Elsevier

Biosensors and Bioelectronics

Volume 216, 15 November 2022, 114639
Biosensors and Bioelectronics

Highly sensitive protein detection by aptamer-based single-molecule kinetic fingerprinting

https://doi.org/10.1016/j.bios.2022.114639Get rights and content

Abstract

Sensitive assays of protein biomarkers play critical roles in clinical diagnostics and biomedical research. Such assays typically employ immunoreagents such as monoclonal antibodies that suffer from several drawbacks, including relatively tedious production, significant batch-to-batch variability, and challenges in site-specific, stoichiometric modification with fluorophores or other labels. One proposed alternative to such immunoreagents, nucleic acid aptamers generated by systematic evolution of ligand by exponential enrichment (SELEX), can be chemically synthesized with much greater ease, precision, and reproducibility than antibodies. However, most aptamers exhibit relatively poor affinity, yielding low sensitivity in the assays employing them. Recently, single molecule recognition through equilibrium Poisson sampling (SiMREPS) has emerged as a platform for detecting proteins and other biomarkers with high sensitivity without requiring high-affinity detection probes. In this manuscript, we demonstrate the applicability and advantages of aptamers as detection probes in SiMREPS as applied to two clinically relevant biomarkers, VEGF165 and IL-8, using a wash-free protocol with limits of detection in the low femtomolar range (3–9 fM). We show that the kinetics of existing RNA aptamers can be rationally optimized for use as SiMREPS detection probes by mutating a single nucleotide in the conserved binding region or by shortening the aptamer sequence. Finally, we demonstrate the detection of endogenous IL-8 from human serum at a concentration below the detection limit of commercial ELISAs.

Introduction

Proteins are useful biomarkers for differentiating between healthy and diseased states in clinical diagnostics (Simren et al., 2020). Sensitive and accurate quantification of proteins is the key for early-stage diagnosis of diseases. The long-standing gold-standard method for protein quantification in biofluids is the enzyme-linked immunosorbent assay (ELISA) (Engvall and Perlmann, 1971). However, the success of ELISA and its digital version (Rissin et al., 2010) in high-sensitivity applications depends on the so-called sandwich assay format, which imposes a nontrivial requirement of two antibodies that can simultaneously bind the same target protein at distinct epitopes with high affinity. In addition to batch-to batch variations in the production of antibody, it is tedious and challenging to generate specific, high affinity monoclonal antibodies, especially against non-immunogenic and toxic proteins (Toh et al., 2015). Moreover, these techniques cannot differentiate between specific signal from the protein of interest and non-specific signal from binding to assay surfaces or matrix (Chatterjee et al., 2020; Cohen and Walt, 2019).

A potential alternative to conventional antibody probes is provided by aptamers, synthetic single-stranded DNA (ssDNA) or RNA oligonucleotides that fold into unique three-dimensional structures and bind their targets specifically (Proske et al., 2005). Compared to antibodies, aptamers have several advantages. First, the generation of aptamers by systematic evolution of ligand by exponential enrichment (SELEX) can be performed faster and at lower cost than generating an antibody against a target protein by hybridoma or phage display technology (Gray et al., 2020; Tuerk and Gold, 1990). Moreover, solid-phase chemical synthesis permits aptamers to be synthesized more easily and more reproducibly than antibodies (Wang et al., 2011). Other advantages of aptamers include their non-toxicity and non-immunogenicity (Fish et al., 2003), reduced steric hindrance due to their smaller sizes (Lee et al., 2006), ease of modification, and thermal stability (Liss et al., 2002).

Due to the potential advantages of aptamers, ELISA-derived assays such as the-enzyme linked apta-sorbent assay (ELASA) have been developed (Drolet et al., 1996; Martin et al., 2013; Ramos et al., 2007; Zhang et al., 2018). However, these assays often have low sensitivity (limit of detection, LOD ∼ 1 pM) and specificity (Toh et al., 2015; Zhang et al., 2018). To overcome this limitation, the SOMAscan assay utilizes modified aptamers called SOMAmers (slow off-rate modified aptamers) as recognition elements, permitting relatively high sensitivity and specificity in protein detection (median LOD for 1129 proteins ∼ 40 fM) (Gold et al., 2010). However, this assay requires expensive synthetic nucleotide modifications, and, like conventional immunoassays, involves multistep sample handling procedures including stringent washing steps, and is unable to differentiate between specific and nonspecific binding (Gold et al., 2010; Rohloff et al., 2014).

Recently, Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) has emerged as a powerful technique for the ultrasensitive and specific detection of protein and other biomarkers, with LODs in the aM to low fM range (Chatterjee et al., 2020; Li et al., 2022; Mandal et al., 2021). The high sensitivity of SiMREPS results from its use of binding and dissociation kinetics to distinguish signal of specific binding to the target from that of nonspecific binding to the assay surfaces or matrix. To achieve this for proteins, a detection antibody with relatively fast dissociation kinetics (koff ∼0.05–0.5 s−1) is needed to permit repeated interrogation of single target molecules and generate characteristic kinetic fingerprints within a reasonably short acquisition time (e.g., 2 min per field of view) without sacrificing sensitivity (Chatterjee et al., 2020).

Despite this success, there are several challenges associated with using antibodies as SiMREPS detection probes. While the success rate for generating protein-SiMREPS probes by phage display has been high, likely due to the relaxed affinity requirement compared to conventional immunoreagents (Chatterjee et al., 2020), developing recombinant antibody reagents by phage display nonetheless involves nontrivial time requirements and cost. While kinetics can be further manipulated using reaction conditions such as salt concentration or temperature (Chatterjee et al., 2020), it is difficult to rationally alter antibody sequences to achieve desired changes in binding or dissociation kinetics. Furthermore, antibodies often exhibit nonspecific adsorption to assay surfaces, even when passivated by reagents such as polyethylene glycol or Tween 20 (Hua et al., 2014). Although the kinetic analysis approach of SiMREPS allows one to filter out mild or moderate nonspecific binding, severe nonspecific binding can still interfere with the analysis and potentially exclude otherwise kinetically suitable antibodies from consideration (Chatterjee et al., 2020). Finally, fluorescent labeling of antibodies is typically performed in a non-regioselective manner (e.g., nonspecific labeling of lysine amines with N-hydroxysuccinimidyl ester reagents), and can result in loss of binding activity, increased kinetic heterogeneity, or aggregation of the antibody (McCormack et al., 1996; Vira et al., 2010).

Motivated by the advantages of aptamers as well as the tolerance of SiMREPS for lower-affinity detection probes than conventional immunoassays, in the present study we demonstrate the applicability of aptamers as detection probes in SiMREPS assays of two clinically relevant protein biomarkers: VEGF165 and IL-8. We show that previously reported aptamers can be rationally optimized for use as dynamically binding SiMREPS probes by incorporating small sequence modifications during chemical synthesis, such as a single nucleotide substitution in the conserved region or the shortening of adjacent helical stems. Thus, unlike antibodies, aptamer sequences can be relatively easily modulated to achieve faster kinetics and, hence, faster data acquisition and better distinction between signal and background kinetic fingerprints. Furthermore, the use of chemical synthesis permits site-specific and stoichiometric fluorophore labelling of these aptamers at sites distal from the antigen-binding site, maintaining binding affinity as well as creating a simpler two-state fluorescence intensity behavior than nonspecifically labeled antibodies provide. Finally, we demonstrate the ultrasensitive detection of spiked-in VEGF165 and endogenous human IL-8 in serum matrices using a wash-free protocol, yielding limits of detection in the low femtomolar range (3.1 fM or 0.026 pg/mL for IL-8 detection and 8.9 fM or 0.340 pg/mL for VEGF165 detection).

Section snippets

Oligonucleotides and proteins

All 2′-fluoropyrimidine-modified and 3′-Cy5-labelled RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT, www.idtdna.com) with high-performance liquid chromatography (HPLC) purification. All oligonucleotide sequences are shown in Fig. 1D and Supporting Information Table S1. They were suspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to a final concentration of 100 μM and then aliquoted and stored at −80 °C. Recombinant human IL-8 (Ser28-Ser99, catalog# BR-1098) was

Aptamer based kinetic fingerprinting assay design

Our aptamer-based protein SiMREPS assays use two probes: a capture antibody, typically an IgG, with slow dissociation kinetics; and a low-affinity RNA aptamer with rapid dissociation kinetics (koff ∼0.05–0.5 s−1) (Fig. 1A). The capture antibody is modified with biotin to permit surface immobilization of the target protein via a streptavidin bridge to a biotin-PEG (polyethylene glycol)-coated coverslip, while the aptamer is modified with an organic fluorophore either at the 3′ end or 5’ end to

Conclusions

In this study, we have demonstrated the applicability of fluorophore-labelled aptamers as rationally tunable probes for the highly sensitive and specific detection of two clinically relevant protein biomarkers, VEGF165 and IL-8, by SiMREPS. As with previously reported protein-SiMREPS assays using Fab antibody detection probes (Chatterjee et al., 2020), aptamer-based SiMREPS achieves low-femtomolar (sub-pg/mL) LODs in animal and human serum, exceeding the sensitivity of commercial ELISAs by more

CRediT authorship contribution statement

Tanmay Chatterjee: Conceptualization, Investigation, Writing – original draft, Writing – review & editing, Visualization, Validation, Formal analysis, Methodology. Alexander Johnson-Buck: Conceptualization, Writing – review & editing, Software, Supervision. Nils G. Walter: Conceptualization, Supervision, Funding acquisition, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.G.W and A.J.-B are cofounder of aLight Sciences, Inc., which seeks to commercialize the SiMREPS technology. A.J.-B is an employee of aLight Sciences, Inc.; N.G.W and A.J.-B are co-inventors of patent applications related to the SiMREPS technology.

Acknowledgements

This work was funded by aLight Sciences, Inc. Antibodies (VEGF165 monoclonal antibody) and recombinant antigens (human IL-8) were provided by Bio-Rad Laboratories, Inc. SiMREPS analysis software was developed in part using funding from a Michigan Economic Development Corporation MTRAC for Life Sciences grant to N.G.W. and A.J-B, as well as NIH grants R21 CA204560 and R33 CA229023 to N.G.W.

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