Study On Thrombin Electrochemical Aptasensor Based On Nanocomposite And Enzyme Amplification

With the merits of fast response, high sensitivity, low cost and so on, aptamer-based electrochemical biosensors (electrochemical aptasensors) have been received much more attention in the field of electrochemical analysis. Recently, different kinds of nanomaterials with large surface area and good catalytic performance, as well as various enzyme-based amplification strategies have been employed in many electrochemical aptasensors for the design of sensitive interface of biosensors and the construction of analysing test platform. Thrombin (TB), as a kind of bioactive protein, plays a crucial role in many procoagulant and coagulation related reactions in human body, and exhibits great application value in biomedicine and clinical disease diagnosis. Therefore, it is of very practical meaning to quantitatively detect TB in a simple and sensitive way. Inspired by these points, three kinds of electrochemical aptasensors for TB detection were developed based on a series of nanocomposites and enzyme catalysis for signal amplification, and their electrochemical analytical performance were to some extent improved. Besides, the three detection systems showed good selectivity and sensitivity for TB detection, providing prospective and may potentially provide effective methods for the diagnosis of certain diseases in clinic.Part 1 A TB electrochemical aptasensor based on exonuclease-catalyzed target recycling and enzyme-catalytic amplificationAn electrochemical aptasensor for detecting TB using exonuclease-catalyzed target recycling and enzyme-catalysis was proposed in this work. Firstly, a large amount of alcohol dehydrogenase (ADH) was encapsulated into 3-(mercaptopropyl)- trimethoxysilane (MPTS) sol with a 3-D network to form MPTS sol-ADH biocomposite, which was further immobilized onto the bare glass carbon electrode (GCE) modified with gold nanoparticles (AuNPs) through the Au-S bond. Then, the AuNPs layer was further coated on the modified electrode, forming AuNPs-MPTS sol-ADH nanocomposites. Afterwards, streptavidin (SA) was dropped onto the electrode surface. Through biotin-streptavidin conjunction, the hybrid double stand DNA labeled with methylene blue (MB) as the redox mediator (MB-dsDNA) was loaded onto the electrode. Finally, the mixture of TB and RecJf exonuclease was introduced. Because of the specific binding between TB and TB aptamer (TBA), the TB-TBA composite was formed with the dissociation of dsDNA. Then, the RecJf exonuclease selectively degraded TBA, releasing TB and catalyzing the target recycling. In addition, through the catalysis of ADH and AuNPs, the electrochemical signal was amplified, resulting in the improvement of the sensitivity of the proposed aptasensor. A linear range from 0.5 pmol·L-1 to 100 nmol·L-1 with a detection limit of 0.17 pmol·L-1 for the TB aptasensor was achieved.Part 2 A TB electrochemical aptasensor based on hemin/G-quadruplex as signal label and AuNPs-ADH-graphene nanocomposite (GSs) as nanocarriersIn the present work, we developed a pseudo triple-enzyme cascade amplified electrochemical aptasensor for TB detection using hemin/G-quadruplex as signal label and AuNPs-ADH-GSs nanocomposite as nanocarrier. Firstly, GSs with large surface area was used as a platform for the immobilization of large amount of ADH in GSs. Then, AuNPs were immobilized on the ADH-GSs nanocomposite through the Au-N bond between AuNPs and ADH, forming the AuNPs-ADH-GSs bionanocomposite. Subsequently, hemin was intercalated into the amino- terminated TBA with the production of hemin/G-quadruplex, which was further immobilized onto AuNPs-ADH-GSs bionanocomposite. Furthermore, bovine serum albumin (BSA) was employed to block the remaining active sites of AuNPs with the formation of hemin/G-quadruplex labeled AuNPs-ADH-GSs bionanocomposite (the second aptamer TBA II complex). Through "sandwich" reaction, TBA Ⅱ was captured onto the electrode surface, resulting in an electrochemical signal. Through the triple catalysis of ADH and hemin/ G-quadruplex acted as NADH oxidase and HRP-mimicking DNAzyme, the electrochemical signal was obviously amplified, leading to the improved sensitivity of the proposed aptasensor with 0.3 pmol·L-1 detection limit.Part 3 A TB electrochemical aptasensor based on the synergetic catalysis of enzyme and porous Au@Pd core-shell nanostructuresIn this work, an amplified electrochemical TB aptasensor based on synergetic catalysis of glucose oxidase (GOx), porous Au@Pd core-shell nanostructures and hemin/G-quadruplex has been developed. Porous Au@Pd core-shell nanostructures not only possess excellent catalytic performance as peroxidase mimics, but also have large surface area, which endow the ability to immobilize large amounts of amino terminated TBA (NH2-TBA) and redox active toluidine blue (Tb). Besides, hemin was intercalated into NH2-TBA to form hemin/G-quadruplex with favorable peroxidase-mimicking properties. Subsequently, GOx acted as the blocking reagent to block the remaining active sites on the porous Au@Pd core-shell nanostructures, achieving the secondary TB aptamer complex (hemin/G-quadruplex-Au@Pd-Tb-GOx bioconjugates). Through the "sandwich" reaction of the NH2-TBA on the modified electrode surface, target TB and the secondary TBA composite, a detectable electrochemical signal was obtained, which was further amplified by the synergetic catalysis of GOx, porous Au@Pd core-shell nanostructures and hemin/G-quadruplex. As a result, the sensitivity of the proposed aptasensor for TB was improved with a detection limit of 0.037 pmol·L-1.