Electronic Immunoassay Using Enzymatic Metallization on Microparticles

Abstract

We present here an inexpensive method for generating a sensitive direct electronic readout in bead-based immunoassays without the use of any intermediate optical instrumentation (e.g., lasers, photomultipliers, etc.). Analyte binding to capture antigen-coated beads or microparticles is converted to probe-directed enzymatically amplified silver metallization on microparticle surfaces. Individual microparticles are then rapidly characterized in a high-throughput manner via single-bead multifrequency electrical impedance spectra captured using a simple and inexpensive microfluidic impedance spectrometry system we develop here, where they flow through a three-dimensional (3D)-printed plastic microaperture sandwiched between plated through-hole electrodes on a printed circuit board. Metallized microparticles are found to have unique impedance signatures distinguishing them from unmetallized ones. Coupled with a machine learning algorithm, this enables a simple electronic readout of the silver metallization density on microparticle surfaces and hence the underlying analyte binding. Here, we also demonstrate the use of this scheme to measure the antibody response to the viral nucleocapsid protein in convalescent COVID-19 patient serum.

Figure 1

(A) Key steps for immunobinding and enzymatic metallization using a model assay. Protein A/G is attached as an antigen to carboxyl groups by NHS-EDC chemistry. HRP-conjugated antibodies bind to immobilized antigens on the bead surface. The HRP enzyme catalyzes metal deposition. (B) SEM image of a carboxyl-functionalized bead as received from the vendor. (C, D) SEM images of beads after metal deposition, with PBS and mIgG-HRP as probes, respectively.

Figure 2

(A) Microfluidic impedance sensing in a tapered aperture. (B) Magnitude of the electric field at the aperture. (C) SEM image of the 3D-printed aperture. (D) Components of the microfluidic system. (E) Equivalent circuit and schematic of the electronic measurement. (F) Example measured impedance “waveform” of a passing polystyrene bead. The peak impedance change, ΔZ_p, is indicated.

Figure 3

(A, B) Peak impedance (ΔZ_p) and phase change (Δϕ_p) at two selected frequencies for different bead sizes and corresponding simulation results. (C, D) Impedance and phase change spectrum for different bead sizes. All aggregate measurements reported here are for 60 beads each.

Figure 4

(A, B) Peak impedance change magnitude (ΔZ_p) spectra of nonmetallized and metallized beads. (C) Mean ΔZ_p spectrum of nonmetallized and metallized beads. (D, E) Peak impedance phase change (Δϕ_p) spectra of nonmetallized and metallized beads. (F) Mean Δϕ_p spectrum of nonmetallized and metallized beads.

Figure 5

(A) Peak impedance change magnitude (ΔZ_p) spectra of beads with impedance magnitudes less than 0 at 150 khz, 380 kHz, or 2 MHz. (B) Subpopulation from panel A of impedance magnitude spectra with a large decrease from 45 to 150 kHz. (C) Subpopulations in panels A and B overlaid with the remaining population. (D, E, F) Peak phase change (Δϕ_p) spectra corresponding to populations in panels A, B, and C, respectively.

Figure 6

(A) Steps for creating training bead sets. (B, C) SEM images of high-metal and low-metal training beads, respectively. (D) Features used in the LDA classifier. (E) Projection of the principal component. (F) Serial dilution curve (0–200 nM) for obtaining the limit of detection of BEAD-EM for the HRP-labeled probe. The metallization metric for low- (blue dotted line) and high-metal (red dotted line) training sets and the 0 nM probe concentration (black dotted line) is shown. The fitted 4PL curve is also shown. (G, H) SEM images of bead trials at varying probe concentrations. (I) Limit of detection of fluorescent probes measured by traditional flow cytometry. 0 nM trial is shown by the black dotted line. The fitted 4PL curve is also shown.

Figure 7

(A) Key steps for the immunoassay for identifying antinucleocapsid IgG in serum from COVID+ patients and healthy prepandemic serum. (B, C) Resulting bead metallization from COVID+ and patient serum, respectively. (D) Features used in the LDA classifier. (E) Projection of the principal component of COVID+ and healthy samples. (F) Final diagnostic measure for COVID+ or healthy serum classification.