Single-Response Duplexing of Electrochemical Label-Free Biosensor from the Same Tag

Abstract

Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low-cost ultra-dense electrochemical chips, the unprecedented single-response multiplexing of typical label-free biosensors is reported. Using a cheap, handheld one-channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi-reference electrodes, Au (0 V) and Ag/AgCl ink (+0.2 V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non-spatially resolved single-response multiplex strategies. As proof of concept, peptide-tethered immunosensors are demonstrated to provide the duplex detection of COVID-19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high-throughput and multi-analyte platforms, as it can be tailored to other biosensing devices and formats.

Keywords: accuracy; multiplexed detection; serology; single‐channel potentiostat; square wave voltammetry; steric hindrance; throughput.

Figure 1

One‐tag electrochemical duplexing. Schematic representation considering Fe^3+/2+ probe and peptide‐tethered biosensors to detect IgG^S. The spatially resolved binding events between the peptide and IgG^S on WE, which are illustrated in an enlarged view, can be assessed from distinguishable SWV peaks due to the distinct Φ of Au (Φ _Au) and Ag/AgCl (Φ _Ag) QREs. The directions of WEs and QREs are indicated by red and blue arrows, respectively. Orange circles indicate the tag and an image shows the charge‐transfer pathway of Fe^3+/2+, that is, OSP. M means metal. Scale bar: 6 mm.

Figure 2

Vertical meshed devices. A) Major microfabrication steps, covering the fabrication of WE fingers on a wafer, their coating with SU‐8, and the patterning of top QREs. The detection areas and spacing between WE and QRE were photolithographically defined by SU‐8. The layers composing the chip and the cross‐sectional view of a sensor are also illustrated. Apart from the 4 × 45 µm as considered in these images, 800 µm WEs were also tested. B) Chips with 830 (1) and 48 sensors (2), along with the amplified stereoscopy image of a sensor with 4 × 45 µm WEs (3). Scale bars: 10 mm (1) and 50 µm (3). C) Characterization of the plasma‐induced effects on WE and SU‐8 by AFM‐IR. Picture showing chips exposed to plasma (1), image by scanning electron microscopy (SEM) of a sensor with the four areas of WE (yellow) and SU‐8 (white) scrutinized by AFM‐IR being highlighted (2), and average IR spectra on WE (3) and QRE (4) before (red) and after (cyan) plasma exposition. Scale bar: 50 µm (2). Contact angles for water drops after 0, 15, and 30 days exposed to plasma are also shown, along with an SEM image of plasma‐treated SU‐8 (scale bar: 100 nm). D) Chip with 48 sensors and illustration of a generic potentiostat circuit, with the contact pads of Au QRE and WE to trigger the highlighted under‐droplet (10 µL) sensor. Scale bar: 6 mm. E) Preliminary electrochemical CV analyses of 1 mmol L⁻¹ Fe^3+/2+ (1) to assess reproducibility using 4 × 45 µm WEs (2). The colors indicate the anodic current peaks (unit: nA) collected by the 48 sensors (2). The WEs and QREs are indicated by numbers 1–16 and letters a–c, respectively. This representation was used throughout the manuscript.

Figure 3

Electrochemical analyses to investigate mass transport on the vertical chips and their reversibility. A) CV analyses of 1 mmol L⁻¹ Fe^3+/2+ using 4 × 45 µm WEs and varying υ to assess the diffusion regimes. Generic illustrations of the semi‐infinite linear diffusion (1) and quasi‐stationary regimes (2), voltammograms at different υ as highlighted (3), and plots of anodic (I _a) and cathodic (I _c) current peaks versus υ ^1/2 (4). Inset shows a stereoscopy image of the sensor (3); scale bar: 50 µm. The currents (4) are related to linear (I _al and I _cl) and quasi‐stationary regimes (I _aq and I _cq). B) Nyquist plots to 1 mmol L⁻¹ Fe^3+/2+ using 4 × 45 µm WEs. The semicircles are hypothesized to represent redox reactions on Au WE (*) and SU‐8 (**). Z″ and Z′ mean imaginary and real impedances, respectively. In the equivalent circuit, CPE 1 and CPE 2 are constant phase elements that express the electric double layer capacitances on Au and SU‐8, respectively; R _Ω means uncompensated resistance. C) SECM‐based current mapping of 1 mmol L⁻¹ FcMeOH over the sensor with 4 × 45 µm WEs (1) and approach curves on Au, glass, and SU‐8 (2). The color bar means the currents in nA (1), whereas the current values (i _i) in (2) were normalized by the stationary state current (i _ss). Insets illustrate the phenomena noted in each case (2). D) SICM plots obtained in bulk and SU‐8 surface to 10 mmol L⁻¹ KCl using 4 × 45 µm WEs. Inset illustrates the ensuing interface phenomena for negatively charged SU‐8, with red and blue circles indicating K⁺ and C⁻ ions, respectively. E) CV analyses of 1 mmol L⁻¹ Fe^3+/2+ utilizing 800 µm WEs at specific υ values as highlighted. Inset shows a stereoscopy image of the sensor, with the bars stressed by arrows signaling the SU‐8 ring width. The dimension bar means 150 µm. F) Nyquist plots to 1 mmol L⁻¹ Fe^3+/2+ using 800 µm WEs. The same circuit in (B) was applied here.

Figure 4

SERM analyses with 800 µm WEs and QREs consisting of Au and Ag/AgCl. A) Chip with the same probe, that is, 2 mmol L⁻¹ Fe^3+/2+, dropped on WEs versus Au (orange) and Ag/AgCl QRE (cyan asterisk) to afford SERM (1) and duplex SWV scan (red line) (2). The colored peaks mean the individual analyses using either Au or Ag/AgCl QREs, as stressed. Scale bar: 5 mm (1). B) FEM simulations for 2 mmol L⁻¹ Fe^3+/2+ and SERM format (1) and current densities at around the peaks of Fe^3+/2+ versus Au (2) and Ag (3). C) CV analyses and analytical curve for Fe^3+/2+. CV scans attained by duplex assay (red line) and individual analyses (colored scans) versus Au or Ag/AgCl QRE, as indicated (1), along with duplex SWV scans to different contents (unit: mmol L⁻¹) of probe (2). Inset shows the analytical curves for current densities (j: mA cm²) versus Au QRE (orange) and Ag/AgCl QRE (cyan) (2).

Figure 5

Bioassays to standard samples and serum samples for COVID‐19 screening. A) Multiplexed detection of 10 µg mL⁻¹ IgG⁻ and IgG^S using biosensors (Bios.) as stressed by red and cyan asterisks, respectively. B) Analytical curves for IgG^S standards utilizing Au and Ag/AgCl QREs. C) In‐house‐built handheld box (1) carrying potentiostat (2), chip connector, and manual selectors (3) of WEs (right) and QREs (left). The potentiostat can be operated via a smartphone as illustrated. Scale bar: 3 cm (1). D) Currents for bare WEs in relation to both QREs (as represented by Au and Ag/AgCl) and after incubation in positive (Pi; vs Au QRE) and negative sera (Ni; vs Ag/AgCl QRE). E) Currents for bare WEs (vs Au and Ag/AgCl QREs) and after exposure to Pi and Ni samples in two cycles of duplex analyses, that is, Pi on Au QRE and Ni on Ag/AgCl QRE (1) followed by the opposite situation (2). (F) Resulting ΔI for positive (PS) and negative sera (NS) using Au (1) and Ag/AgCl (2) QREs.