Fig 1: Blind comparison of CRP concentrations (A) and cTnI concentrations (B) estimated using the shotgun protein-tagging assay against a reference immunoassay for 12 patient samples (asterisks represent samples where cTnI levels were lower than the immunoassay LOD, i.e., <2 pg/mL). Error bars represent standard deviations from two independent measurements on two different electrodes.
Fig 2: Representative Langmuir–Freundlich fits from online multiplexed assays for (A) CRP and (B) cTnI both spiked in 1% human serum in 0.1 M MES buffer. Insets represents linear semi-log correlation with correlation coefficients (R2) of 0.978 for CRP and 0.993 for cTnI. Error bars represent standard deviations between three independent measurements on three different electrodes.
Fig 3: Langmuir–Freundlich fits from offline multiplexed protein assays as detected from mixed samples of both proteins at a CRP-responsive sensor (A) and a cTnI-responsive sensor in dilute serum (B). Respective LODs are 1.0 pg/mL for CRP and 0.6 pg/mL for cTnI. Insets depict associated linear semi-log correlation plots with correlation coefficients (R2) of 0.95 and 0.94 for CRP and cTn1, respectively. Error bars represent standard deviations between three independent measurements on three different electrodes.
Fig 4: Langmuir–Freundlich fits of (A) CRP spiked in 1% human serum in MES buffer and (C) cTnI spiked in 1% human serum in MES buffer. Representative DPV peaks as function of (B) increasing CRP connection on anti-CRP-decorated GCE sensors and (D) cTnI at anti-cTnI sensors. Bottom DPV voltammograms in (B) and (D) are the background signals from incubation with unspiked 1% HS. Error bars represent standard deviations between three independent measurements at three different electrodes. Insets in (A) and (C) are associated linear semi-log correlation trends with correlation coefficients (R2) of 0.99 and 0.97, respectively.
Fig 5: Electrochemical specificity analysis for (A) anti-CRP-modified electrodes and (B) anti-cTnI-modified electrodes in offline single protein analyses. The response (current density) to a large excess of interfering species is <10% of the target-specific signal at anti-CRP interfaces and <20% across both surfaces with all interferents, indicative of good specificity and low cross-reactivity (without the need for complex surface chemistry). Insets show the response of the anti-CRP-modified electrode (A) and the anti-cTnI-modified electrode (B) upon repetitive exposure to MB-labeled 1% fetal bovine serum. Such analyses confirm that the signal is target-specific with minimal (consistently less than the assay LOD (blank + 3 × SD)) contribution from interfering species. Error bars represent standard deviation from three independent measurements at three different electrodes. Note that specificity is yet further improved under flow (Figure S8).
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