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J Colloid Interface Sci ; 559: 1-12, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31605780


Experimental and computational approaches are utilized to investigate the influence of electrostatic fields on the binding force between human coagulation protein thrombin and its DNA aptamer. The thiolated aptamer was deposited onto gold substrate located in a liquid cell filled with binding buffer, then the thrombin-functionalized atomic force microscopy (AFM) probe was repeatedly brought into contact with the aptamer-coated surface under applied electrical potentials of -100, 0, and 100 mV respectively. Force drops during the pull-off process were measured to determine the unbinding forces between thrombin and aptamer in a range of loading rates spanning from ~3 × 102 to ~1 × 104 pN/s. The results from experiments showed that both of the binding strength and propensity of the complex are drastically diminished under positive electrode potential, whereas there is no influence on the molecular binding from negative electrode potential. We also used a theoretical analysis to explain the nature of electrostatic potential and field inside the aptamer-thrombin layer, which in turn could quantify the influence of the electrostatically repulsive force on a thrombin molecule that promotes dissociation from the aptamer due to positive electrode potential, and achieve good agreement with the experimental results. The study confirms the feasibility of electrostatic modulation upon the binding interaction between thrombin and aptamer, and implicates an underlying application perspective upon nanoscale manipulation of the stimuli responsive biointerface.

Aptâmeros de Nucleotídeos/química , Trombina/química , Técnicas Biossensoriais/métodos , Eletricidade , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Fenômenos Mecânicos , Modelos Moleculares , Ligação Proteica , Eletricidade Estática , Propriedades de Superfície
Biosens Bioelectron ; 126: 88-95, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30396022


Nanoporous alumina membranes have become a ubiquitous biosensing platform for a variety of applications and aptamers are being increasingly utilized as recognition elements in protein sensing devices. Combining the advantages of the two, we report label-free sensitive detection of human α-thrombin by an aptamer-functionalized nanoporous alumina membrane using a four-electrode electrochemical cell. The sensor response to α-thrombin was determined in the presence of a high concentration (500 µM) of human serum albumin (HSA) as an interfering protein in the background. The sensor sensitivity was also characterized against γ-thrombin, which is a modified α-thrombin lacking the aptamer binding epitope. The detection limit, within an appreciable signal/noise ratio, was 10 pM of α-thrombin in presence of 500 µM HSA. The proposed scheme involves the use of minimum reagents/sample preparation steps, has appreciable response in presence of high concentrations of interfering molecules and is readily amenable to miniaturization by association with existing-chip based electrical systems for application in point-of-care diagnostic devices.

Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , Nanoporos , Trombina/análise , Eletrodos , Desenho de Equipamento , Humanos , Limite de Detecção , Membranas Artificiais , Nanoporos/ultraestrutura , Albumina Sérica/análise
Sci Rep ; 6: 37449, 2016 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-27874042


The binding/unbinding of the human thrombin and its 15-mer single stranded DNA aptamer, under the application of external stimulus in the form of electrostatic potential/electric field, is investigated by a combination of continuum analysis and atomistic molecular dynamics simulation. In agreement with the experiments that demonstrate the influence of electrostatic potential on the thrombin/aptamer complex, our computations show that the application of positive electric field successfully unbinds the thrombin from the aptamer. Results from umbrella sampling simulations reveal that there is a decrease in the free energy of binding between the thrombin and aptamer in presence of positive electric fields. Hydrogen bonding and non-bonded interaction energies, and hence the free energy of binding, between the thrombin and its aptamer reduce as the applied electric field is shifted from negative to positive values. Our analyses demonstrate that application of electrical stimulus modifies the molecular interactions within the complex and consequently, electrical field can be used to modulate the association between the thrombin and its aptamer.

Aptâmeros de Nucleotídeos/metabolismo , Trombina/metabolismo , Aptâmeros de Nucleotídeos/química , Cristalografia por Raios X , DNA de Cadeia Simples , Estimulação Elétrica , Eletrodos , Humanos , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Eletricidade Estática , Termodinâmica , Trombina/química