Antigen-Mimic Nanoparticles in Ultrasensitive on-Chip Integrated Anti-p53 Antibody Quantification

Abstract

As a tumor-suppressing protein, p53 plays a crucial role in preventing cancer development. Its utility as an early cancer detection tool is significant, potentially enabling clinicians to forestall disease advancement and improve patient prognosis. In response to the pathological overexpression of this antigen in tumors, the prevalence of anti-p53 antibodies increases in serum, in a manner quantitatively indicative of cancer progression. This spike can be detected through techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation. In this study, we present an electrochemical approach that supports ultrasensitive and highly selective anti-p53 autoantibody quantification without the use of an immuno-modified electrode. We specifically employ antigen-mimicking and antibody-capturing peptide-coated magnetic nanoparticles, along with an AC magnetic field-promoted sample mixing, prior to the presentation of Fab-captured targets to simple lectin-modified sensors. The subfemtomolar assays are highly selective and support quantification from serum-spiked samples within minutes.

Keywords: antigen-mimicking peptide; cancer detection; electrochemical enzyme-amplified assay; nanoparticle-assisted immunoisolation; p53.

Figure 1

Schematic depiction of the nanoparticle-assisted immunocapture and downstream electrochemical enzyme-amplified assay for the DO-1 antibody quantification. The synthesized antigen-mimic was covalently tethered to IONP surfaces, along with HRP (bottom left). An AC magnetic field was employed to enhance mixing of the capture IONPs with 100 μL samples within a microfluidic channel (upper left). After immunocapture, the target–nanoparticle complexes were manually transferred to 200 μL microplate wells, encompassing Con A-modified screen-printed carbon electrode (SPCE) interfaces (upper right). In the presence of the tetramethylbenzidine substrate (TMB) and H₂O₂, a concentration-dependent SWV response is then generated (bottom right).

Figure 2

(a) SPR peptide specificity assessments of the synthesized DO-1 sequence (CQETFSDLWKLLPENNVL) and control peptides, when exposed to 10 μg/mL DO-1 antibody. Control peptide 1 (SPDDIEQWFT) was a nontarget p53 epitope (amino acids 46–55 on the p53 protein). Control peptide 2 (CPPPPEKEKEK) served as a non-p53 control. (b) Comparative binding affinity assessment between Con A and DO-1 antibody as assessed by electrochemical impedance spectroscopy (EIS, blue) and SPR (red). SPR analyses were at Con A-saturated gold chips, with a BSA-saturated surface as the reference. EIS measurements were conducted at Con A-modified SPCEs in a 5 mM [Fe(CN)₆]^3/4– PBS solution (pH = 7.4). The impedimetric and SPR responses were both fitted to Langmuir–Freundlich isotherms (R²_SPR = 0.993 vs R²_EIS = 0.999, K_{d,ConA-DO-1antibody} = 13.6 ± 2.3 nM from the plot mean). Impedimetric error bars represent the standard deviation from three individual electrodes (n = 3).

Figure 3

(a) Comparative SWV voltammetric responses to 50.0 ng/mL DO-1 antibody versus serum under three incubation conditions (10 min static recruitment), 10 min shaking, or magnetic mixing (10 min MAC immunoisolation) demonstrating the advantages associated with magnetic field-assisted recruitment. Static recruitment involved incubating samples with bioreceptive IONPs for 10 min. Shaking employed a standard lab vortex mixer. MAC immunoisolation was performed within a microfluidic channel (see Experimental Section) under an optimized AC magnetic field (see below). (b) The effect of nanoparticle oscillation frequency (up to 2.0 Hz) and AC magnetic field strength (adjusted by electromagnetic potential) on microfluidic DO-1 antibody immunoisolation. Recruitment efficiency was normalized to 100% using the SWV current response of bioreceptive IONPs capturing 1 ng/mL DO-1 antibody as acquired under optimized microfluidic isolation conditions (i.e., electromagnetic potential = 11.5 V; nanoparticle oscillation frequency = 2.0 Hz; flow rate = 50 μL/min).

Figure 4

SWV assessment of the effect of IONP surface composition on sensing performance when exposed to 10 μg/mL DO-1 antibody. The red line represents a quadratic correlation between IONP peptide coverage and the resulting electrochemical signal with an R² of 0.95. The black square indicates the equivalent surface coverage and peak current of the full antigen protein. SWV measurements were conducted in 100 μL of TMB substrate solution, ranging from +1.0 V to −0.5 V (vs Ag/AgCl) with an amplitude of 20 mV, a potential step of 5 mV, and a frequency of 50 Hz. All error bars represent the standard deviation across three individual recruitments (n = 3).

Figure 5

(a) Voltammetric response of Con A-modified SPCE interfaces to the target DO-1 antibody as recruited by DO-1 peptide/HRP-IONPs (black) and control, nonreceptive HRP-IONPs (red), respectively (black line representing a linear fitting, R² = 0.99). Two regimes (healthy control and disease) were defined using literature values, extrapolated to the calibration curve. (b) Comparison of sensor response to target antibody (immunoisolation under the specified optimized MAC conditions) for the target specific peptide (i.e., DO-1 peptide) and full antigen capture particles. All error bars depict the standard deviation from three individual electrodes (n = 3).

Figure 6

Recovery analysis across clinically relevant concentrations of DO-1 antibody spiked into 100 μL of 1.0% human serum. All error bars represent the standard deviation across three individual electrodes (n = 3).