E-DNA scaffold sensors and the reagentless, single-step, measurement of HIV-diagnostic antibodies in human serum

Antibodies are critical diagnostic biomarkers, yet quantitative measurement at the point of care remains limited. ELISA, the current gold standard for antibody quantification, offers excellent analytical performance and parallelization but is slow, multi-step, and requires specialized equipment. Lateral flow immunoassays are rapid and simple but largely qualitative and poorly multiplexed. A technology that combines ELISA-like quantification and multiplexing with point-of-care convenience could significantly improve serological diagnostics. The authors previously developed reagentless, single-step E-DNA scaffold sensors that are quantitative, easily multiplexed, rapid (<10 min), and simple. The platform uses a gold electrode with a DNA strand bearing a 3' redox reporter and a 5' thiol for surface attachment. Hybridization to a complementary strand presenting an antibody-binding epitope forms a rigid double-helical scaffold tethered by a flexible linker. In the absence of target, the reporter approaches the electrode to enable electron transfer; antibody binding imposes steric hindrance that reduces electron transfer, producing a signal quantitatively related to target concentration. Prior work characterized analytical performance using monoclonal antibodies; here the study evaluates clinical performance using human serum, focusing on HIV-diagnostic antibodies against gp41 epitopes, and compares E-DNA sensors with commercial ELISA and lateral flow assays.

- Clinical samples: 15 anonymized, commercially sourced human serum samples (10 putatively HIV-positive, 5 putatively HIV-negative). - Comparator assays: Commercial ELISA (HIV1/2 Ab ELISA Kit) and commercial lateral flow immunoassay (UniGold Recombigen HIV-1/2). ELISA readout at 450 nm absorbance; lateral flow band intensities quantified using ImageJ (Analyze/Plot Profile). Workflow/time documented: ELISA >2 h with >20 steps; lateral flow ~10 min, 2 steps, qualitative with a narrow read window (10–12 min). - E-DNA scaffold sensor construction: Gold electrodes modified with a thiolated DNA anchor strand bearing a 3' redox reporter and 5' thiol for immobilization. A complementary oligonucleotide (PNA-epitope chimera) hybridizes to form a rigid duplex scaffold presenting an HIV gp41 epitope at the distal end. Square wave voltammetry monitors signaling current; antibody binding reduces electron transfer due to steric hindrance. - Targeted HIV gp41 epitopes: Three epitopes spanning a range of immunogenicities selected from literature: 483 (LVCKSGKIVCT; high immunogenicity), 2F5 (ELDKWAASWNC; low), and 4E10 (NWDFTIVMIVKRIKKK; near-zero). In parts of the text, the highly immunogenic epitope is referred to as 4B3; this corresponds to epitope 483. - Novel ELISA mimicking E-DNA architecture: Streptavidin-coated 96-well plates presenting a biotinylated DNA anchor hybridized to the complementary PNA-epitope chimera; detection via HRP-labeled secondary antibody with colorimetric readout at 450 nm. Used to validate immunogenicity of selected epitopes using the same serum set. - E-DNA assay conditions: Serum typically diluted 1:80 in PBS for screening. For analytical titrations, the most reactive HIV-positive serum for 2F5 (UID: 403384) was serially diluted while maintaining overall serum at 1:40 by supplementing with HIV-negative serum and PBS to assess dynamic range and effective titers. Performance compared against the novel ELISA under analogous conditions. - Data analysis: Signal change upon antibody binding quantified as percentage decrease in square wave voltammetry peak current. ROC analysis performed to compare clinical performance among ELISA, E-DNA, and lateral flow (reported AUC values).

- Ground-truth performance of commercial assays on 15 samples: ELISA and lateral flow each achieved 90% clinical sensitivity and 100% specificity; both flagged the same putatively HIV-positive sample (UID: 200760) as negative. - Epitope immunogenicity (novel ELISA with clinical sera): Epitope 483 (high immunogenicity) yielded positive responses in 9/10 HIV-positive and 0/5 HIV-negative; epitope 2F5 (low) positive in 2/10 HIV-positive; epitope 4E10 (near-zero) showed no significant signal in any sample. - E-DNA clinical screening: Sensors displaying the high-immunogenic epitope (483/“4B3”) detected antibodies in 9/10 HIV-positive samples (the same outlier negative by ELISA/LFIA). Sensors targeting 4E10 showed no significant response, matching ELISA. At 1:80 serum dilution, sensors targeting 2F5 did not detect positives seen by ELISA (2/10), indicating lower analytical sensitivity for this weak epitope under these conditions. - Signal magnitudes: Average E-DNA signal change for the high-immunogenic epitope in HIV-positive sera ~15%. Demonstrative addition of 10 nM target antibody can yield ~30% current decrease. - ROC analysis (AUC): ELISA 0.96; E-DNA scaffold sensor 0.92; lateral flow immunoassay 0.90. E-DNA outperforms lateral flow in clinical discriminability and approaches ELISA. - Analytical sensitivity/dynamic range (titer at ~10% of saturating response): ELISA: epitope 483 starts ~1:72,000; epitope 2F5 ~1:960; epitope 4E10 none. E-DNA: epitope 483 starts ~1:14,400; epitope 2F5 ~1:40; epitope 4E10 none. ELISA exhibits lower detection limits, but E-DNA still provides quantitative, clinically relevant results rapidly and in a single step. - Operational performance: E-DNA achieves required clinical performance (targeting 90% sensitivity and 100% specificity in this set), quantitative readout, two or fewer steps, <12 min, and no fixed read window, combining ELISA-like quantification/multiplexing with point-of-care convenience.

The study addresses the need for rapid, quantitative point-of-care serology by demonstrating that E-DNA scaffold sensors can match the clinical performance of established assays while offering major workflow advantages. Using authentic HIV-positive/negative sera, E-DNA sensors targeting gp41 epitopes provided clinical sensitivity and specificity comparable to ELISA and lateral flow, with ROC performance intermediate between them and superior to lateral flow. Although ELISA retains superior analytical sensitivity due to enzymatic amplification and wash steps, this did not translate into superior clinical classification on the tested cohort. The E-DNA platform delivers quantitative results in minutes, with minimal steps and without strict timing constraints, directly addressing limitations of lateral flow assays (qualitative output, subjective band reading, narrow read window) and the complexity of ELISA. The epitope-specific findings underscore that clinical utility depends on immunogenicity: high-immunogenic epitopes (483) yield robust detection, whereas low/non-immunogenic epitopes (2F5, 4E10) challenge analytical sensitivity, particularly at practical serum dilutions. Overall, E-DNA scaffold sensors show strong potential to improve point-of-care serology by merging quantification and multiplexing with speed and ease of use.

E-DNA scaffold sensors enable rapid, reagentless, single-step, quantitative detection of HIV-diagnostic antibodies directly in human serum, achieving clinical sensitivity and specificity comparable to commercial ELISAs and lateral flow assays while providing faster, simpler operation and quantitative output. Despite ELISA’s superior analytical sensitivity, E-DNA’s clinical performance, speed (<12 min), minimal steps, and lack of fixed read windows make it well-suited for point-of-care diagnostics. Future work should include larger, more diverse clinical cohorts; optimization to enhance analytical sensitivity for weakly immunogenic epitopes (e.g., assay architecture, reporter/measurement parameters, signal amplification strategies compatible with one-step use); expanded multiplexing across additional HIV epitopes and other pathogens; and validation in real-world clinical workflows.

- Small sample size (15 sera) limits generalizability and precise estimates of sensitivity/specificity. - Epitope dependence: Strong performance hinges on highly immunogenic epitopes (e.g., 483); detection of low/non-immunogenic targets (2F5, 4E10) was limited, with E-DNA failing to detect 2F5 positives at 1:80 dilution. - Analytical sensitivity is lower than ELISA, shifting dynamic ranges to higher concentrations and potentially missing low-titer antibodies without optimization. - Potential discrepancy in specificity at certain operating conditions (e.g., a reported response in one HIV-negative sample for the high-immunogenic epitope) indicates thresholding and dilution conditions may affect classification. - Study relies on commercial designation of sample HIV status and small reference set; one "putatively positive" sample may have been misclassified.