Katarzynska, Katarzyna Cypryk, Agnieszka Los-Stegienta, Jolanta Slowikowska-Hilczer, Renata Walczak-Jedrzejowska, Jacek Zielinski, Jerzy Gebicki
This review summarizes studies dedicated to the assessment of microvascular function based on microcirculatory oscillations monitored by the Flow-Mediated Skin Fluorescence (FMSF) technique. Two approaches are presented. The first approach uses oscillatory parameters measured under normoxic conditions, expressed as flowmotion (FM), vasomotion (VM), and the normoxia oscillatory index (NOI). These parameters have been used for the identification of impaired microcirculatory oscillations associated with intense physical exercise, post-COVID syndrome, psychological stress, and erectile dysfunction. The second approach involves characterization of the microcirculatory response to hypoxia based on the measurement of hypoxia sensitivity (HS). The HS parameter is used to characterize microvascular complications in diabetes, such as diabetic kidney disease and diabetic foot ulcers. Based on research conducted by the authors of this review, the FMSF parameter ranges characterizing microvascular function are presented. The diagnostic approach to assessing microvascular function based on flowmotion monitored by the FMSF technique has a wide range of applications and the potential to be integrated into widespread medical practice.
{"title":"Assessment of Microvascular Function Based on Flowmotion Monitored by the Flow-Mediated Skin Fluorescence Technique.","authors":"Andrzej Marcinek, Joanna Katarzynska, Katarzyna Cypryk, Agnieszka Los-Stegienta, Jolanta Slowikowska-Hilczer, Renata Walczak-Jedrzejowska, Jacek Zielinski, Jerzy Gebicki","doi":"10.3390/bios14100459","DOIUrl":"https://doi.org/10.3390/bios14100459","url":null,"abstract":"<p><p>This review summarizes studies dedicated to the assessment of microvascular function based on microcirculatory oscillations monitored by the Flow-Mediated Skin Fluorescence (FMSF) technique. Two approaches are presented. The first approach uses oscillatory parameters measured under normoxic conditions, expressed as flowmotion (FM), vasomotion (VM), and the normoxia oscillatory index (NOI). These parameters have been used for the identification of impaired microcirculatory oscillations associated with intense physical exercise, post-COVID syndrome, psychological stress, and erectile dysfunction. The second approach involves characterization of the microcirculatory response to hypoxia based on the measurement of hypoxia sensitivity (HS). The HS parameter is used to characterize microvascular complications in diabetes, such as diabetic kidney disease and diabetic foot ulcers. Based on research conducted by the authors of this review, the FMSF parameter ranges characterizing microvascular function are presented. The diagnostic approach to assessing microvascular function based on flowmotion monitored by the FMSF technique has a wide range of applications and the potential to be integrated into widespread medical practice.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11505855/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Seok Hyeon Kim, Yujun Kim, Seokjoon Kim, Eun Sung Lee, Byung Seok Cha, Ki Soo Park
Mercury ions (Hg2+) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection method for Hg2+ using a multicomponent nucleic acid enzyme (MNAzyme)-assisted nucleic acid lateral flow assay (NALFA). The MNAzyme, which was engineered to contain thymine-thymine mismatches, is responsive only to the presence of Hg2+ and exerts efficient cleavage activity on substrates that can be captured by the NALFA strip, and thus the proposed system enables the visual detection of Hg2+ in the NALFA strip. Our assay demonstrated sufficient detection sensitivity and specificity to meet the WHO standards, offering a good practical alternative for rapid environmental and public health monitoring.
{"title":"MNAzyme-Assisted Nucleic Acid Lateral Flow Assay for Cost-Effective, On-Site Mercury Detection.","authors":"Seok Hyeon Kim, Yujun Kim, Seokjoon Kim, Eun Sung Lee, Byung Seok Cha, Ki Soo Park","doi":"10.3390/bios14100454","DOIUrl":"https://doi.org/10.3390/bios14100454","url":null,"abstract":"<p><p>Mercury ions (Hg<sup>2+</sup>) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effective, on-site detection method for Hg<sup>2+</sup> using a multicomponent nucleic acid enzyme (MNAzyme)-assisted nucleic acid lateral flow assay (NALFA). The MNAzyme, which was engineered to contain thymine-thymine mismatches, is responsive only to the presence of Hg<sup>2+</sup> and exerts efficient cleavage activity on substrates that can be captured by the NALFA strip, and thus the proposed system enables the visual detection of Hg<sup>2+</sup> in the NALFA strip. Our assay demonstrated sufficient detection sensitivity and specificity to meet the WHO standards, offering a good practical alternative for rapid environmental and public health monitoring.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11505659/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Priyanka Balyan, Shekhar Gupta, Sai Kiran Mavileti, Shyam S Pandey, Tamaki Kato
Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye-peptide conjugate (SQ-3 PC) for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes SQ-1, SQ-2, and SQ-3 along with a dye-peptide conjugate SQ-3-PC as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye-peptide conjugates in water, utilizing SQ-3 and SQ-3 PC respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye-peptide conjugates in water utilizing SQ-3 and SQ-3 PC, respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe SQ-3 PC demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of SQ-3 PC led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that SQ-3 PC works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.
胰蛋白酶已被认为是结直肠癌、胃癌和胰腺癌等多种肿瘤的潜在生物标记物,这凸显了它在疾病诊断中的重要性。为了满足对快速、经济、实时检测方法的需求,我们提出了一种创新策略,利用设计和合成近红外敏感染料-肽共轭物(SQ-3 PC),通过荧光开/关传感灵敏、选择性地监测胰蛋白酶活性。目前的研究涉及设计和合成三种非对称方碱染料 SQ-1、SQ-2 和 SQ-3,以及作为胰蛋白酶特异性探针的染料-肽共轭物 SQ-3-PC,并对其进行光物理表征。分别利用 SQ-3 和 SQ-3 PC 对单独的染料及其相应的染料-肽共轭物在水中进行的吸收光谱研究表明,与在二甲基亚砜溶液中观察到的结果相比,染料的聚集性增强,荧光淬灭明显。分别利用 SQ-3 和 SQ-3 PC 对水中的染料和相应的染料-肽共轭物进行的吸收光谱研究表明,与在二甲基亚砜溶液中观察到的结果相比,不仅染料聚集增强,而且荧光淬灭也很明显。由于聚集诱导淬灭(AIQ)和荧光共振能量转移(FRET)的共同作用,胰蛋白酶特异性探针 SQ-3 PC 在水中的荧光淬灭效率达到 61.8%。研究发现,FRET 比 AIQ 起主导作用。胰蛋白酶介导的 SQ-3 PC 水解可使淬灭的荧光快速有效地恢复(30 分钟内增加 5 倍)。染料发射最大值处荧光的浓度变化表明,SQ-3 PC 是一种胰蛋白酶特异性荧光生物传感器,其线性关系可达 30 nM,检测限和定量限分别为 1.07 nM 和 3.25 nM。
{"title":"NIR-Sensitive Squaraine Dye-Peptide Conjugate for Trypsin Fluorogenic Detection.","authors":"Priyanka Balyan, Shekhar Gupta, Sai Kiran Mavileti, Shyam S Pandey, Tamaki Kato","doi":"10.3390/bios14100458","DOIUrl":"https://doi.org/10.3390/bios14100458","url":null,"abstract":"<p><p>Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye-peptide conjugate (<b>SQ-3 PC</b>) for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes <b>SQ-1</b>, <b>SQ-2</b>, and <b>SQ-3</b> along with a dye-peptide conjugate <b>SQ-3-PC</b> as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye-peptide conjugates in water, utilizing <b>SQ-3</b> and <b>SQ-3 PC</b> respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye-peptide conjugates in water utilizing <b>SQ-3</b> and <b>SQ-3 PC</b>, respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe <b>SQ-3 PC</b> demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of <b>SQ-3 PC</b> led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that <b>SQ-3 PC</b> works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11505658/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jason P Ware, Delaney K Shea, Shelby L Nicholas, Ella A Stimson, Jessica L Riesterer, Stuart D Ibsen
Bacterial membrane vesicle (BMV) nanoparticles are secreted naturally by bacteria throughout their lifecycle and are a rich source of biomarkers from the parent bacteria, but they are currently underutilized for clinical diagnostic applications, such as pathogen identification, due to the time-consuming and low-yield nature of traditional recovery methods required for analysis. The recovery of BMVs is particularly difficult from complex biological fluids. Here, we demonstrate a recovery method that uses dielectrophoretic (DEP) forces generated on electrokinetic microfluidic chips to isolate and analyze BMVs from human plasma. DEP takes advantage of the natural difference in dielectric properties between the BMVs and the surrounding plasma fluid to quickly and consistently collect these particles from as little as 25 µL of plasma. Using DEP and immunofluorescence staining of the LPS biomarker carried on BMVs, we have demonstrated a lower limit of detection of 4.31 × 109 BMVs/mL. The successful isolation of BMVs from human plasma using DEP, and subsequent on-chip immunostaining for biomarkers, enables the development of future assays to identify the presence of specific bacterial species by analyzing BMVs from small amounts of complex body fluid.
{"title":"Recovery and Analysis of Bacterial Membrane Vesicle Nanoparticles from Human Plasma Using Dielectrophoresis.","authors":"Jason P Ware, Delaney K Shea, Shelby L Nicholas, Ella A Stimson, Jessica L Riesterer, Stuart D Ibsen","doi":"10.3390/bios14100456","DOIUrl":"https://doi.org/10.3390/bios14100456","url":null,"abstract":"<p><p>Bacterial membrane vesicle (BMV) nanoparticles are secreted naturally by bacteria throughout their lifecycle and are a rich source of biomarkers from the parent bacteria, but they are currently underutilized for clinical diagnostic applications, such as pathogen identification, due to the time-consuming and low-yield nature of traditional recovery methods required for analysis. The recovery of BMVs is particularly difficult from complex biological fluids. Here, we demonstrate a recovery method that uses dielectrophoretic (DEP) forces generated on electrokinetic microfluidic chips to isolate and analyze BMVs from human plasma. DEP takes advantage of the natural difference in dielectric properties between the BMVs and the surrounding plasma fluid to quickly and consistently collect these particles from as little as 25 µL of plasma. Using DEP and immunofluorescence staining of the LPS biomarker carried on BMVs, we have demonstrated a lower limit of detection of 4.31 × 10<sup>9</sup> BMVs/mL. The successful isolation of BMVs from human plasma using DEP, and subsequent on-chip immunostaining for biomarkers, enables the development of future assays to identify the presence of specific bacterial species by analyzing BMVs from small amounts of complex body fluid.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11505931/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Herein, we report a ratio-based electrochemical biosensor for the detection of interleukin-6 (IL-6). We electropolymerized methylene blue (MB) on the surface of screen-printed carbon electrodes; introduced an internal reference signal probe; modified the carboxylate multi-walled carbon nanotubes on the electrode surface to increase the electrochemically active area; and finally linked the amino-modified IL-6 aptamer to the electrode surface through the Schiff base reaction, with bovine serum albumin (BSA) added to mask non-specific adsorption. After adding IL-6 to the samples, the signal of IMB remained almost unchanged, while the signal of I[Fe(CN)6]3-/4- decreased with increasing IL-6 concentration. Thus, a novel ratiometric electrochemical sensor with a linear range of 0.001~1000.0 ng/mL and a low detection limit of 0.54 pg/mL was successfully developed. The sensor had high repeatability, stability, sensitivity, and practicability. It provides a new method for constructing proportional electrochemical sensors and detecting IL-6.
{"title":"Ratiometric Electrochemical Detection of Interleukin-6 Using Electropolymerized Methylene Blue and a Multi-Walled Carbon-Nanotube-Modified Screen-Printed Carbon Electrode.","authors":"Zhuo Liu, Fengyu Liu, Chaofan Wang, Hongjuan Li, Yongqian Xu, Shiguo Sun","doi":"10.3390/bios14100457","DOIUrl":"https://doi.org/10.3390/bios14100457","url":null,"abstract":"<p><p>Herein, we report a ratio-based electrochemical biosensor for the detection of interleukin-6 (IL-6). We electropolymerized methylene blue (MB) on the surface of screen-printed carbon electrodes; introduced an internal reference signal probe; modified the carboxylate multi-walled carbon nanotubes on the electrode surface to increase the electrochemically active area; and finally linked the amino-modified IL-6 aptamer to the electrode surface through the Schiff base reaction, with bovine serum albumin (BSA) added to mask non-specific adsorption. After adding IL-6 to the samples, the signal of I<sub>MB</sub> remained almost unchanged, while the signal of I[Fe(CN)<sub>6</sub>]<sup>3-/4-</sup> decreased with increasing IL-6 concentration. Thus, a novel ratiometric electrochemical sensor with a linear range of 0.001~1000.0 ng/mL and a low detection limit of 0.54 pg/mL was successfully developed. The sensor had high repeatability, stability, sensitivity, and practicability. It provides a new method for constructing proportional electrochemical sensors and detecting IL-6.</p>","PeriodicalId":48608,"journal":{"name":"Biosensors-Basel","volume":null,"pages":null},"PeriodicalIF":4.9,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11506342/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142510854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}