This study integrated sample partition, incubation, and continuous fluorescence detection on a single microfluidic chip for droplet-based digital Loop-Mediated Isothermal Amplification (LAMP) of nucleic acids. This integration eliminated the need to transfer reactions between different platforms, avoiding sample contamination and loss. Prior to the reaction, filling the channels with an oil phase and adding a glass cover slip on top of the chip overcame the problem of bubble generation in the channels during the LAMP reaction due to heating. Additionally, using two fluorescence intensity thresholds enabled simultaneous detection and counting of positive and negative droplets within a single fluorescence detection channel. The chip can partition approximately 6000 droplets from a 5 µL sample within 10 min, with a droplet diameter of around 110 µm and a coefficient of variation (CV) value of 0.82%. Staphylococcus aureus was quantified via the proposed platform. The results demonstrated a highly accurate correlation coefficient (R = 0.9998), and the detection limit reached a concentration of 1.7 × 102 copies/µL. The entire process of the droplet digital LAMP reaction, from droplet generation to incubation to quantitative results, took a maximum of 70 min.
{"title":"Integrated Droplet-Based Digital Loop-Mediated Isothermal Amplification Microfluidic Chip with Droplet Generation, Incubation, and Continuous Fluorescence Detection","authors":"Yen-Heng Lin, Yuan-Ting Hung, Wei Chang, Chiuan-Chian Chiou","doi":"10.3390/bios14070334","DOIUrl":"https://doi.org/10.3390/bios14070334","url":null,"abstract":"This study integrated sample partition, incubation, and continuous fluorescence detection on a single microfluidic chip for droplet-based digital Loop-Mediated Isothermal Amplification (LAMP) of nucleic acids. This integration eliminated the need to transfer reactions between different platforms, avoiding sample contamination and loss. Prior to the reaction, filling the channels with an oil phase and adding a glass cover slip on top of the chip overcame the problem of bubble generation in the channels during the LAMP reaction due to heating. Additionally, using two fluorescence intensity thresholds enabled simultaneous detection and counting of positive and negative droplets within a single fluorescence detection channel. The chip can partition approximately 6000 droplets from a 5 µL sample within 10 min, with a droplet diameter of around 110 µm and a coefficient of variation (CV) value of 0.82%. Staphylococcus aureus was quantified via the proposed platform. The results demonstrated a highly accurate correlation coefficient (R = 0.9998), and the detection limit reached a concentration of 1.7 × 102 copies/µL. The entire process of the droplet digital LAMP reaction, from droplet generation to incubation to quantitative results, took a maximum of 70 min.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"38 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141573764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tao Yan, Fan Weng, Yang Ming, Shijie Zhu, Miao Zhu, Chunsheng Wang, Changfa Guo, Kai Zhu
Bioanalysis based on optical imaging has gained significant progress in the last few decades. Luminescence probes are capable of detecting, monitoring, and tracing particular biomolecules in complex biological systems to figure out the roles of these molecules in organisms. Considering the rapid development of luminescence probes for bio-applications and their promising future, we have attempted to explore the working principles and recent advances in bio-applications of luminescence probes, in the hope of helping readers gain a detailed understanding of luminescence probes developed in recent years. In this review, we first focus on the current widely used luminescence probes, including fluorescence probes, bioluminescence probes, chemiluminescence probes, afterglow probes, photoacoustic probes, and Cerenkov luminescence probes. The working principles for each type of luminescence probe are concisely described and the bio-application of the luminescence probes is summarized by category, including metal ions detection, secretion detection, imaging, and therapy.
{"title":"Luminescence Probes in Bio-Applications: From Principle to Practice","authors":"Tao Yan, Fan Weng, Yang Ming, Shijie Zhu, Miao Zhu, Chunsheng Wang, Changfa Guo, Kai Zhu","doi":"10.3390/bios14070333","DOIUrl":"https://doi.org/10.3390/bios14070333","url":null,"abstract":"Bioanalysis based on optical imaging has gained significant progress in the last few decades. Luminescence probes are capable of detecting, monitoring, and tracing particular biomolecules in complex biological systems to figure out the roles of these molecules in organisms. Considering the rapid development of luminescence probes for bio-applications and their promising future, we have attempted to explore the working principles and recent advances in bio-applications of luminescence probes, in the hope of helping readers gain a detailed understanding of luminescence probes developed in recent years. In this review, we first focus on the current widely used luminescence probes, including fluorescence probes, bioluminescence probes, chemiluminescence probes, afterglow probes, photoacoustic probes, and Cerenkov luminescence probes. The working principles for each type of luminescence probe are concisely described and the bio-application of the luminescence probes is summarized by category, including metal ions detection, secretion detection, imaging, and therapy.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"175 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141573763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics.
适用于分析各种生化和免疫化学标记物的简单分析装置非常受欢迎，可以在标准医院之外提供实验室诊断。本研究的重点是构建一个易于重复的自己动手制作的 ELISA 读板器生物传感器装置，该装置由一般可用的廉价部件组装而成。该比色生物传感器基于标准的 96 孔微孔板、3D 打印部件和作为检测器的智能手机摄像头。当使用 RGB 颜色模型的 B 通道进行校准时，检测限为 19 pg/mL。该检测方法与酶联免疫吸附法的相关性良好，在标准生物样本中未观察到明显的基质效应或样本中预期的蛋白质干扰。这项研究的结果将为开发简单的分析设备提供参考，这些设备可通过 3D 打印技术轻松复制，并可在普通电子产品上找到。
{"title":"A 3D-Printed Do-It-Yourself ELISA Plate Reader as a Biosensor Tested on TNFα Assay","authors":"Miroslav Pohanka, Ondřej Keresteš, Jitka Žáková","doi":"10.3390/bios14070331","DOIUrl":"https://doi.org/10.3390/bios14070331","url":null,"abstract":"Simple analytical devices suitable for the analysis of various biochemical and immunechemical markers are highly desirable and can provide laboratory diagnoses outside standard hospitals. This study focuses on constructing an easily reproducible do-it-yourself ELISA plate reader biosensor device, assembled from generally available and inexpensive parts. The colorimetric biosensor was based on standard 96-well microplates, 3D-printed parts, and a smartphone camera as a detector was utilized here as a tool to replace the ELISA method, and its function was illustrated in the assay of TNFα as a model immunochemical marker. The assay provided a limit of detection of 19 pg/mL when the B channel of the RGB color model was used for calibration. The assay was well correlated with the ELISA method, and no significant matrix effect was observed for standard biological samples or interference of proteins expected in a sample. The results of this study will inform the development of simple analytical devices easily reproducible by 3D printing and found on generally available electronics.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141573766","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zhuotao Hu, Yingchao Hu, Lu Huang, Wei Zhong, Jianfeng Zhang, Dengyun Lei, Yayi Chen, Yao Ni, Yuan Liu
The continued advancement of organic electronic technology will establish organic electrochemical transistors as pivotal instruments in the field of biological detection. Here, we present a comprehensive review of the state-of-the-art technology and advancements in the use of organic electrochemical transistors as biosensors. This review provides an in-depth analysis of the diverse modification materials, methods, and mechanisms utilized in organic electrochemical transistor-structured biosensors (OETBs) for the selective detection of a wide range of target analyte encompassing electroactive species, electro-inactive species, and cancer cells. Recent advances in OETBs for use in sensing systems and wearable and implantable applications are also briefly introduced. Finally, challenges and opportunities in the field are discussed.
{"title":"Recent Progress in Organic Electrochemical Transistor-Structured Biosensors","authors":"Zhuotao Hu, Yingchao Hu, Lu Huang, Wei Zhong, Jianfeng Zhang, Dengyun Lei, Yayi Chen, Yao Ni, Yuan Liu","doi":"10.3390/bios14070330","DOIUrl":"https://doi.org/10.3390/bios14070330","url":null,"abstract":"The continued advancement of organic electronic technology will establish organic electrochemical transistors as pivotal instruments in the field of biological detection. Here, we present a comprehensive review of the state-of-the-art technology and advancements in the use of organic electrochemical transistors as biosensors. This review provides an in-depth analysis of the diverse modification materials, methods, and mechanisms utilized in organic electrochemical transistor-structured biosensors (OETBs) for the selective detection of a wide range of target analyte encompassing electroactive species, electro-inactive species, and cancer cells. Recent advances in OETBs for use in sensing systems and wearable and implantable applications are also briefly introduced. Finally, challenges and opportunities in the field are discussed.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141548715","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sezen Harmankaya, Hacı Ahmet Deveci, Ahmet Harmankaya, Fatma Hazan Gül, Necip Atar, Mehmet Lütfi Yola
In this work, a new surface plasmon resonance (SPR) sensor based on sulphur-doped titanium dioxide (S-TiO2) nanostructures and molecularly imprinted polymer (MIP) was presented for thiram (THI) determination in milk samples. Firstly, the S-TiO2 nanomaterial with a high product yield was prepared by using a facile sol–gel hydrolysis technique with a high product yield. After that, UV polymerization was carried out for the preparation of the THI-imprinted SPR chip based on S-TiO2 using a mixture including ethylene glycol dimethacrylate (EGDMA) as the cross-linker, N,N′-azobisisobutyronitrile (AIBN) as the initiator, and methacryloylamidoglutamicacid (MAGA) as the monomer. The reliability of the sensor preparation procedure has been successfully proven by characterization studies of the prepared nanomaterials and SPR chip surfaces through spectroscopic, microscopic, and electrochemical methods. As a result, the prepared SPR sensor showed linearity in the range of 1.0 × 10−9–1.0 × 10−7 M with a detection limit (LOD) of 3.3 × 10−10 M in the real samples, and a sensor technique for THI determination with high sensitivity, repeatability, and selectivity can be included in the literature.
{"title":"Thiram Determination in Milk Samples by Surface Plasmon Resonance Based on Molecularly Imprinted Polymers and Sulphur-Doped Titanium Dioxide","authors":"Sezen Harmankaya, Hacı Ahmet Deveci, Ahmet Harmankaya, Fatma Hazan Gül, Necip Atar, Mehmet Lütfi Yola","doi":"10.3390/bios14070329","DOIUrl":"https://doi.org/10.3390/bios14070329","url":null,"abstract":"In this work, a new surface plasmon resonance (SPR) sensor based on sulphur-doped titanium dioxide (S-TiO2) nanostructures and molecularly imprinted polymer (MIP) was presented for thiram (THI) determination in milk samples. Firstly, the S-TiO2 nanomaterial with a high product yield was prepared by using a facile sol–gel hydrolysis technique with a high product yield. After that, UV polymerization was carried out for the preparation of the THI-imprinted SPR chip based on S-TiO2 using a mixture including ethylene glycol dimethacrylate (EGDMA) as the cross-linker, N,N′-azobisisobutyronitrile (AIBN) as the initiator, and methacryloylamidoglutamicacid (MAGA) as the monomer. The reliability of the sensor preparation procedure has been successfully proven by characterization studies of the prepared nanomaterials and SPR chip surfaces through spectroscopic, microscopic, and electrochemical methods. As a result, the prepared SPR sensor showed linearity in the range of 1.0 × 10−9–1.0 × 10−7 M with a detection limit (LOD) of 3.3 × 10−10 M in the real samples, and a sensor technique for THI determination with high sensitivity, repeatability, and selectivity can be included in the literature.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dopamine (DA), ascorbic acid (AA), and uric acid (UA) are crucial neurochemicals, and their abnormal levels are involved in various neurological disorders. While electrodes for their detection have been developed, achieving the sensitivity required for in vivo applications remains a challenge. In this study, we proposed a synthetic Au24Cd nanoenzyme (ACNE) that significantly enhanced the electrochemical performance of metal electrodes. ACNE-modified electrodes demonstrated a remarkable 10-fold reduction in impedance compared to silver microelectrodes. Furthermore, we validated their excellent electrocatalytic activity and sensitivity using five electrochemical detection methods, including cyclic voltammetry, differential pulse voltammetry, square-wave pulse voltammetry, normal pulse voltammetry, and linear scanning voltammetry. Importantly, the stability of gold microelectrodes (Au MEs) modified with ACNEs was significantly improved, exhibiting a 30-fold enhancement compared to Au MEs. This improved performance suggests that ACNE functionalization holds great promise for developing micro-biosensors with enhanced sensitivity and stability for detecting small molecules.
多巴胺（DA）、抗坏血酸（AA）和尿酸（UA）是重要的神经化学物质，它们的异常水平与各种神经系统疾病有关。虽然用于检测它们的电极已经开发出来，但要达到体内应用所需的灵敏度仍是一个挑战。在这项研究中，我们提出了一种合成的 Au24Cd 纳米酶（ACNE），它能显著提高金属电极的电化学性能。与银微电极相比，ACNE 改性电极的阻抗明显降低了 10 倍。此外，我们还使用了五种电化学检测方法，包括循环伏安法、差分脉冲伏安法、方波脉冲伏安法、正常脉冲伏安法和线性扫描伏安法，验证了它们出色的电催化活性和灵敏度。重要的是，用 ACNE 修饰的金微电极（Au MEs）的稳定性显著提高，与 Au MEs 相比提高了 30 倍。性能的提高表明，ACNE 功能化为开发具有更高灵敏度和稳定性的小分子检测微生物传感器带来了巨大前景。
{"title":"Au24Cd Nanoenzyme Coating for Enhancing Electrochemical Sensing Performance of Metal Wire Microelectrodes","authors":"Jia-Yi Chen, Shuang Huang, Shuang-Jie Liu, Zheng-Jie Liu, Xing-Yuan Xu, Meng-Yi He, Chuan-Jie Yao, Tao Zhang, Han-Qi Yang, Xin-Shuo Huang, Jing Liu, Xiao-Dong Zhang, Xi Xie, Hui-Jiuan Chen","doi":"10.3390/bios14070328","DOIUrl":"https://doi.org/10.3390/bios14070328","url":null,"abstract":"Dopamine (DA), ascorbic acid (AA), and uric acid (UA) are crucial neurochemicals, and their abnormal levels are involved in various neurological disorders. While electrodes for their detection have been developed, achieving the sensitivity required for in vivo applications remains a challenge. In this study, we proposed a synthetic Au24Cd nanoenzyme (ACNE) that significantly enhanced the electrochemical performance of metal electrodes. ACNE-modified electrodes demonstrated a remarkable 10-fold reduction in impedance compared to silver microelectrodes. Furthermore, we validated their excellent electrocatalytic activity and sensitivity using five electrochemical detection methods, including cyclic voltammetry, differential pulse voltammetry, square-wave pulse voltammetry, normal pulse voltammetry, and linear scanning voltammetry. Importantly, the stability of gold microelectrodes (Au MEs) modified with ACNEs was significantly improved, exhibiting a 30-fold enhancement compared to Au MEs. This improved performance suggests that ACNE functionalization holds great promise for developing micro-biosensors with enhanced sensitivity and stability for detecting small molecules.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"67 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141506397","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Minja Mladenović, Stefan Jarić, Mirjana Mundžić, Aleksandra Pavlović, Ivan Bobrinetskiy, Nikola Ž. Knežević
Mesoporous silica nanoparticles (MSNs) exhibit highly beneficial characteristics for devising efficient biosensors for different analytes. Their unique properties, such as capabilities for stable covalent binding to recognition groups (e.g., antibodies or aptamers) and sensing surfaces, open a plethora of opportunities for biosensor construction. In addition, their structured porosity offers capabilities for entrapping signaling molecules (dyes or electroactive species), which could be released efficiently in response to a desired analyte for effective optical or electrochemical detection. This work offers an overview of recent research studies (in the last five years) that contain MSNs in their optical and electrochemical sensing platforms for the detection of cancer biomarkers, classified by cancer type. In addition, this study provides an overview of cancer biomarkers, as well as electrochemical and optical detection methods in general.
{"title":"Biosensors for Cancer Biomarkers Based on Mesoporous Silica Nanoparticles","authors":"Minja Mladenović, Stefan Jarić, Mirjana Mundžić, Aleksandra Pavlović, Ivan Bobrinetskiy, Nikola Ž. Knežević","doi":"10.3390/bios14070326","DOIUrl":"https://doi.org/10.3390/bios14070326","url":null,"abstract":"Mesoporous silica nanoparticles (MSNs) exhibit highly beneficial characteristics for devising efficient biosensors for different analytes. Their unique properties, such as capabilities for stable covalent binding to recognition groups (e.g., antibodies or aptamers) and sensing surfaces, open a plethora of opportunities for biosensor construction. In addition, their structured porosity offers capabilities for entrapping signaling molecules (dyes or electroactive species), which could be released efficiently in response to a desired analyte for effective optical or electrochemical detection. This work offers an overview of recent research studies (in the last five years) that contain MSNs in their optical and electrochemical sensing platforms for the detection of cancer biomarkers, classified by cancer type. In addition, this study provides an overview of cancer biomarkers, as well as electrochemical and optical detection methods in general.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"237 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wen Sun, Jianhua Wang, Jin Chen, Xiwei Huang, Xin Rao, Jiangtao Su, Yuqiao Huang, Boyu Zhang, Lingling Sun
Cell dielectric property measurement holds significant potential for application in cell detection and diagnosis due to its label-free and noninvasive nature. In this study, we developed a biosensor designed to measure the permittivity of liquid samples, particularly cell suspensions at the nanoliter scale, utilizing microwave and millimeter wave coplanar waveguides in conjunction with a microchannel. This biosensor facilitates the measurement of scattering parameters within a frequency domain ranging from 1 GHz to 110 GHz. The obtained scattering parameters are then converted into dielectric constants using specific algorithms. A cell capture structure within the microchannel ensures that cell suspensions remain stable within the measurement zone. The feasibility of this biosensor was confirmed by comparison with a commercial Keysight probe. We measured the dielectric constants of three different cell suspensions (HepG2, A549, MCF-7) using our biosensor. We also counted the number of cells captured in multiple measurements for each cell type and compared the corresponding changes in permittivity. The results indicated that the real part of the permittivity of HepG2 cells is 0.2–0.8 lower than that of the other two cell types. The difference between A549 and MCF-7 was relatively minor, only 0.2–0.4. The fluctuations in the dielectric spectrum caused by changes in cell numbers during measurements were smaller than the differences observed between different cell types. Thus, the sensor is suitable for measuring cell suspensions and can be utilized for label-free, noninvasive studies in identifying biological cell suspensions.
{"title":"Biosensor with Microchannel for Broadband Dielectric Characterization of Nanoliter Cell Suspensions up to 110 GHz","authors":"Wen Sun, Jianhua Wang, Jin Chen, Xiwei Huang, Xin Rao, Jiangtao Su, Yuqiao Huang, Boyu Zhang, Lingling Sun","doi":"10.3390/bios14070327","DOIUrl":"https://doi.org/10.3390/bios14070327","url":null,"abstract":"Cell dielectric property measurement holds significant potential for application in cell detection and diagnosis due to its label-free and noninvasive nature. In this study, we developed a biosensor designed to measure the permittivity of liquid samples, particularly cell suspensions at the nanoliter scale, utilizing microwave and millimeter wave coplanar waveguides in conjunction with a microchannel. This biosensor facilitates the measurement of scattering parameters within a frequency domain ranging from 1 GHz to 110 GHz. The obtained scattering parameters are then converted into dielectric constants using specific algorithms. A cell capture structure within the microchannel ensures that cell suspensions remain stable within the measurement zone. The feasibility of this biosensor was confirmed by comparison with a commercial Keysight probe. We measured the dielectric constants of three different cell suspensions (HepG2, A549, MCF-7) using our biosensor. We also counted the number of cells captured in multiple measurements for each cell type and compared the corresponding changes in permittivity. The results indicated that the real part of the permittivity of HepG2 cells is 0.2–0.8 lower than that of the other two cell types. The difference between A549 and MCF-7 was relatively minor, only 0.2–0.4. The fluctuations in the dielectric spectrum caused by changes in cell numbers during measurements were smaller than the differences observed between different cell types. Thus, the sensor is suitable for measuring cell suspensions and can be utilized for label-free, noninvasive studies in identifying biological cell suspensions.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ehsan Bahrami Moghadam, Nam Nguyen, Yixi Wang, Patrick C. Cirino
Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives.
{"title":"Directed Evolution of Protein-Based Sensors for Anaerobic Biological Activation of Methane","authors":"Ehsan Bahrami Moghadam, Nam Nguyen, Yixi Wang, Patrick C. Cirino","doi":"10.3390/bios14070325","DOIUrl":"https://doi.org/10.3390/bios14070325","url":null,"abstract":"Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from Yersinia pseudotuberculosis to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in E. coli, show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"156 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141506399","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Each application of neurostimulators requires unique stimulation parameter specifications to achieve effective stimulation. Balancing the current magnitude with stimulation resolution, waveform, size, and channel count is challenging, leading to a loss of generalizability across broad neural interfaces. To address this, this paper proposes a highly scalable, programmable neurostimulator with a System-on-Chip (SOC) capable of 32 channels of independent stimulation. The compliance voltage reaches up to ±22.5 V. A pair of 8-bit current-mode DACs support independent waveforms for source and sink operations and feature a user-selectable dual range for low-current intraparenchymal microstimulation with a resolution of 4.31 μA/bit, as well as high current stimulation for spinal cord and DBS applications with a resolution of 48.00 μA/bit, achieving a wide stimulation range of 12.24 mA while maintaining high-resolution biological stimulation. A dedicated communication protocol enables full programmable control of stimulation waveforms, effectively improving the range of stimulation parameters. In vivo electrophysiological experiments successfully validate the functionality of the proposed stimulator. This flexible stimulator architecture aims to enhance its generality across a wide range of neural interfaces and will provide more diverse and refined stimulation strategies.
{"title":"A Scalable, Programmable Neural Stimulator for Enhancing Generalizability in Neural Interface Applications","authors":"Meng Yin, Xiao Wang, Liuxindai Zhang, Guijun Shu, Zhen Wang, Shoushuang Huang, Ming Yin","doi":"10.3390/bios14070323","DOIUrl":"https://doi.org/10.3390/bios14070323","url":null,"abstract":"Each application of neurostimulators requires unique stimulation parameter specifications to achieve effective stimulation. Balancing the current magnitude with stimulation resolution, waveform, size, and channel count is challenging, leading to a loss of generalizability across broad neural interfaces. To address this, this paper proposes a highly scalable, programmable neurostimulator with a System-on-Chip (SOC) capable of 32 channels of independent stimulation. The compliance voltage reaches up to ±22.5 V. A pair of 8-bit current-mode DACs support independent waveforms for source and sink operations and feature a user-selectable dual range for low-current intraparenchymal microstimulation with a resolution of 4.31 μA/bit, as well as high current stimulation for spinal cord and DBS applications with a resolution of 48.00 μA/bit, achieving a wide stimulation range of 12.24 mA while maintaining high-resolution biological stimulation. A dedicated communication protocol enables full programmable control of stimulation waveforms, effectively improving the range of stimulation parameters. In vivo electrophysiological experiments successfully validate the functionality of the proposed stimulator. This flexible stimulator architecture aims to enhance its generality across a wide range of neural interfaces and will provide more diverse and refined stimulation strategies.","PeriodicalId":100185,"journal":{"name":"Biosensors","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141520696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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