Pub Date : 2024-09-26DOI: 10.1016/j.ab.2024.115682
Sahar Mortezagholi , Faezeh Maghsood , Sorour Shojaeian , Fazel Shokri , Mohammad Mehdi Amiri , Ahmad Ghorbani , Mahdi Shabani , Amir-Hassan Zarnani
Placenta-Specific Protein 1 (PLAC1) is essential for normal placental and embryonic development. It is widely expressed in various types of cancer cells. We produced a panel of anti-mouse plac1 monoclonal antibodies (mAbs) with different applications. Two recombinant proteins were produced containing either the extracellular domain (ED) plus tetanus toxin P2, P30, pan-DR epitope (PADRE), and KDEL3 (main plac1) or ED plus KDEL3 (control plac1). Recombinant proteins were used for immunization and screening. Positive clones were selected by ELISA and flow cytometry. Purified mAbs were tested by ELISA, WB, flow cytometry, immunohistochemistry (IHC), and immunofluorescent (IF). A combination of bioinformatics tools was used to predict the target epitope(s) of the mAbs. Eight anti-mouse plac1 mAbs (all IgG1/κ1) were generated, all reacting with high affinity in ELISA. Seven clones recognized plac1 in both reduced and non-reduced Western blots, while one only recognized the non-reduced form. Cross-inhibition ELISA revealed that all mAbs recognized overlapping epitopes with a shared motif except for 5C9. Four clones reacted with the native antigen in flow cytometry, but none were functional in IF or IHC staining. The produced multifunctional mAbs can be used to investigate different aspects of PLAC1 biology in reproduction and cancer.
{"title":"Production and characterization of a panel of anti-mouse placenta-specific protein 1 (plac1) monoclonal antibodies","authors":"Sahar Mortezagholi , Faezeh Maghsood , Sorour Shojaeian , Fazel Shokri , Mohammad Mehdi Amiri , Ahmad Ghorbani , Mahdi Shabani , Amir-Hassan Zarnani","doi":"10.1016/j.ab.2024.115682","DOIUrl":"10.1016/j.ab.2024.115682","url":null,"abstract":"<div><div>Placenta-Specific Protein 1 (PLAC1) is essential for normal placental and embryonic development. It is widely expressed in various types of cancer cells. We produced a panel of anti-mouse plac1 monoclonal antibodies (mAbs) with different applications. Two recombinant proteins were produced containing either the extracellular domain (ED) plus tetanus toxin P2, P30, pan-DR epitope (PADRE), and KDEL3 (main plac1) or ED plus KDEL3 (control plac1). Recombinant proteins were used for immunization and screening. Positive clones were selected by ELISA and flow cytometry. Purified mAbs were tested by ELISA, WB, flow cytometry, immunohistochemistry (IHC), and immunofluorescent (IF). A combination of bioinformatics tools was used to predict the target epitope(s) of the mAbs. Eight anti-mouse plac1 mAbs (all IgG1/κ1) were generated, all reacting with high affinity in ELISA. Seven clones recognized plac1 in both reduced and non-reduced Western blots, while one only recognized the non-reduced form. Cross-inhibition ELISA revealed that all mAbs recognized overlapping epitopes with a shared motif except for 5C9. Four clones reacted with the native antigen in flow cytometry, but none were functional in IF or IHC staining. The produced multifunctional mAbs can be used to investigate different aspects of PLAC1 biology in reproduction and cancer.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142339518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.ab.2024.115679
Aye Myat Myat Thinn , Wei Wang , Qing Chen
Binding kinetics of drug and its target protein is crucial for the efficacy and safety of the drug. Using surface plasmon resonance (SPR) technology, we performed a competitive SPR chaser assay, a method to study biomolecular interactions with very slow dissociation rate constants (kd < 1E-4 s−1). This report described the principle and the experimental setup of the chaser assay, which involves using a competitive probe (chaser) to detect changes in target occupancy by a test molecule over time. We demonstrated the applicability of the chaser assay for both small and large molecules and compared the results with conventional SPR kinetic analysis and other methods. We suggest that the chaser assay is a useful and robust technique to characterize very tight biomolecular interactions, and that it can also be used to study cooperativity in ternary complex formation.
{"title":"Competitive SPR chaser assay to study biomolecular interactions with very slow binding dissociation rate constant","authors":"Aye Myat Myat Thinn , Wei Wang , Qing Chen","doi":"10.1016/j.ab.2024.115679","DOIUrl":"10.1016/j.ab.2024.115679","url":null,"abstract":"<div><div>Binding kinetics of drug and its target protein is crucial for the efficacy and safety of the drug. Using surface plasmon resonance (SPR) technology, we performed a competitive SPR chaser assay, a method to study biomolecular interactions with very slow dissociation rate constants (<em>k</em><sub><em>d</em></sub> < 1E-4 s<sup>−1</sup>). This report described the principle and the experimental setup of the chaser assay, which involves using a competitive probe (chaser) to detect changes in target occupancy by a test molecule over time. We demonstrated the applicability of the chaser assay for both small and large molecules and compared the results with conventional SPR kinetic analysis and other methods. We suggest that the chaser assay is a useful and robust technique to characterize very tight biomolecular interactions, and that it can also be used to study cooperativity in ternary complex formation.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142339517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-25DOI: 10.1016/j.ab.2024.115683
Li Wan , Li Feng , Meiling Wang , Yanhui Yang , Pinxiu Pan , Shuhua Gao
There is an urgent need for novel strategies to accurately and reliably detect pathogenic bacteria to address the global epidemic of antibiotic resistance. This study proposes an innovative approach combining dual aptamer-based target recognition and proximity ligation assay (PLA) triggered DNAzyme recycling cleavage. This method allows for the precise identification and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA). The fluorescence probe labeled with a fluorophore is modified on gold nanoparticles (AuNPs), resulting in the quenching of the fluorescent signal by the AuNPs. The interaction between MRSA and two aptamers leads to forming a Mg2+-dependent DNAzyme. The DNAzyme cleaves the fluorescence probe, causing the fluorescent fragment to detach from the surface of the AuNPs, in which the quenched fluorescence signal in the fluorescence probe reappears. The DNAzyme-assisted cleavage and rebinding process generates a processive strolling along the surface track of AuNPs. Consequently, the fluorescence intensity experiences a substantial recovery. A strong linear correlation is observed between the fluorescence intensity and MRSA concentration within 50 cfu/mL to 106 cfu/mL. We believe that implementing the novel integrated strategy will broaden the range of bacterial detection methods in the battlefield environment and stimulate the creation of potential new drugs in the future.
{"title":"Proximity ligation-triggered DNAzyme for selective fluorescent aptasensing of methicillin-resistant Staphylococcus aureus","authors":"Li Wan , Li Feng , Meiling Wang , Yanhui Yang , Pinxiu Pan , Shuhua Gao","doi":"10.1016/j.ab.2024.115683","DOIUrl":"10.1016/j.ab.2024.115683","url":null,"abstract":"<div><div>There is an urgent need for novel strategies to accurately and reliably detect pathogenic bacteria to address the global epidemic of antibiotic resistance. This study proposes an innovative approach combining dual aptamer-based target recognition and proximity ligation assay (PLA) triggered DNAzyme recycling cleavage. This method allows for the precise identification and reliable detection of methicillin-resistant <em>Staphylococcus aureus</em> (MRSA). The fluorescence probe labeled with a fluorophore is modified on gold nanoparticles (AuNPs), resulting in the quenching of the fluorescent signal by the AuNPs. The interaction between MRSA and two aptamers leads to forming a Mg<sup>2+</sup>-dependent DNAzyme. The DNAzyme cleaves the fluorescence probe, causing the fluorescent fragment to detach from the surface of the AuNPs, in which the quenched fluorescence signal in the fluorescence probe reappears. The DNAzyme-assisted cleavage and rebinding process generates a processive strolling along the surface track of AuNPs. Consequently, the fluorescence intensity experiences a substantial recovery. A strong linear correlation is observed between the fluorescence intensity and MRSA concentration within 50 cfu/mL to 10<sup>6</sup> cfu/mL. We believe that implementing the novel integrated strategy will broaden the range of bacterial detection methods in the battlefield environment and stimulate the creation of potential new drugs in the future.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142339519","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-24DOI: 10.1016/j.ab.2024.115681
Micaela Riscado , Rita Carapito , Cláudio J. Maia , Chantal Pichon , Mara G. Freire , Mattia Sponchioni , Fani Sousa
The development of RNA-based drugs is highly pursued due to the possibility of creating viable and effective therapies. However, their translation to clinical practice strongly depends on efficient technologies to produce substantial levels of these biomolecules, with high purity and high quality. RNAs are commonly produced by chemical or enzymatic methods, displaying these limitations. In this sense, recombinant production arises as a promising, cost-effective method, allowing large-scale production of RNA. Rhodovulum sulfidophilum (R. sulfidophilum), a marine purple bacterium, offers the advantage of RNA secretion into the extracellular medium, which contains low levels of RNases and other impurities. Therefore, RNA recovery can be simplified compared to standard extraction protocols involving cell lysis, resulting in a more clarified sample and an improved downstream process. In this work, R. sulfidophilum was transformed with a plasmid DNA encoding pre-miR-29b-1, which is known to be involved in the Alzheimer's disease pathway. After production, the pre-miR-29b-1 was recovered through different extraction methods to verify the most advantageous one. A protocol for extracellular RNA recovery was then established, revealing to be a simpler and less time-consuming method, reducing around 16 h in execution time and the quantity of reagents needed when compared to other established methods. The new strategy brings the additional advantage of eliminating the toxic organic compounds routinely used in intracellular RNA extractions. Overall, the optimized process described here, using isopropanol as the precipitation agent, offers a greener, safer, and faster alternative for recombinant RNA recovery and concentration, with an extracellular RNA recovery of 7 μg/mL and target pre-miRNA-29b-1 recovery of 0.7 μg/L of medium.
{"title":"A new approach for extracellular RNA recovery from Rhodovulum sulfidophilum","authors":"Micaela Riscado , Rita Carapito , Cláudio J. Maia , Chantal Pichon , Mara G. Freire , Mattia Sponchioni , Fani Sousa","doi":"10.1016/j.ab.2024.115681","DOIUrl":"10.1016/j.ab.2024.115681","url":null,"abstract":"<div><div>The development of RNA-based drugs is highly pursued due to the possibility of creating viable and effective therapies. However, their translation to clinical practice strongly depends on efficient technologies to produce substantial levels of these biomolecules, with high purity and high quality. RNAs are commonly produced by chemical or enzymatic methods, displaying these limitations. In this sense, recombinant production arises as a promising, cost-effective method, allowing large-scale production of RNA. <em>Rhodovulum sulfidophilum (R. sulfidophilum)</em>, a marine purple bacterium, offers the advantage of RNA secretion into the extracellular medium, which contains low levels of RNases and other impurities. Therefore, RNA recovery can be simplified compared to standard extraction protocols involving cell lysis, resulting in a more clarified sample and an improved downstream process. In this work, <em>R. sulfidophilum</em> was transformed with a plasmid DNA encoding pre-miR-29b-1, which is known to be involved in the Alzheimer's disease pathway. After production, the pre-miR-29b-1 was recovered through different extraction methods to verify the most advantageous one. A protocol for extracellular RNA recovery was then established, revealing to be a simpler and less time-consuming method, reducing around 16 h in execution time and the quantity of reagents needed when compared to other established methods. The new strategy brings the additional advantage of eliminating the toxic organic compounds routinely used in intracellular RNA extractions. Overall, the optimized process described here, using isopropanol as the precipitation agent, offers a greener, safer, and faster alternative for recombinant RNA recovery and concentration, with an extracellular RNA recovery of 7 μg/mL and target pre-miRNA-29b-1 recovery of 0.7 μg/L of medium.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142327122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hypochlorite and ascorbic acid (AA), play an indispensable role in numerous physiological activities. Herein, a ratiometric colorimetric sensing strategy for the determination of hypochlorite and AA was developed via the catalytic oxidation and reduction of 3,3′,5,5′-tetramethylbenzidine (TMB). Interestingly, in the presence of Fe3O4-MOF-5(Fe) and hypochlorite, TMB complexes in acidic environments were oxidized to blue oxidized TMB and further diazotized to produce yellow-green diazotized TMB, resulting in the hypochlorite concentration-dependent ratiometric variation for the absorbance at 652 and 450 nm (A450/A652). Moreover, the diazotized TMB was restored to colorless TMB due to the reducibility of AA, and the detection limit of hypochlorite and AA were 0.027 and 0.677 μM, respectively. The ratiometric colorimetric sensing platform offered higher sensitivity and better selectivity because of the specific hypochlorite-induced reaction and the excellent peroxidase-like activity of Fe3O4-MOF-5(Fe). The proposed novel strategy provided the guidance to develop sensors for successive detection of hypochlorite and AA in complicated samples.
次氯酸盐和抗坏血酸（AA）在多种生理活动中发挥着不可或缺的作用。本文通过催化 3,3′,5,5′-四甲基联苯胺（TMB）的氧化和还原，开发了一种测定次氯酸盐和抗坏血酸的比色法传感策略。有趣的是，在 Fe3O4-MOF-5(Fe)和次氯酸盐存在的情况下，TMB 复合物在酸性环境中被氧化成蓝色氧化 TMB，并进一步重氮化生成黄绿色重氮化 TMB，从而导致 652 纳米和 450 纳米吸光度（A450/A652）随次氯酸盐浓度的比率变化。此外，由于 AA 的还原性，重氮化 TMB 恢复为无色 TMB，次氯酸盐和 AA 的检测限分别为 0.027 和 0.677 μM。由于次氯酸盐诱导反应的特异性和 Fe3O4-MOF-5(Fe) 卓越的过氧化物酶样活性，比色法传感平台具有更高的灵敏度和更好的选择性。所提出的新策略为开发用于连续检测复杂样品中次氯酸盐和 AA 的传感器提供了指导。
{"title":"A novel ratiometric colorimetric sensor for detecting hypochlorite and ascorbic acid based on cascade reaction","authors":"Miaowen Sun, Yanzhu Liu, Lingge Shi, Haomiao Dou, Feiyan Ma, Guangda Xu, Longshan Zhao","doi":"10.1016/j.ab.2024.115678","DOIUrl":"10.1016/j.ab.2024.115678","url":null,"abstract":"<div><div>Hypochlorite and ascorbic acid (AA), play an indispensable role in numerous physiological activities. Herein, a ratiometric colorimetric sensing strategy for the determination of hypochlorite and AA was developed via the catalytic oxidation and reduction of 3,3′,5,5′-tetramethylbenzidine (TMB). Interestingly, in the presence of Fe<sub>3</sub>O<sub>4</sub>-MOF-5(Fe) and hypochlorite, TMB complexes in acidic environments were oxidized to blue oxidized TMB and further diazotized to produce yellow-green diazotized TMB, resulting in the hypochlorite concentration-dependent ratiometric variation for the absorbance at 652 and 450 nm (A<sub>450</sub>/A<sub>652</sub>). Moreover, the diazotized TMB was restored to colorless TMB due to the reducibility of AA, and the detection limit of hypochlorite and AA were 0.027 and 0.677 μM, respectively. The ratiometric colorimetric sensing platform offered higher sensitivity and better selectivity because of the specific hypochlorite-induced reaction and the excellent peroxidase-like activity of Fe<sub>3</sub>O<sub>4</sub>-MOF-5(Fe). The proposed novel strategy provided the guidance to develop sensors for successive detection of hypochlorite and AA in complicated samples.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142320143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-21DOI: 10.1016/j.ab.2024.115677
Nanfei Yang , Yuncheng Bei , Yahong Huang , Wei Zheng , Jiehua Ma , Jiangqiong Ke
Serum Cystatin C (CysC) is an impressive marker for early diagnosis of renal dysfunction. In this work, we established a novel electrochemical immunosensor based on Fe3O4/AuNPs-MWCNTs@PDA nanocomposite for the detection of CysC. The Fe3O4/AuNPs-MWCNTs@PDA nanozyme complex by polydopamine encapsulation can not only carry massive detection antibodies, but also bind the electroactive substance toluidine blue (TB) through electrostatic adsorption. By immobilizing AuNPs onto the electrode to bind the capture antibody (Ab1), we constructed a sandwich electrochemical immunosensor with low cost, high sensitivity, and repeatability. The detection range is 3.9–125.0 ng/mL with a significant linear relationship between the current peak difference (ip) and logarithm of the CysC concentration. Moreover, the detection limit of the immunosensor is 0.157 ng/mL. We have successfully utilized this novel immunosensor to detect CysC in human serum samples, and these results have implications for its potential use in clinical application.
{"title":"An electrochemical immunosensor for sensitive and rapid detection of cystatin C based on Fe3O4/AuNPs-MWCNTs@PDA nanocomposite","authors":"Nanfei Yang , Yuncheng Bei , Yahong Huang , Wei Zheng , Jiehua Ma , Jiangqiong Ke","doi":"10.1016/j.ab.2024.115677","DOIUrl":"10.1016/j.ab.2024.115677","url":null,"abstract":"<div><div>Serum Cystatin C (CysC) is an impressive marker for early diagnosis of renal dysfunction. In this work, we established a novel electrochemical immunosensor based on Fe<sub>3</sub>O<sub>4</sub>/AuNPs-MWCNTs@PDA nanocomposite for the detection of CysC. The Fe<sub>3</sub>O<sub>4</sub>/AuNPs-MWCNTs@PDA nanozyme complex by polydopamine encapsulation can not only carry massive detection antibodies, but also bind the electroactive substance toluidine blue (TB) through electrostatic adsorption. By immobilizing AuNPs onto the electrode to bind the capture antibody (Ab1), we constructed a sandwich electrochemical immunosensor with low cost, high sensitivity, and repeatability. The detection range is 3.9–125.0 ng/mL with a significant linear relationship between the current peak difference (ip) and logarithm of the CysC concentration. Moreover, the detection limit of the immunosensor is 0.157 ng/mL. We have successfully utilized this novel immunosensor to detect CysC in human serum samples, and these results have implications for its potential use in clinical application.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142279341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Norepinephrine (NE) is the primary catecholamine (CA) of interest in the medical field, as it plays a key role in regulating the hormonal and neurological systems. Some NE concentration dysfunction can lead to a number of serious physical conditions. As a result, quick and sensitive NE detection is most critical in medical technology. Thus, in this research, a molecularly imprinted polymer (MIP) was used to create an electrochemical sensor for the selective detection of NE. Prior to this, functional monomers were chosen through molecular modeling utilizing molecular mechanics and quantum mechanics computations. According to these studies, the 3-aminophenylboronic acid (3-APBA) functional monomer produces the most stable complex with NE in molecular modeling calculations. Based on this, by electropolymerizing 3-APBA in the presence of the template molecule NE, an imprinting polymer film is formed on the screen-printed carbon electrode (SPCE) surface. Stepwise fabrication of imprinted polymer films was examined through differential pulse voltammetry (DPV), cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). The performance of the electrochemical NE sensor removal and rebinding levels of the template was studied and optimized. The selectivity for NE was confirmed by using interference studies of small molecules like dopamine, tyrosine, and serotonin. Under optimum levels, the fabricated MIP sensor had a broad linear range over NE concentrations of 0.1 pM–5 pM; sensitivity: 0.004 mA pM−1; limit of detection: 0.03 pM. It is noteworthy that the newly created MIP sensor was effectively validated for NE detection in plasma samples.
去甲肾上腺素（NE）是医学领域关注的主要儿茶酚胺（CA），因为它在调节荷尔蒙和神经系统方面发挥着关键作用。某些 NE 浓度失调会导致一些严重的身体状况。因此，快速灵敏地检测 NE 在医疗技术中至关重要。因此，在这项研究中，使用分子印迹聚合物（MIP）创建了一种选择性检测 NE 的电化学传感器。在此之前，利用分子力学和量子力学计算，通过分子建模选择了功能单体。根据这些研究，在分子建模计算中，3-氨基苯硼酸（3-APBA）功能单体与 NE 产生的复合物最为稳定。在此基础上，通过在模板分子 NE 存在下对 3-APBA 进行电聚合，在丝网印刷碳电极（SPCE）表面形成压印聚合物薄膜。研究人员通过差分脉冲伏安法（DPV）、循环伏安法（CV）、扫描电子显微镜（SEM）和电化学阻抗谱（EIS）对印迹聚合物薄膜的逐步制备过程进行了检验。研究并优化了电化学 NE 传感器的去除性能和模板的再结合水平。通过对多巴胺、酪氨酸和血清素等小分子的干扰研究，证实了 NE 的选择性。在最佳水平下，所制造的 MIP 传感器在 0.1 pM 至 5 pM 的 NE 浓度范围内具有较宽的线性范围；灵敏度：0.004 mA pM-1；检测限：0.03 pM：0.03 pM。值得注意的是，新制作的 MIP 传感器在检测血浆样品中的 NE 方面得到了有效验证。
{"title":"Theoretical screening and electrochemical sensor for determination of norepinephrine using a molecularly imprinted poly (3-amiophenylboronic acid)","authors":"Karthikeyan Murugesan , Marimuthu Dhinesh Kumar , Ganesan Kaniraja , Periyasamy Ananthappan , Vairathevar Sivasamy Vasantha , Chandran Karunakaran","doi":"10.1016/j.ab.2024.115676","DOIUrl":"10.1016/j.ab.2024.115676","url":null,"abstract":"<div><div>Norepinephrine (NE) is the primary catecholamine (CA) of interest in the medical field, as it plays a key role in regulating the hormonal and neurological systems. Some NE concentration dysfunction can lead to a number of serious physical conditions. As a result, quick and sensitive NE detection is most critical in medical technology. Thus, in this research, a molecularly imprinted polymer (MIP) was used to create an electrochemical sensor for the selective detection of NE. Prior to this, functional monomers were chosen through molecular modeling utilizing molecular mechanics and quantum mechanics computations. According to these studies, the 3-aminophenylboronic acid (3-APBA) functional monomer produces the most stable complex with NE in molecular modeling calculations. Based on this, by electropolymerizing 3-APBA in the presence of the template molecule NE, an imprinting polymer film is formed on the screen-printed carbon electrode (SPCE) surface. Stepwise fabrication of imprinted polymer films was examined through differential pulse voltammetry (DPV), cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). The performance of the electrochemical NE sensor removal and rebinding levels of the template was studied and optimized. The selectivity for NE was confirmed by using interference studies of small molecules like dopamine, tyrosine, and serotonin. Under optimum levels, the fabricated MIP sensor had a broad linear range over NE concentrations of 0.1 pM–5 pM; sensitivity: 0.004 mA pM<sup>−1</sup>; limit of detection: 0.03 pM. It is noteworthy that the newly created MIP sensor was effectively validated for NE detection in plasma samples.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142279342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In recent years, important efforts have been made to elucidate the mechanisms of epigenetic regulation, and one of the most studied epigenetic modifications was DNA methylation/demethylation. In this study, the voltammetric behaviour of 5-hydroxymethylcytosine was studied in the pH range of 2.00–11.00 using pencil graphite electrodes by differential pulse and square wave voltammetry. The effect of buffer solutions, scan rate, square wave voltammetry parameters, and stripping conditions on the voltammetric responses of 5-hydroxymethylcytosine were performed. The electrochemical oxidation process of 5-hydroxymethylcytosine on the pencil graphite electrode was realized under adsorption control. In human urine, by square wave stripping voltammetry, 5-hydroxymethylcytosine was quantified in a concentration range of 1.00 × 10−5 M-2.00 × 10−4 M. The proposed method was tested in the presence of cytosine in human urine. The recovery value of 5-hydroxymethylcytosine was found to be 99.57 %.
{"title":"Electrochemical sensor for the analysis of 5-hydroxymethylcytosine in the presence of cytosine using pencil graphite electrode","authors":"Selva Bilge , Manolya Müjgan Gürbüz , Sibel A. Ozkan , Burcu Dogan Topal","doi":"10.1016/j.ab.2024.115674","DOIUrl":"10.1016/j.ab.2024.115674","url":null,"abstract":"<div><p>In recent years, important efforts have been made to elucidate the mechanisms of epigenetic regulation, and one of the most studied epigenetic modifications was DNA methylation/demethylation. In this study, the voltammetric behaviour of 5-hydroxymethylcytosine was studied in the pH range of 2.00–11.00 using pencil graphite electrodes by differential pulse and square wave voltammetry. The effect of buffer solutions, scan rate, square wave voltammetry parameters, and stripping conditions on the voltammetric responses of 5-hydroxymethylcytosine were performed. The electrochemical oxidation process of 5-hydroxymethylcytosine on the pencil graphite electrode was realized under adsorption control. In human urine, by square wave stripping voltammetry, 5-hydroxymethylcytosine was quantified in a concentration range of 1.00 × 10<sup>−5</sup> M-2.00 × 10<sup>−4</sup> M. The proposed method was tested in the presence of cytosine in human urine. The recovery value of 5-hydroxymethylcytosine was found to be 99.57 %.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142274858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1016/j.ab.2024.115672
Tao Han , Cheng Zhang , Hui Yu , Jinghong Li
The integration of fiber optics and plasmonic sensors is promising to improve the practical usability over conventional bulky sensors and systems. To achieve high sensitivity, it typically requires fabrication of well-defined plasmonic nanostructures on optical fibers, which greatly increases the cost and complexity of the sensors. Here, we present a fiber-optic sensor system by using chemical absorption of gold nanoparticles and a replaceable configuration. By functioning gold nanoparticles with aptamers or antibodies, we demonstrate the applications in chemical sensing using two different modes. Measuring shift in resonance wavelength enables the Pb2+ detection with a high linearity and a limit of detection of 0.097 nM, and measuring absorption peak amplitude enables the detection of E. coli in urinary tract infection with a dynamic range between 103 to 108 CFU/mL. The high sensitivity, simple fabrication and disposability of this sensing approach could pave the way for point-of-care testing with fiber-optic plasmonic sensors.
{"title":"A disposable fiber-optic plasmonic sensor for chemical sensing","authors":"Tao Han , Cheng Zhang , Hui Yu , Jinghong Li","doi":"10.1016/j.ab.2024.115672","DOIUrl":"10.1016/j.ab.2024.115672","url":null,"abstract":"<div><p>The integration of fiber optics and plasmonic sensors is promising to improve the practical usability over conventional bulky sensors and systems. To achieve high sensitivity, it typically requires fabrication of well-defined plasmonic nanostructures on optical fibers, which greatly increases the cost and complexity of the sensors. Here, we present a fiber-optic sensor system by using chemical absorption of gold nanoparticles and a replaceable configuration. By functioning gold nanoparticles with aptamers or antibodies, we demonstrate the applications in chemical sensing using two different modes. Measuring shift in resonance wavelength enables the Pb<sup>2+</sup> detection with a high linearity and a limit of detection of 0.097 nM, and measuring absorption peak amplitude enables the detection of <em>E. coli</em> in urinary tract infection with a dynamic range between 10<sup>3</sup> to 10<sup>8</sup> CFU/mL. The high sensitivity, simple fabrication and disposability of this sensing approach could pave the way for point-of-care testing with fiber-optic plasmonic sensors.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142274859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Our study delved into the intricate dynamics of antifungal susceptibility testing for Candida spp., employing a Design of Experiments approach. We systematically investigated the influence of pH, temperature, inoculum size, and glucose concentration on both growth patterns and inhibitory concentrations of Candida spp. Our findings underscore the nuanced interplay between these factors, revealing significant impacts on susceptibility outcomes. Notably, even minor adjustments in these parameters yielded substantial variations in growth and inhibitory concentrations, underscoring the critical importance of meticulous control over growth conditions in antifungal susceptibility testing protocols. Each Candida isolates exhibited unique susceptibility profiles, necessitating tailored culture conditions for accurate testing. Our study sheds light on the variability inherent in Candida spp. growth patterns and emphasizes the need for standardized protocols to ensure consistency across laboratories. By leveraging the design of experiments, our research provides a systematic framework for unraveling the complexities of antifungal susceptibility testing, offering valuable insights for optimizing testing protocols and informing clinical decision-making in antifungal treatment. These findings represent a significant step towards enhancing the efficacy and reliability of antifungal susceptibility testing in clinical practice.
{"title":"Unraveling the complexities of antifungal susceptibility testing in Candida spp.: Insights from design of experiments","authors":"Ânderson Ramos Carvalho , Luana Candice Genz Bazana , Marco Flôres Ferrão , Alexandre Meneghello Fuentefria","doi":"10.1016/j.ab.2024.115675","DOIUrl":"10.1016/j.ab.2024.115675","url":null,"abstract":"<div><p>Our study delved into the intricate dynamics of antifungal susceptibility testing for <em>Candida</em> spp., employing a Design of Experiments approach. We systematically investigated the influence of pH, temperature, inoculum size, and glucose concentration on both growth patterns and inhibitory concentrations of <em>Candida</em> spp. Our findings underscore the nuanced interplay between these factors, revealing significant impacts on susceptibility outcomes. Notably, even minor adjustments in these parameters yielded substantial variations in growth and inhibitory concentrations, underscoring the critical importance of meticulous control over growth conditions in antifungal susceptibility testing protocols. Each <em>Candida</em> isolates exhibited unique susceptibility profiles, necessitating tailored culture conditions for accurate testing. Our study sheds light on the variability inherent in <em>Candida</em> spp. growth patterns and emphasizes the need for standardized protocols to ensure consistency across laboratories. By leveraging the design of experiments, our research provides a systematic framework for unraveling the complexities of antifungal susceptibility testing, offering valuable insights for optimizing testing protocols and informing clinical decision-making in antifungal treatment. These findings represent a significant step towards enhancing the efficacy and reliability of antifungal susceptibility testing in clinical practice.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142238574","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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