Fluorescence-Linked Aptamer Assay for SARS-CoV-2 Spike-Protein: A Step-by-Step Performance Analysis in Clinical Samples

Abstract

The COVID-19 pandemic has been a main concern over the last two years and has become one of the most important crises in the history of human health. Today, there is still a need for affordable and reliable diagnostic tests for massive disease monitoring. Previously, a set of highly specific DNA-aptamers (C7/C9) binding to the SARS-CoV-2 Spike (S) protein were isolated but its performance in clinical samples remained to be tested. Here, 242 samples were collected through three different methods and subjected to florescence-linked aptamer assays (FLAA) based on C7/C9 aptamers through two readout protocols. Then, a step-by-step statistical approach which included agreement tests, proportion comparisons and binomial and multinomial logistic regressions was used to predict optimal conditions for the novel C7/C9 FLAA test. RTqPCR threshold cycles, symptoms onset and processing time were influential factors on FLAA test results. Naturally occurring mutations on S were also detected and analyzed. Aminoacidic substitutions D614G and T732A appeared relevant for aptamer recognition although further studies are necessary. The methodology presented here is the first step to determine the performance and diagnosis across a range of clinical contexts and it might serve as a base for a complete analysis applicable to other designs of new diagnostic tests.

Aptamers and Nanobodies as New Bioprobes for SARS-CoV-2 Diagnostic and Therapeutic System Applications

“…The LOD of N protein in this digital method was 33.28 pg/mL, showing a 300 times lower detection limit than the traditional ELISA method [ 78 ]. Fluorescence-linked aptamer assays (FLAA) have been reported as a sandwich-type detection system using fluorescence modification directly on an aptamer without enzymatic linkage [ 77 , 104 ]. As the fluorescence sensor utilized inorganic material, gold nanostar (AuNS), which changed the shape of AuNPs, detected specific SARS-CoV-2 S protein or SARS-CoV-2 pseudo-typed baculovirus in 10 min through distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy (an LOD of 130 fg/mL for antigen and 8 particles/mL for virus) [ 82 ].…”

Recent Advances in Biological Applications of Aptamer-Based Fluorescent Biosensors

“…This sandwich-type detection strategy exhibits high sensitivity and specificity and is suitable for applying the lateral flow strip type kit for on-site diagnosis [37]. Franco-Urquijo et al reported an aptasensor that can diagnose SARS-CoV-2 through the FLAA test [38]. They designed a sandwich-type detection system using C7/C9 aptamers that can specifically bind to the SARS-CoV-2 spike (S) protein.…”

“…(1) they can be easily modified into an aptamer, (2) they are inexpensive, and (3) a variety of natural and synthetic fluorophores are well known and can be selected depending on the purpose [58][59][60]. Previously developed organic dyes, fluorescein and rhodamine, are still used as fluorophores in aptasensors [30,32,35,38,54,57]. However, these previous fluorescent organic dyes have problems such as hydrophobicity, photobleaching, and being affected by pH changes [61].…”

Novel enzyme-linked aptamer-antibody sandwich assay and hybrid lateral flow strip for SARS-CoV-2 detection

“…1 ). While previous studies have successfully developed SARS-CoV-2 antibody-based analysis methods for N protein detection and aptamer-based analysis methods for spike protein (S) detection in nasopharyngeal samples or antibody detection in serum samples [ 14 , 15 ], none of these methods have demonstrated sufficient sensitivity for effective prevention and control. This research introduces a more convenient and cost-effective aptamer-antibody hybrid method for aiding in the prevention and control of SARS-CoV-2.…”