silicon and thrombin-aptamer

A reusable and straightforward aptasensor with the implementation of open-ended porous silicon (OEPSi) membranes was introduced for thrombin detection. When passing through the nanochannels of OEPSi integrated in a microfluidic cell, thrombin in sample solution could be captured by thrombin-binding aptamers (TBA) immobilized along the inner walls. The formation of thrombin-aptamer complex causes refractive index changes which can be measured by reflective interferometric Fourier transform spectroscopy (RIFTS). And this flow-through configuration with OEPSi has proven more efficient in capturing thrombin than the flow-over configuration with closed-ended PSi. For higher sensitivity, we investigated how the pore size, ionic strength, pH and aptamers affected the thrombin-aptamer interaction in nanopores. Under optimized conditions, the limits of detection (LOD) for thrombin detection in the buffer and serum were ∼6.70 nM and ∼8.21 nM respectively and a wide linear detection range (10-1000 nM) was observed. More importantly, this work reveals the sensitivity of the label-free biosensor can be significantly improved by attaching newly designed aptamers with two thrombin-binding sites. This phenomenon also indicates the potential of aptamer probes in adjusting effective pore size and enhancing the interaction between aptamers and targets through meticulous sequence design. Furthermore, the proposed strategy has been applied in thrombin detection in clinic samples successfully, which was verified by Enzyme-Linked Immunosorbent Assays (ELISA).

Topics: Aptamers, Nucleotide; Biosensing Techniques; Equipment Design; Humans; Limit of Detection; Membranes, Artificial; Porosity; Silicon; Thrombin

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

Topics: Antibodies, Monoclonal; Aptamers, Nucleotide; Biosensing Techniques; Equipment Design; Humans; Limit of Detection; Optics and Photonics; Protein Binding; Silicon; Thrombin

A fiber-optic biosensor using an aptamer receptor has been developed for the measurement of thrombin. An antithrombin DNA aptamer was immobilized on the surface of silica microspheres, and these aptamer beads were distributed in microwells on the distal tip of an imaging fiber. A different oligonucleotide bead type prepared using the same method as the aptamer beads was also included in the microwells to measure the degree of nonspecific binding. The imaging fiber was coupled to a modified epifluorescence microscope system, and the distal end of the fiber was incubated with a fluorescein-labeled thrombin (F-thrombin) solution. Nonlabeled thrombin could be detected using a competitive binding assay with F-thrombin. The aptamer beads selectively bound to the target and could be reused without any sensitivity change. The fiber-optic microarray system has a detection limit of 1 nM for nonlabeled thrombin, and each test can be performed in ca. 15 min including the regeneration time.

Topics: Animals; Aptamers, Nucleotide; Biosensing Techniques; Cattle; DNA; Dose-Response Relationship, Drug; Fiber Optic Technology; Humans; Microscopy, Fluorescence; Microscopy, Video; Oligonucleotides; Optical Fibers; Protein Binding; Sensitivity and Specificity; Serum Albumin; Silicon; Thrombin