Detection of aptamer-protein interactions using QCM and electrochemical indicator methods

Electrochemical indicator methylene blue and differential pulse voltammetry allowing to determine charge transfer from electrode surface to the thrombin bounded on a DNA aptamer and thus to detect thrombin (1) with high selectivity in comparison with nonspecific binding caused by human IgG (2) or human serum albumin (HSA) (3). We report novel method of detection thrombin-aptamer interaction based on measurement the charge consumption from the electrode covered by DNA aptamers to an electrochemical indicator methylene blue (MB), that is bounded to a thrombin. The binding of thrombin to an aptamers has been detected also by QCM method in flow measuring cell. We showed that using MB it is possible to detect thrombin with high sensitivity and selectivity. © 2004 Elsevier Ltd. All rights reserved

Development of a highly specific amine-terminated aptamer functionalized surface plasmon resonance biosensor for blood protein detection

This paper presents a generally applicable approach for the highly specific detection of blood proteins. Thrombin and thrombin-binding aptamers are chosen for demonstration purposes. The sensor was prepared by immobilizing amine-terminated aptamers onto a gold modified surface using a two-step self-assembled monolayer (SAM) immobilization technique and the physical detection is performed using Surface Plasmon Resonance (SPR). The developed sensor has an optimal detectable range of 5–1000 nM and the results show the sensor has good reversibility, sensitivity and selectivity. Furthermore, the sensor shows the potential of being improved and standardized for direct detection of other blood proteins for clinical applications

Electrochemical aptasensor for human osteopontin detection using a DNA aptamer selected by SELEX

A DNA aptamer with affinity and specificity for human osteopontin (OPN), a potential breast cancer biomarker, was selected using the SELEX process, considering its homology rate and the stability of its secondary structures. This aptamer exhibited a satisfactory affinity towards OPN, showing dissociation constants lower than 2.5 nM. It was further used to develop a simple, label-free electrochemical aptasensor against OPN. The aptasensor showed good sensitivity towards OPN in standard solutions, being the square wave voltammetry (SWV), compared to the cyclic voltammetry, the most sensitive technique with detection and quantification limits of 1.4 ± 0.4 nM and 4.2 ± 1.1 nM, respectively. It showed good reproducibility and acceptable selectivity, exhibiting low signal interferences from other proteins, as thrombin, with 2.610 times lower current signals-off than for OPN. The aptasensor also successfully detected OPN in spiked synthetic human plasma. Using SWV, detection and quantification limits (1.3 ± 0.1 and 3.9 ± 0.4 nM) within the OPN plasma levels reported for patients with breast cancer (0.44.5 nM) or with metastatic or recurrent breast cancer (0.98.4 nM) were found. Moreover, preliminary assays, using a sample of human plasma, showed that the aptasensor and the standard ELISA method quantified similar OPN levels (2.2 ± 0.7 and 1.7 ± 0.1 nM, respectively). Thus, our aptasensor coupled with SWV represents a promising alternative for the detection of relevant breast cancer biomarkers.The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and from project BioTecNorte (project number NORTE-01-0145-FEDER-000004). This work was also financially supported by Project POCI-01–0145-FEDER-006984 – Associate Laboratory LSRE-LCM and by Project UID/QUI/00616/2013 – CQ-VR both funded by FEDER - Fundo Europeu de Desenvolvimento Regional through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia, Portugal. S. Meirinho also acknowledges the research grant provided by Project UID/EQU/50020/2013.info:eu-repo/semantics/publishedVersio

Voltammetric aptasensors for protein disease biomarkers detection: a review

"Available online 24 May 2016"An electrochemical aptasensor is a compact analytical device where the bioreceptor (aptamer) is coupled to a transducer surface to convert a biological interaction into a measurable signal (current) that can be easily processed, recorded and displayed. Since the discovery of the Systematic Evolution of Ligands by Enrichment (SELEX) methodology, the selection of aptamers and their application as bioreceptors has become a promising tool in the design of electrochemical aptasensors. Aptamers present several advantages that highlight their usefulness as bioreceptors such as chemical stability, cost effectiveness and ease of modification towards detection and immobilization at different transducer surfaces. In this review, a special emphasis is given to the potential use of electrochemical aptasensors for the detection of protein disease biomarkers using voltammetry techniques. Methods for the immobilization of aptamers onto electrode surfaces are discussed, as well as different electrochemical strategies that can be used for the design of aptasensors.The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit, from Project POCI-01-0145- FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia and project ref. RECI/BBB-EBI/ 0179/2012 (project number FCOMP-01-0124-FEDER-027462) and S. Meirinhos's doctoral grant (ref SFRH/BD/65021/2009) funded by Fundação para a Ciência e a Tecnologia

Constant Current Chronopotentiometry as a Method for Detection of Singlet Oxygen Protein Damage

Proteins are one of the major targets for oxidative damage in the cell.Indirect non-radical oxidation of the protein via formation and subsequent reaction with single oxygen (O-1(2))is one of the major processes.In this work we studied the single oxygen(O-1(2))-mediated oxidation of bovine serum albumin (BSA) by constant current chronopotentiometry in combination with mercury electrode.Our chronopotentiometric data show that photo-oxidized BSA was more susceptible to the electric field-induced denaturation than non-oxidized native BSA. Our method utilizing intrinsic electrochemical signal of proteins provides a sensitive detection methods for minor damages in various proteins

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Detection of aptamer-protein interactions using QCM and electrochemical indicator methods

Electrochemical indicator methylene blue and differential pulse voltammetry allowing to determine charge transfer from electrode surface to the thrombin bounded on a DNA aptamer and thus to detect thrombin (1) with high selectivity in comparison with nonspecific binding caused by human IgG (2) or human serum albumin (HSA) (3). We report novel method of detection thrombin-aptamer interaction based on measurement the charge consumption from the electrode covered by DNA aptamers to an electrochemical indicator methylene blue (MB), that is bounded to a thrombin. The binding of thrombin to an aptamers has been detected also by QCM method in flow measuring cell. We showed that using MB it is possible to detect thrombin with high sensitivity and selectivity. © 2004 Elsevier Ltd. All rights reserved

Detection of aptamer-protein interactions using QCM and electrochemical indicator methods

Electrochemical indicator methylene blue and differential pulse voltammetry allowing to determine charge transfer from electrode surface to the thrombin bounded on a DNA aptamer and thus to detect thrombin (1) with high selectivity in comparison with nonspecific binding caused by human IgG (2) or human serum albumin (HSA) (3). We report novel method of detection thrombin-aptamer interaction based on measurement the charge consumption from the electrode covered by DNA aptamers to an electrochemical indicator methylene blue (MB), that is bounded to a thrombin. The binding of thrombin to an aptamers has been detected also by QCM method in flow measuring cell. We showed that using MB it is possible to detect thrombin with high sensitivity and selectivity. © 2004 Elsevier Ltd. All rights reserved