D-shaped plastic optical fibre aptasensor for fast thrombin detection in nanomolar range

Abstract

The development of optical biosensors for the rapid and costless determination of clinical biomarkers is of paramount importance in medicine. Here we report a fast and low-cost biosensor based on a plasmonic D-shaped plastic optical fibre (POF) sensor derivatized with an aptamer specific for the recognition of thrombin, the target marker of blood homeostasis and coagulation cascade. In particular, we designed a functional interface based on a Self Assembled Monolayer (SAM) composed of short Poly Ethylene Glycol (PEG) chains and biotin-modified PEG thiol in ratio 8:2 mol:mol, these latter serving as baits for the binding of the aptamer through streptavidin-chemistry. The SAM was studied by X-ray Photoelectron Spectroscopy (XPS) analysis, static contact angle (CA), Surface Plasmon Resonance (SPR) in POFs, and fluorescence microscopy on gold surface. The optimized SAM composition enabled the immobilization of about 112 ng/cm2 of aptamer. The thrombin detection exploiting POF-Aptasensor occurred in short times (5–10 minutes), the reached Limit of Detection (LOD) was about 1 nM, and the detection range was 1.6–60 nM, indicating the POF-Aptasensor well addresses the needs for a low-cost, simple to use and to realize, rapid, small size and portable diagnostic platform.

SARS-CoV-2 spike protein detection through a plasmonic D-shaped plastic optical fiber aptasensor

“…Through an opportune calibration curve (reported in Fig. S2 ) the aptamer density resulted in 89.3 ± 36.9 ng/cm 2 , in good agreement with previous data [ 38 ]. Moreover, an additional experiment was performed varying the aptamer concentration from 1 up to 100 μM, confirming that 10 μM was enough to cover the surface (see Fig.…”

“…Besides the characterization of the aptamer layer on POF platform previously reported [ 38 ], here we focused on the evaluation of the stability of the platform over time, testing different conservation conditions both in terms of solutions and temperatures. First, the stability of the SAM layer was analyzed, testing different conditions, as reported in experimental section.…”

“…S4 ). Secondly, we tested the aging of the aptamer layer: for this purpose, an anti-THR aptamer was used applying an immune-chemiluminescence method previously used [ 38 ]. Figure 1 B showed that, up to about 12 days, the aptamer platform was able to recognize thrombin in an unaltered way (inside error bars) in PBS solution, but also in the other conditions namely PBS+20%DMSO and dried, as reported in Fig.…”

“…5 μg/ml of Cy3-SA solution in phosphate buffer for one hour was applied and after washing, the surfaces were analyzed with a fluorescence microscope. The effect of aging on the aptamer layer was instead tested using an aptamer specific for thrombin recognition immobilized with the same protocol evaluating its ability to bind thrombin by means of a chemiluminescence protocol [ 38 ]. The aptamer-treated surfaces were aged in PBS buffer (PBS) or PBS + 20% DMSO (PBS+DMSO) or dried (DRY) at 4 °C for several days.…”

“…In this work, we immobilized an aptamer specific for the recognition of the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein, published by Song with an affinity constant (K d ) of 5.8 nM [ 37 ], on a SPR D-shaped POF probe, previously used in other bio-chemical applications [ 36 ]. The aptameric sequence was here immobilized on a short PEG interface [ 38 ] and a chemical-physical and functional characterization was performed by X-ray Photoelectron Spectroscopy (XPS), static contact angle (CA), atomic force microscopy (AFM), fluorescence microscopy, and plasmonic study. SARS-CoV-2 spike protein binding was optically measured without any amplification systems.…”