Aptamer-based fluorescent solid-phase thrombin assay using a silver-coated glass substrate and signal amplification by glucose oxidase

Abstract

We describe an aptamer-based solid-state biosensor for the fluorometric determination of thrombin. The surface of silver-coated glass was modified with the thrombin-binding aptamer 1 (TBA 1) of the sequence 5′-HS-TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3′. A second (and biotinylated) thrombin -binding aptamer (TBA 2) with the sequence 5′-biotin-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′ was applied as the signaling aptamer. Following binding of thrombin by TBA 1 on the surface, TBA 2 is added and then binds to the thrombin on the surface of the silver-coated glass to form the thrombin-aptamer complex. The biotin groups on TBA 2 are then coated with streptavidin, and biotin-labeled glucose oxidase (biotinGOx) is added to bind to streptavidin. The quantity of GOx immobilized in this way is directly related to the quantity of thrombin bound on the surface. Following cleavage of the aptamer with DNase I, glucose is added and oxidized by GOx to yield H 2 O 2 . Horseradish peroxidase is added and causes the oxidation of 3-p-hydroxyphenylpropanoic acid to yield a fluorescent product. The intensity of the blue fluorescence is directly related to the thrombin concentration in the 300 pM to 6500 pM range, and the detection limit is as low as 82 pM. The assay has good selectivity and practicability.

A sensitive colorimetric DNA biosensor for specific detection of the HBV gene based on silver‐coated glass slide and G‐quadruplex‐hemin DNAzyme

“…Then, the quartz glass slides were dipped into the mixture at 25°C for 5 min. Finally, shining silver film was formed on the surface of glass slide …”

“…All the experiments were performed following the relevant laws and institutional guidelines. Before the recovery experiment in human sera, each sample was removed small molecules by Amicon filtration device 10 000 …”

“…[16][17][18][19] Oligonucleotide immobilization can be used to separate bound biomolecules from free molecules, and several immobilization methods have been reported for the functionalization of surfaces such as glass, electrode, nano-magnetic microsphere, and nanoparticles. [20][21][22][23][24][25] An idea sensing platform is being developed by using the concept of silver coated glass slide(SCGS) which is an attractive separation material, because thiol-functionalized oligonucleotides can self-assemble on the silver surface through Ag-S bonds, [26][27][28][29] and SCGS is easily prepared by the silver mirror reaction. [30][31][32][33] In this article, by using SCGS as an idea separation material, we describe a simple, sensitive colorimetric DNA biosensor for the determination of the target DNA which selected from the HBV gene (15)…”

“…Finally, shining silver film was formed on the surface of glass slide. 8,29 2.3 | Immobilization of capture DNA on the SCGS Capture DNA(Oligo-1) can be immobilized on the SCGS according to the reported methods. 34 The SCGS was placed in Tris-HAc buffer which contained 1.0 μM Oligo-1.…”

“…Oligonucleotide immobilization can be used to separate bound biomolecules from free molecules, and several immobilization methods have been reported for the functionalization of surfaces such as glass, electrode, nano‐magnetic microsphere, and nanoparticles . An idea sensing platform is being developed by using the concept of silver coated glass slide(SCGS) which is an attractive separation material, because thiol‐functionalized oligonucleotides can self‐assemble on the silver surface through Ag‐S bonds, and SCGS is easily prepared by the silver mirror reaction …”