Aptamer-Based Detection of Plasma Proteins by an Electrochemical Assay Coupled to Magnetic Beads

Abstract

The DNA thrombin aptamer has been extensively investigated, and the coupling of this aptamer to different transduction principles has demonstrated the wide applicability of aptamers as bioreceptors in bioanalytical assays. The goal of this work was to design an aptamer-based sandwich assay with electrochemical detection for thrombin analysis in complex matrixes, using a simple target capturing step by aptamer-functionalized magnetic beads. The conditions for the aptamer immobilization and for the protein binding have been first optimized by surface plasmon resonance, and then transferred to the electrochemical-based assay performed onto screen-printed electrodes. The assay was then applied to the analysis of thrombin in buffer, spiked serum, and plasma and high sensitivity and specificity were found. Moreover, thrombin was generated in situ in plasma by the conversion of its precursor prothrombin, and the formation of thrombin was followed at different times. The concentrations detected by the electrochemical assay were in agreement with a simulation software that mimics the formation of thrombin over time (thrombogram). The proposed work demonstrates that the high specificity of aptamers together with the use of magnetic beads are the key features for aptamer-based analysis in complex matrixes, opening the possibility of a real application to diagnostics or medical investigation.

Highly sensitive detection of human thrombin in serum by affinity capillary electrophoresis/laser-induced fluorescence polarization using aptamers as probes

“…The RSDs (n = 3) along the whole concentration range were <10% for both assays. Both in standard solution and spiked serum, the obtained LODs are much lower than that the previously reported values of 0.5 nM (sandwich assay) [24], 9.8 nM (APCE/LIF assay) [29], and 2 nM (APCE/LIF assay) [18] based on thrombin aptamers.…”

“…Aptamers possess some advantageous properties over antibodies, such as ease-of-synthesis, ease-of-labeling and excellent stability. Alternatives to antibodies, aptamers have been pursued as affinity probes or ligands for protein detection in several methods [22][23][24]. It is also likely to act as a class of molecules that rival antibodies in both therapeutic and diagnostic applications [25].…”

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

“…37 Concentrations in the upper picomolar range indicate thrombin-related diseases with coagulation irregularities whereas normal levels can be found at μM down to nM range. 38 Thus, label-free and fast sensors able to measure from nanomolar down to the picomolar range are important to recognize and distinguish healthy from disease thrombin levels.…”

Self-bending symmetric cusp beams

“…2 Nevertheless, the high nonspecific background encountered in complex media can be efficiently diminished by changing the detection format with double-site interaction, in particular, using sandwich assays. [3][4][5][6][7][8][9][10][11][12][13] Alternatively, the proximity between two aptamers that bind different thrombin epitopes has been exploited to collect a specific signal of the target. [14][15][16][17] Enhanced sensitivity and reduced reaction time have been achieved with bivalent aptamer structures in which distinct aptamers are conjugated with an optimal linker, 18,19 or co-conjugated to the sensor surface with an optimal density.…”