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. 2026 Feb 5;11(6):10425–10436. doi: 10.1021/acsomega.5c11711

High-Sensitivity DNA Aptasensors for Detecting Salivary Biomarker S100A7 in Heart Failure

Tomas Schwenke †, Micaela B Sequeira †, Roxane Mutschler †, Chamindie Punyadeera ‡, Alexander Beliaev §,∥, Kirill Alexandrov †,§,*, Elena Eremeeva †,*
PMCID: PMC12917830  PMID: 41726651

Abstract

Early detection of heart failure (HF) is vital for improving patient outcomes, lowering hospital readmission rates, and enabling prompt treatment. We present the first high-affinity DNA aptamer for the salivary HF biomarker S100A7 and its application in highly sensitive, noninvasive diagnostic tests. Iterative truncation of the initial 82-nt aptamer (17–82) produced a 43-nt core (17–43) with a binding affinity of 27 nM, which was further enhanced to 5.5 nM through dimerization. Biochemical and mutational studies confirmed that 17–43 adopts a G-quadruplex structure, which is essential for S100A7 recognition and resistance to enzymatic degradation in human saliva. Incorporating 17–43 into sandwich aptamer-ELISA and hybrid aptamer–antibody ELISA assays allowed detection of recombinant S100A7 in human saliva with limits of detection (LOD) of 7.4 ng mL–1 (0.6 nM) and 29 pg/mL (2.2 pM), respectivelyoutperforming commercial immunoassays in both sensitivity and dynamic range. The hybrid assay maintained its full performance after 2.5 months of room temperature storage. Additionally, a biolayer interferometry (BLI) sensor with 17–43 quantified S100A7 in patient saliva (n = 3), achieving a LOD of 3.2 ng mL–1 (0.3 nM) with a total assay time of less than 20 min. The aptamer’s stability, high specificity, and versatility across biosensing platforms establish it as a promising tool for noninvasive heart failure diagnostics, laying the groundwork for portable aptamer-based biosensors for multiplexed monitoring of HF biomarkers.

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Introduction

Heart failure (HF) is a chronic cardiovascular disorder currently affecting about 64 million people worldwide. Its prevalence has steadily increased over the past decade, driven by population aging; rising rates of obesity, diabetes, and hypertension; and improved survival after acute cardiac events. − HF occurs when a weakened heart muscle can no longer pump blood effectively, leading to symptoms such as fatigue, shortness of breath, and fluid retention. ,, Despite advances in treatment and prevention, HF remains a leading cause of illness and death, with roughly 50% mortality within five years of diagnosis. As a result, the burden of HF on healthcare systems is growing. ,

Current diagnostic methods for HF, including echocardiography and electrocardiography, while reliable, require specialized equipment and trained personnel, limiting accessibility for early and ongoing monitoring. Biomarker-based testing using B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) immunoassays provides valuable diagnostic information. Still, it relies on invasive blood collection, restricting its use for frequent patient monitoring. , The ability to detect HF using saliva-based diagnostics offers a promising noninvasive alternative. , Saliva collection is noninvasive, simple, and eliminates the risk of infection associated with blood sampling. Moreover, saliva shares over 25% of its biomolecular content with blood, making it a viable medium for disease diagnostics. , Early detection of HF, particularly at asymptomatic stages, enables timely intervention, thereby reducing morbidity, mortality, and hospitalizations.

Several salivary biomarkers have been explored for HF diagnosis, including NT-proBNP, galectin-3, kallikrein-1, and S100A7. ,,, Among these, S100A7 has demonstrated strong diagnostic relevance, with salivary concentrations reported to be about twice as high in HF patients as in healthy individuals (4.5 μg mL–1 vs 2.1 μg mL–1, respectively). S100A7 is a pro-inflammatory protein associated with various pathophysiological conditions, including inflammatory skin diseases, Alzheimer’s disease, systemic sclerosis, and multiple cancers. − Recently developed antibody-based ELISA assays for salivary S100A7 detection exhibit a sensitivity limit of 12 ng mL–1 (∼1 nM). Although this sensitivity meets diagnostic requirements, the high production cost, thermal instability, and susceptibility to degradation of antibodies limit their practical application. Hence, the development of a robust, low-cost biosensor for S100A7 detection with enhanced sensitivity and extended shelf life under ambient conditions could provide a practical alternative for HF patient monitoring.

In this study, we investigated the potential of S100A7-specific DNA aptamers for HF diagnostics. − Unlike antibodies, aptamers are generated entirely in vitro through the Systematic Evolution of Ligands by EXponential Enrichment (SELEX), enabling selection against a broad range of molecular targets, including toxic and nonimmunogenic compounds that are often inaccessible to traditional antibody generation. Aptamers are entirely synthetic, eliminating the need for animal use and allowing for rapid, scalable, and cost-effective production. Their chemical and thermal stabilities support long-term storage and use in resource-limited settings. At the same time, their conformational flexibilitythe ability to fold into well-defined three-dimensional structures upon target bindingenhances target binding and facilitates integration into biosensing platforms. Aptamers are highly compatible with diverse detection technologies, including portable point-of-care (POC) colorimetric, electrochemical, and optical sensors, such as lateral flow assays and smartphone-based systems. − These properties make aptamers promising candidates for the development of sensitive and affordable diagnostic tools for on-site HF monitoring.

Here, we report the first aptamer with a high affinity and selectivity for S100A7. Using this aptamer, we developed sensitive assays for quantitative S100A7 detection in saliva: an aptamer-antibody hybrid ELISA with a limit of detection (LOD) of 29 pg mL–1 (2.2 pM) in 0.1% human saliva, and a biolayer interferometry assay (BLI) achieving an LOD of 3.2 ng mL–1 (0.25 nM) in 2.5% human saliva within 20 min. This work demonstrates the feasibility of aptamer-based diagnostics for monitoring heart failure and represents an important step toward the development of rapid, noninvasive cardiovascular biosensors.

Results

In Vitro Selection of S100A7-Specific Aptamers

To generate S100A7-specific aptamers, we employed a SELEX strategy based on magnetic bead separation of target–DNA complexes (Figures A and S1). The process included a negative selection step against streptavidin beads to remove nonspecific binders, followed by positive selection on S100A7-coated beads. Enrichment was monitored across ten rounds, after which the pooled library was subjected to NGS analysis (Figure S2 and Tables S1–S3). NGS identified a dominant aptamer family, with aptamer 17 representing >50% of the pool (Figure B). Eight of the most abundant sequences were evaluated by direct ELISA, and aptamers 17 and 151 showed the most potent and specific binding to S100A7 (Figure S3).

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In vitro selection and characterization of the S100A7-specific aptamer. (A) Schematic of the SELEX process used to isolate S100A7-binding aptamers. (B) Predicted secondary and tertiary structures of aptamer 17, generated using NUPACK and AlphaFold3. (C–E) Characterization of aptamer-protein interaction by three complementary assays: direct ELISA (C), ITC (D), and BLI (E). In direct ELISA, S100A7 was immobilized on microplate wells, incubated with a FAM-labeled aptamer, and detected with an HRP-conjugated anti-FAM antibody and TMB substrate. (D) ITC was performed by titrating the aptamer solution into the S100A7 protein in a cell at 20 °C. (E) In the BLI assay, a biotinylated aptamer was immobilized on streptavidin-coated sensor tips and exposed to a series of S100A7 concentrations. Detailed experimental data are shown in Figure S4 and the Experimental Section in the Supporting Information.

The affinity of aptamer 17 for S100A7 was determined using direct ELISA, isothermal titration calorimetry (ITC), and biolayer interferometry (BLI), resulting in K D values of 80, 78, and 1.6 nM, respectively (Figures C and S4). Despite the method-dependent variations in absolute values, all three techniques consistently indicate high-nanomolar to low-nanomolar affinity. Thermodynamic analysis by ITC indicated a strong, favorable interaction, driven predominantly by a large enthalpic contribution (ΔH = −18 kcal mol–1), resulting in a free energy of binding (ΔG) of −9.54 kcal mol–1. BLI analysis further revealed that aptamer 17 has rapid association kinetics (k on = 2.3 × 105 M–1s–1) and slow dissociation (k off = 3.7 × 10–4 s–1). These thermodynamic and kinetic profiles are advantageous for biosensor development as they ensure aptamer-target complex stability during multiple washing steps, a key requirement for robust analytical assays.

Optimization of the Aptamer Structure and Properties

To define the minimal S100A7-binding motif, we combined NGS data with secondary-structure prediction to design a panel of truncation variants derived from aptamer 17 (Figures A and S5). , NGS analysis revealed that the nucleotide variability increased toward the 3′ end (positions >50 displayed the highest mutation frequencies), suggesting this region might be dispensable. To test this, the full-length aptamer 17 (82 nucleotides (nt), hereafter 17–82) and a series of truncated variants were analyzed for binding to S100A7 and control proteins using direct ELISA and ITC (Figures B,C and S6). Truncation of the 3′ end not only preserved binding but improved affinity, reducing the dissociation constant from 63 to 27 nM and yielding a 43-nt variant selected for further analysis. Notably, the 40-nt randomized region alone (17–40) failed to bind S100A7, indicating that the predicted 5′–3′ terminal stemincluding part of the primer regionis critical for stabilizing the S100A7-binding domain and enhancing interaction affinity. No significant improvement was observed in subsequent stem-region mutagenesis or further truncations (Figure S7). Furthermore, a scrambled variant of aptamer 17–43 (17–43-SCR; Figure S8), containing identical nucleotide composition but in randomized order, showed no detectable affinity toward S100A7, further confirming the sequence-specific nature of the aptamer-target interaction.

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Optimization of the S100A7-specific full-length aptamer 17 (hereafter 17–82). (A) Sequence alignment of truncated variants of aptamer 17–82 (52, 47, 45, and 43 nt) and the 40-nt sequence corresponding to the randomized region of the initial library. Primer sequences are underlined; mutations introduced into the original aptamer sequence are shown in red. Nucleotide variations identified by NGS within the 40-nt random region of the 17–82 cluster are highlighted in gray. (B) ELISA assay signals for aptamer 17–82 variants incubated with S100A7 and control antigens. (C) ITC analysis of the binding affinities between S100A7 and truncated variants (D) Structural model of the S100A7 homodimer (PDB: 1PSR), which guided the design of dimeric 17–43 versions. (E) Dimeric 17–43 variants connected using poly-T or poly-A linkers of 5, 10, 12, and 14 nucleotides. (F) Results of ITC analysis of dimeric 17–43 aptamers binding to S100A7. (G) Summary of dimer binding affinities determined by ITC as a function of linker length. (H) Schematic overview of the 17–82 optimization strategy. All aptamer sequences are provided in Table S1 of the Supporting Information.

S100A7 is an obligatory homodimer in which the Ca2+-binding loops are separated by approximately 28 Åa distance that corresponds to roughly eight DNA base pairs. − We, therefore, tested whether dimerization of the selected sequence would create a molecule capable of binding both S100A7 subunits and thereby increasing the avidity of the complex. To this end, we synthesized dimeric versions of aptamer 17–43, joined by poly-T or poly-A linkers of 5, 10, 12, and 14 nucleotides, to determine the optimal spacing between individual binding units (Figures D–H and S9). ITC analysis revealed that the optimal linker length was 10-nt, with poly-T (17–43-T10) and poly-A (17–43-A10) exhibiting K D values of 5.5 and 8.3 nM, respectively, whereas the shortest 5-nt poly-T linker completely abolished binding.

Prediction of Aptamer-S100A7 Complex Structure

To elucidate the secondary and tertiary structures of aptamer 17 variants and their interaction with S100A7, detailed structural and sequence analyses were performed. We hypothesized that the aptamer adopts a G-quadruplex (G4) secondary structure, characterized by stacked guanine tetrads stabilized by coordination with monovalent cations. This hypothesis is supported by the guanine-rich composition of aptamer 17, which is often indicative of structures in which potassium (and to a lesser extent sodium) ions coordinate the O6 atoms of guanine residues. ,

ITC was used to evaluate the effect of the ionic composition on the folding and target binding of the 17–82, 17–43, and 17–43-T10 aptamer variants. Complex formation was strictly dependent on potassium ions (Table S4), supporting the formation of a G4 structure. Comparative buffer studies revealed that binding affinity was higher in phosphate-buffered saline (PBS) than in Tris-based buffers and that the presence of magnesium further enhanced aptamer binding in both systems for all variants tested.

To confirm that aptamer 17 indeed forms a G4 structure, we performed binding assays with the G-quadruplex-specific nanobody SG4. The apparent K D values for interactions of 17–82 and 17–43 with SG4 were approximately 2 orders of magnitude higher than those of well-characterized G4-forming sequences, such as MycG4 and the ochratoxin A aptamer 3O32. , The mutated MycG4 control (mMycG4), which cannot form a G4 structure, failed to bind SG4 under the assay conditions (Figures A and S10A).

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Prediction of S100A7-specific aptamer structure. (A) Binding curves of the G-quadruplex-specific nanobody SG4 to various oligonucleotides measured by FLAG-ELISA. Previously characterized G-quadruplex-forming sequences (MycG4 and 3O32) were included as positive controls, and a mutant mMycG4 sequence, incapable of forming a G-quadruplex, served as a negative control. Dissociation constants (K D) are indicated; “ND” denotes not determined K D. Error bars represent standard deviations (SD) from 3–5 independent replicates. (B) Identification of aptamer 17 residues critical for G4 formation and S100A7 interactions. Mutations resulting in complete loss of binding are highlighted in red, those causing partial loss of affinity in yellow, and those with little or no effect in green. G-quartets forming the G-quadruplex core are indicated in purple, and nucleotides predicted by AlphaFold3 to directly interact with S100A7 are highlighted in blue. The interaction analysis was performed using ITC and direct ELISA (Figures S10–S13). (C, D) AlphaFold3 prediction of the tertiary structures of aptamer 17–43 alone (C) and in complex with the S100A7 homodimer (D).

To identify nucleotides critical for G4 formation and S100A7 recognition, we performed comprehensive single-nucleotide mutagenesis. Guanine bases within each predicted G-quartet were systematically substituted with cytosine (C1–C6 mutations; Figures B and S11). With the exception of mutation C6, located outside the predicted G-quartets, all substitutions abolished binding, confirming the essential role of guanine tetrads for aptamer functionality. We also examined whether stabilizing G-quartets by cytosine→guanine substitutions could enhance binding, but this approach did not yield improvement and, in some cases, disrupted target recognition (G1–G3 mutations, Figure S12).

Finally, we performed structural modeling of the aptamer and its complex with S100A7 using AlphaFold3. Aptamer 17–43 was predicted to interact primarily with the N-terminal region of S100A7, specifically engaging residues Q4 and R7 of both S100A7 monomers. These residues likely form hydrogen bonds and electrostatic interactions with nucleotides T17, T18, C29, and T30, thereby stabilizing the aptamer–protein complex (Figure B–D). We tested this prediction by mutating pyrimidine nucleotides at the predicted interface (mutations labeled A1–A6: T/C → A, Figure S13). Mutations at T17, T18, C29, and T30 completely abolished binding, confirming their critical role in direct S100A7 interaction, whereas substitutions at T16 (A1) and C28 (A4) had a minimal effect. Consistent with the structural model (Figure D), these bases are oriented away from the S100A7 surface.

Stability of Aptamers in Human Saliva

Next, we evaluated the stability of 17–82 and 17–43 aptamer variants in human saliva to assess their suitability for saliva-based heart failure diagnostics. Fluorescein-labeled aptamers were incubated in 25% unprocessed or membrane-filtered human saliva (molecular weight cutoff, MWCO, 30, 50, or 100 kDa), and integrity at 0, 0.5, 1, and 2 h was assessed by urea-PAGE (Figures and S14). After 2 h at room temperature in unprocessed saliva, 88% of aptamer 17–82 and 99% of the truncated 17–43 remained intact. Filtration through a 100 kDa MWCO membrane yielded similar results (93% and 99% intact aptamer for 17–82 and 17–43, respectively), whereas filtration at 30 or 50 kDa further improved stability to levels approaching those in the PBS control (∼100%). These findings are consistent with the molecular weight range (30–45 kDa) of common nucleases, including DNase I, which are effectively removed by lower MWCO filtration.

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Analysis of 17–82 and 17–43 aptamer stability in human saliva. Aptamers were incubated in PBS or PBS supplemented with 25% unfiltered or molecular-weight–filtered saliva (30, 50, or 100 kDa cutoffs) for up to 2 h (Figure S14).

The higher stability of truncated aptamer 17–43 compared with the full-length 17–82 can be attributed to its G-quadruplex motif, which confers increased structural rigidity and nuclease resistance by minimizing unstructured, enzyme-accessible regions present in the parent 17–82 aptamer (Figure H). The robust stability of 17–43 in unfiltered saliva over a 2 h time frame indicates that it is well suited for practical diagnostic applications, particularly since diagnostic assays typically use saliva at lower concentrations (≤5%).

Development of Aptamer-Based Sandwich ELISA Assay (Aptamer-ELISA)

Given the dimeric nature of the S100A7 protein, we tested whether aptamer 17 variants could serve as both capture and detection elements in a sandwich aptamer-ELISA format (Figures A, S15 andS16). In this assay, biotinylated capture aptamers were immobilized on streptavidin-coated plates, incubated with S100A7, and detected with fluorescein-labeled aptamers using horseradish peroxidase (HRP)-conjugated anti-FITC antibody. Assay specificity was confirmed using the scrambled control aptamer 17–43-SCR (Figure S15D); no detectable signal was observed for any combination of scrambled aptamers, even at high S100A7 concentrations (up to 3000 nM), demonstrating that signal generation arose exclusively from specific aptamer–target interactions (Figure S16A).

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Aptamer-based and hybrid aptamer-antibody ELISA assays for S100A7 detection in human saliva. (A) Schematic of the sandwich aptamer-ELISA employing biotinylated capture and reporter aptamers. (B) Analytical performance of the aptamer-ELISA using aptamers 17–43 and 17–82, with recombinant S100A7 spiked into 5% human saliva (see Figure S16 for extended data). (C) Schematic of the aptamer-antibody hybrid ELISA, combining a biotinylated capture aptamer and an HRP-conjugated S100A7 antibody. (D) Comparative performance of hybrid ELISA using aptamer variants 17–82, 17–43, 17–43-T10, and scrambled control 17–43-SCR. (E) Robustness of aptamer 17–43-based hybrid ELISA at varying saliva concentrations (full saliva-background analysis in Figure S18). (F) Limit of detection (LOD) for hybrid ELISA assays using aptamers 17–82 (left) and 17–43 (right), with S100A7 spiked into 0.1–1% saliva. (G) Performance of a commercial S100A7 ELISA kit. Error bars represent mean ± SD from 3–10 independent replicates.

We assessed the feasibility of detecting S100A7 in human saliva using full-length (17–82) and truncated (17–43) aptamers, which combine high affinity with cost efficiency compared with the longer dimeric 17–43-T10. Although 17–43-T10 gave the greatest sensitivity, its dual-label synthesis cost remains a limiting factor. Using 5% saliva spiked with recombinant S100A7, we obtained apparent K D values of 51 nM and 60 nM for 17–82 and 17–43, respectively (Figure B). The corresponding limits of detection (LOD) were 11.3 ng mL–1 (0.87 nM) and 7.4 ng mL–1 (0.57 nM), respectively. The assay exhibited a dynamic range of 108-fold for 17–82 and 165-fold for 17–43. Importantly, detection limits remained consistent across saliva concentrations from 0% to 25%, with LODs between 9.8–12 ng mL–1 (17–82) and 7.4–13 ng mL–1 (17–43), indicating minimal matrix interference (Figure S16B). Both assays showed excellent linearity (R 2 > 0.99), with working ranges of 11–1221 ng mL–1 (0.9–100 nM) for 17–82 and 7.4–1221 ng mL–1 (0.6–100 nM) for 17–43.

Development of an Aptamer-Antibody Hybrid ELISA Assay (Hybrid ELISA)

Previously reported antibody-based ELISA for salivary S100A7 achieved an LOD of 12.2 ng mL–1. Our aptamer-only ELISA provided improved sensitivity but remained insufficient for highly sensitive diagnostics. To further enhance analytical performance, we designed a hybrid ELISA combining aptamer-based capture with antibody-based detection.

In this assay, biotinylated aptamers were immobilized onto streptavidin-coated plates as in the aptamer-only assay, and detection of bound S100A7 was achieved using an HRP-conjugated anti-S100A7 antibody (Figures C,D, and S17). Aptamers 17–82, 17–43, and 17–43-T10, along with the scrambled control 17–43-SCR, were tested as capture reagents. The apparent K D values for 17–82, 17–43, and 17–43-T10 were 3.8, 4.1, and 3.6 nM, respectively. Unlike the aptamer-only ELISA (Figure S16A), which showed marked performance differences, the hybrid format revealed no significant advantage for the dimeric aptamer 17–43-T10, suggesting a possible competition between the antibody and the dimeric aptamer.

For subsequent evaluations, we selected the shortest aptamer, 17–43, and demonstrated consistent K D values (3.0–3.9 nM) in human saliva diluted 0–10%, confirming robust performance and minimal matrix interference (Figures E, S17, and S18). Direct comparison of 17–82 and 17–43 in 0.1–1% saliva showed that the aptamer–antibody pairing enhanced sensitivity by more than 2 orders of magnitude over the aptamer-only ELISA, achieving LODs of 16 pg mL–1 (1.2 pM) for 17–82 and 29 pg mL–1 (2.2 pM) for 17–43 in 0.1% saliva (Figures F and S17). The working ranges were 16–2607 pg mL–1 (17–82) and 29–6517 pg mL–1 (17–43), with dynamic ranges of 161 and 224, respectively. These values compare favorably with those of a commercial high-sensitivity immunoassay (LOD 161 pg mL–1; dynamic range 37; Figures G and Table S5). The hybrid ELISA also delivered superior accuracy (R 2 = 0.99 for both aptamers) relative to that of the commercial kit (R 2 = 0.95).

To evaluate long-term stability, hybrid-ELISAs were stored either precoated with capture aptamers (17–82, 17–43, or 17–43-T10) or uncoated for 2.5 months at room temperature or 4 °C (Figure S19). Assays performed after storage showed no loss of sensitivity or dynamic range compared with freshly prepared plates. Precoating shortened the assay time by ∼1.5 h, reducing the total time to 3 h, compared with ∼4–5 h for the commercial kits. Plates could be stored at RT for at least 2.5 months (Tables S5 and S6). Collectively, these results establish aptamer-antibody hybrid ELISA as a robust, cost-efficient, and highly sensitive platform for S100A7 detection in human saliva.

Analysis of Aptamer Specificity within the S100-Protein Family

The S100 proteins constitute a family of homologous, small calcium-binding proteins involved in calcium homeostasis, proliferation, differentiation, and inflammatory responses (Figures A and S20). ,− To assess the specificity of 17–43 toward selected S100 proteins (S100A5, S100A8, and S100A9), we performed ITC analysis. Aptamer 17–43 displayed high-nanomolar affinity for its cognate target S100A7 (K D = 100 nM), moderate affinity for S100A5 (K D = 183 nM), and markedly weaker binding to S100A8 (K D = 1260 nM), while no measurable interaction was detected with S100A9 (Figure B,C).

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Analysis of aptamer specificity toward S100 homologues. (A) Structures of S100-family proteins tested: homodimers S100A7 (PDB: 1PSR) and S100A5 (PDB: 6WN7), and the heterodimer S100A8/A9 (PDB: 7QUV). (B) ITC analysis of aptamer 17–43 binding to S100A5, S100A7, S100A8, and S100A9 was performed at 20 °C in PBSM buffer using 10 μM protein in the cell and 100 μM aptamer in the syringe. (C) Summary of thermodynamic parameters derived from ITC binding isotherms; full thermograms are shown in Figure S20. (D) Sandwich aptamer-ELISA performed by sequential incubation of capture and detection 17–43 aptamers (500 nM each) with serial dilutions of recombinant S100 proteins, followed by detection using an HRP-conjugated anti-FAM antibody. (E) Aptamer–antibody hybrid ELISA combining 17–43 (500 nM) as the capture aptamer with an anti-S100A7 monoclonal antibody (1:2000 dilution) for signal generation under identical assay conditions.

We next evaluated the specificity of both aptamer-only and hybrid sandwich ELISA assays using aptamer 17–43. Despite the measurable affinity for S100A5 and S100A8 observed in ITC, both ELISA formats exhibited exclusive specificity for S100A7, showing no detectable cross-reactivity with other S100 family members (Figure D,E). The enhanced selectivity of sandwich ELISAs likely reflects the requirement for simultaneous dual binding in which the target must engage both capture and detection elements (aptamer or antibody), each recognizing a distinct epitope. This dual-epitope recognition minimizes nonspecific interactions and background noise, accounting for the superior specificity of these assays compared with single-site binding methods such as ITC. Consequently, both the aptamer and hybrid ELISA sensors can be regarded as highly specific and sensitive monoclonal aptamer-based immunoassays for S100A7 detection.

Application of Biolayer Interferometry for Salivary S100A7 Quantification

Encouraged by the excellent performance of our selected aptamers, we explored their use in a BLI assay to enable rapid, real-time quantification of S100A7 (Figures A and S21A). We first measured the binding of biotinylated aptamers 17–82, 17–43, and the dimeric 17–43-T10 to S100A7 under optimized conditions (Figures S21 and S22). BLI analysis yielded K D values of 11 nM for 17–82, 14 nM for 17–43, and 1.9 nM for 17–43-T10 (Figure B). Despite its higher affinity, dimeric 17–43-T10 showed a lower maximal response (Bmax), likely due to steric hindrance.

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Detection of S100A7 using aptamer-based biolayer interferometry. (A) Schematic of the BLI assay for aptamer-based detection of S100A7. (B) Representative BLI assay showing interactions of aptamer variants 17–82, 17–43, and 17–43-T10 with S100A7 in buffer (all raw sensorgrams are provided in Figure S22). (C) BLI sensorgrams of aptamer variants measured in 2.5% filtered human saliva spiked with recombinant S100A7. (D) Linear regression analysis of the response signals from panel (C), used to determine assay parameters for each aptamer variant (full analysis shown in Figure S23). (E) Comparison of BLI responses obtained using aptamer 17–43 for saliva samples from patients with acute heart failure (n = 3) and healthy controls (n = 3). Samples were analyzed in 3–4 technical replicates using untreated saliva diluted to 2.5% in assay buffer. Individual response values for each donor are plotted, showing clear separation between HF (mean 2711 ng mL–1) and control (mean 459 ng mL–1) groups. (F) Comparison of mean S100A7 concentrations detected in saliva from HF patients using BLI, validated against measurements obtained with a commercial high-sensitivity ELISA kit performed concurrently.

To evaluate the assay performance in a physiologically relevant matrix, we spiked S100A7 into 2.5% human saliva across a linear 0–10 nM range (Figure C,D). Under these conditions, apparent KD values increased slightly to 11 nM (17–82), 17 nM (17–43), and 22 nM (17–43-T10). Linear regression analysis defined LOD values of 0.23 nM for 17–82, 0.25 nM for 17–43, and 0.39 nM for 17–43-T10, with corresponding dynamic ranges of 106, 102, and 64, respectively (full analysis in Figures S22 and S23), confirming the sensitivity and robustness of the BLI platform in saliva-containing samples.

Finally, we assessed the potential clinical utility of the 17–43-based BLI sensor using unprocessed saliva from patients with acute heart failure (n = 3) and healthy volunteers (n = 3), collected as described in the Supporting Information. The assay achieved good reproducibility (intra-assay CV 1–11%; interassay CV 3–14%) and showed excellent correlation with the commercial S100A7 ELISA (Figures E,F and S24). Although the cohort size precludes formal diagnostic analysis, HF and control samples exhibited clearly distinct S100A7 levels (mean 2711 vs 459 ng mL–1, respectively), demonstrating feasibility for real-sample quantification. A comparative summary of the analytical performance of the 17–43 aptamer across the ELISA and BLI assay formats is provided in Table .

1. Summary of 17-43-Based ELISA and BLI Sensor Performance in Human Saliva.

assay saliva (%) K D, nM (R 2) LOD, nM (ng mL–1) dynamic range
aptamer-ELISA 5 60.4 (0.99) 0.57 (7.4) 165
hybrid ELISA 0.1 3.1 (0.99) 0.002 (0.029) 224
BLI 2.5 2.1 (0.99) 0.25 (3.2) 102
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Discussion

Heart failure remains a major global health challenge due to the lack of early detection methods, leading to high morbidity, mortality, and hospital readmission rates. , Current diagnostics rely on invasive and resource-intensive techniques, limiting their suitability for routine monitoring. The salivary S100A7 protein has emerged as a potential biomarker for heart failure, and biosensors targeting this marker offer a promising alternative for detection in clinical and point-of-care settings.

In this study, we used the SELEX strategy to identify and characterize the first high-affinity DNA aptamer targeting S100A7. After ten rounds of selection, we observed the enrichment of a dominant sequence, designated aptamer 17–82. This aptamer was subsequently truncated to 43 nucleotides (17–43), which enhanced its binding affinity from 63 to 27 nM. Further dimerization of the 17–43 sequence improved the affinity to 5.5 nM. Experimentally validated structural modeling indicated that 17–43 adopts a G-quadruplex (G4) conformationa structural motif likely responsible for the aptamer’s stability and resistance to degradation in human saliva.

The utility of aptamer 17–43 was tested in a range of binding assays. A sandwich Aptamer-ELISA, employing aptamers as both capture and detection elements, achieved a limit of detection of 7.4 ng mL–1 (0.57 nM) in 5% human saliva (Table ). Incorporation of an S100A7 antibody into the assay lowered the LOD to 29 pg mL–1 (2 pM) in 0.1% human saliva, providing a 6-fold improvement over commercial antibody-based ELISA kits (161 pg mL–1 or 14 pM). Notably, both ELISA formats demonstrated exceptional selectivity, with no cross-reactivity observed for other S100-family proteins, including S100A5, S100A8, and S100A9, and no binding to other saliva components. The hybrid ELISA retained full functionality after 2.5 months of storage, favorably compared with antibody-based assays.

We demonstrated the integration of aptamer 17–43 into a label-free biolayer interferometry (BLI) platform, enabling the detection of S100A7 in saliva. The BLI sensor achieved an LOD of 3.2 ng mL–1 (0.25 nM) in 2.5% saliva, with rapid response kinetics, allowing detection in under 20 min. This approach offers a highly sensitive and quantitative alternative to traditional immunoassays and may be particularly useful for high-throughput screening applications.

In summary, this study provides strong evidence that aptamer-based biosensors are a viable alternative to traditional immunoassays for HF diagnostics. The discovery of a high-affinity S100A7-specific aptamer marks an important step toward the creation of saliva-based HF monitoring tools. Since S100A7 is an inflammatory mediator elevated in various pathophysiological conditions, − aptamer-based tests like those described here could, in theory, be adapted for other disease contexts in which S100A7 is involved; however, exploring these applications is beyond the scope of this study.

While larger clinical studies are needed to fully establish diagnostic accuracy, the results presented clearly demonstrate feasibility and promising analytical potential. Future work should focus on clinical validation with larger, more diverse populations, assessing native S100A7 variability, sensor multiplexing, and integration of these platforms into portable devices for real-time patient monitoring and early disease detection. Developing such aptamer-based systems could greatly improve the accessibility, cost-effectiveness, and precision of cardiovascular diagnosticsultimately leading to better patient outcomes and easing the burden on healthcare systems.

Experimental Section

Oligonucleotides

All DNA oligonucleotides were synthesized by Integrated DNA Technologies with a standard desalting purity (Table S1). Samples were dissolved in molecular biology-grade water, and their concentrations were measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific).

Isothermal Titration Calorimetry (ITC) Experiments

ITC experiments were conducted using a MicroCal PEAQ-ITC instrument (Malvern) at 20 °C, with different buffer compositions detailed in Supporting Table S7. Typically, aptamers were prepared in PBSM, heated to 95 °C for 5 min, and gradually cooled down to 4 °C over 30 min before analysis. Each ITC experiment was performed with 40 μL of 100 μM aptamer solution loaded into the syringe and 280 μL of 10 μM protein solution (S100A7, S100A5, S100A8, or S100A9) loaded into the cell. An initial purge injection of 0.4 μL was followed by 19 successive injections of 2 μL with 150 s intervals between injections, under stirring at 750 rpm. Raw data were corrected for dilution heat and analyzed using MicroCal Analysis software, with data fitted to a single-site binding model.

Biolayer Interferometry (BLI) Analysis

BLI was used to determine the binding affinity (K D) and kinetic parameters (k on and k off) of aptamer variants and to develop a BLI-based sensor for detecting S100A7 in human saliva samples. All experiments were performed using the Octet RED96 system (Fortebio) with streptavidin-coated Octet SA Biosensors (Sartorius) at 25 °C in 96-well black flat-bottom OptiPlate microplates (Revvity Health Sciences Inc.; Cat. No. 6065400), with a total volume of 200 μL per well.

For kinetic measurements, biotinylated aptamers with different 5′- or 3′-biotin tags (/5Biosg/, /5BiotinTEG/, or/3BioTEG/) were renatured at 50 nM in PBSM-TB buffer supplemented with 0.05% Tween-20 and BSA to reduce the level of nonspecific sensor binding. Biosensor tips were equilibrated in PBSM-TB buffer for 60 s (baseline) before being transferred to aptamer solutions (loading step, 300 s). Unbound aptamers were removed by incubating biosensor tips in a PBSM-TB buffer for 60 s (baseline correction).

The association phase was conducted by immersing biosensors in wells containing serial dilutions of recombinant S100A7 (0 to 1000 nM) in PBSM-TB buffer or PBSM-TB buffer supplemented with human S100A7-free saliva (0–10%). The dissociation phase was initiated by dipping biosensors into PBSM-TB buffer with both association and dissociation recorded for 300 s. Binding kinetics were analyzed using Data Analysis HT 9.0 software with a 1:1 global binding model. The K D was determined by plotting S100A7 concentration vs response (nm) and analyzed using GraphPad Prism software.

Stability of Aptamers in Human Saliva

The FAM-labeled 17–82 and 17–43 aptamers (2 μM each) were incubated in either PBS buffer or 25% unfiltered or 30, 50, or 100 K MWCO-filtered human saliva at RT in a 100 μL reaction volume. Aliquots (10 μL) were collected at 0, 0.5, 1, and 2 h, quenched by adding 10 μL of 2× gel loading dye, denatured at 95 °C for 10 min, and stored at −20 °C before analysis via 10% denaturing Urea-PAGE. Samples were visualized using a ChemiDoc MP Imaging System (Bio-Rad, Alexa Fluor 488 filter), and band intensities were quantified using Image Lab Software (Bio-Rad).

A detailed description of the in vitro selection procedure, ELISA assay formats, protein expression and purification, saliva collection protocols, and quantification of assay performance parameters is provided in the Experimental Section of the Supporting Information.

Supplementary Material

ao5c11711_si_001.pdf (3.4MB, pdf)

Acknowledgments

The authors thank Christopher Howard for providing access to the BLI instrument, Victoria Coyne for assistance with NGS data collection, and Zhong Guo and Zhenling Cui for sharing plasmid constructs. We also acknowledge Xi Zhang for generously providing saliva samples. Finally, we are grateful to Patricia Walden and Maria Micaela Fiorito for their invaluable technical support, laboratory assistance, and management. This work was partially funded by ARC Centre of Excellence in Synthetic Biology CE200100029 to K.A. and A.B., ARC Linkage grant 200200916, and NHMRC Investigator grant APP 2033951 to K.A. K.A. gratefully acknowledges the financial support of CSIRO-QUT Synthetic Biology Alliance as well as the support of CSIRO Synthetic Biology and Advanced Engineering Biology Future Science Platforms. This study is funded by the National Health and Medical Research Council (APP 2002576) funding awarded to C.P. C.P. is currently receiving research funding from the National Health and Medical Research Council (APP 2012560), the Australian Research Council (IH240100013 and DP250101156), the Garnett Passe and Rodney Williams Foundation, Gallipoli Medical Research Foundation, Metro North Collaborative Grant Scheme, Tour De Cure and the Royal Brisbane Women Hospital Foundation.

Glossary

Abbreviation

A10

ten-adenosine spacer linker in dimeric aptamer constructs

BLI

bio-layer interferometry

ELISA

enzyme-linked immunosorbent assay

FAM

6-carboxyfluorescein

G4

G-quadruplexex

HF

heart failure

HRP

horseradish peroxidase

ITC

isothermal titration calorimetry

LOD

limit of detection

MWCO

molecular weight cut-off

PBS

phosphate-buffered saline

PDB

protein data bank

POC

point-of-care

SELEX

systematic evolution of ligands by exponential enrichment

T10

ten-thymidine spacer linker in dimeric aptamer constructs

TMB

3,3′,5,5′-tetramethylbenzidine

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11711.

  • Detailed experimental materials and methods, supporting figures and tables referenced in the text, comprehensive protein and oligonucleotide sequence data, and additional information on biophysical and binding analyses (PDF)

⊥.

Chacabuco 960, Quillota 2260000, Valparaíso, Chile

The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. A.B., K.A., and E.E. conceived and designed the research; T.S., M.B.S., R.M., and E.E. performed the experiments; C.P. provided and coordinated saliva sample collection; T.S. and E.E. analyzed and interpreted the data; C.P., K.A., and E.E. wrote and revised the manuscript.

The authors declare no competing financial interest.

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Supplementary Materials

ao5c11711_si_001.pdf (3.4MB, pdf)

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