Ultrasensitive Detection Platform of Disease Biomarkers Based on Recombinase Polymerase Amplification with H-Sandwich Aptamers

Abstract

The detection of trace protein biomarkers is essential in the diagnostic field. Protein detection systems ranging from widely used enzyme-linked immunosorbent assays to simple, inexpensive approaches, such as lateral flow immunoassays, play critical roles in medical and drug research. Despite continuous progress, current systems are insufficient for the diagnosis of diseases that require high sensitivity. In this study, we developed a heterogeneous sandwich-type sensing platform based on recombinase polymerase amplification using DNA aptamers specific to the target biomarker. Only the DNA bound to the target in the form of a heterogeneous sandwich was selectively amplified, and the fluorescence signal of an intercalating dye added before the amplification reaction was detected, thereby enabling high specificity and sensitivity. We applied this method for the detection of protein biomarkers for various infectious diseases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and observed attomolar-level detection of biomarkers and low cross-reactivity between different viruses. We also confirmed detection efficiency of the proposed method using clinical samples. These results demonstrate that the proposed sensing platform can be used to diagnose various diseases requiring high sensitivity, specificity, and accuracy.

Aptamers for SARS‐CoV‐2: Isolation, Characterization, and Diagnostic and Therapeutic Developments

“…Although far less common than S protein aptamers, aptamers for the N protein of SARS‐CoV‐2 have also been discovered. At present, three SELEX studies have been reported (Table 3 and Table S5) [88–90] …”

Recent advances of functional nucleic acid-based sensors for point-of-care detection of SARS-CoV-2

“…A COVID-19 diagnostic approach that integrates RT-RPA and universal lateral flow test strips into an individual microfluidic chip has been reported with fast, highly sensitive, 30-min acquisition results [ 55 ]. Kang et al [ 56 ] reported an ultrasensitive protein biomarker detection platform based on immunoassays combined with RPA. Only the DNA bound to the target in the form of a heterosandwich was selectively amplified with high specificity and sensitivity.…”

Nanoelectrokinetic-assisted lateral flow assay for COVID-19 antibody test

“…Research is underway to improve the sensitivity and specificity of LFAs to achieve more accurate and high-performance POCT. Such investigations have primarily focused on assay optimization (reagent and receptor) ( Dighe et al, 2022b , Dighe et al, 2022a ; Garg et al, 2021a , Garg et al, 2021b ; Grant et al, 2020b , Grant et al, 2020a ; Yu et al, 2020b , Yu et al, 2020a ; Zhang et al, 2021b , Zhang et al, 2021a ), signal amplification (chemical enhancement and electrochemistry and fluorescence reader) ( Cheng et al, 2017a , Cheng et al, 2017b ; Wang et al, 2017a , Wang et al, 2017b , 2021b ), and sample enrichment (magnetic separation and electrokinetic pre-concentration) ( Kang et al, 2021b , Kang et al, 2021a ; Wang et al, 2021aa , Wang et al, 2021ba , Wang et al, 2021ab , Wang et al, 2021bb ; Zhou et al, 2021a , Zhou et al, 2021b ) to achieve high sensitivity and selectivity ( Dempsey and Rathod, 2018b , Dempsey and Rathod, 2018a ; Han et al, 2020a , Han et al, 2020b ; Jia et al, 2018b , Jia et al, 2018a ; Li et al, 2019a , Li et al, 2019b ; Loynachan et al, 2018b , Loynachan et al, 2018a ; Mu et al, 2019b , Mu et al, 2019a ; Niu et al, 2020a , Niu et al, 2020b , Niu et al, 2021a , Niu et al, 2021b ; Ojaghi et al, 2018a , Ojaghi et al, 2018b ; Xu et al, 2014a , Xu et al, 2014b ). As one of the sample enrichment techniques, our group described a nanoelectrokinetic (NEK)-based method for sample enrichment on paper, illustrating that bovine serum albumin (BSA) can be preconcentrated by up to five times the initial concentration in serum ( Han et al, 2018b , Han et al, 2018a ; Jeong et al, 2018b , Jeong et al, 2018a ).…”