Sensing of COVID-19 Antibodies in Seconds via Aerosol Jet Nanoprinted Reduced-Graphene-Oxide-Coated 3D Electrodes

Abstract

Rapid diagnosis is critical for the treatment and prevention of diseases. An advanced nanomaterial-based biosensing platform that detects COVID-19 antibodies within seconds is reported. The biosensing platform is created by 3D nanoprinting of three-dimensional electrodes, coating the electrodes by nanoflakes of reduced-graphene-oxide (rGO), and immobilizing specific viral antigens on the rGO nanoflakes. The electrode is then integrated with a microfluidic device and used in a standard electrochemical cell. When antibodies are introduced on the electrode surface, they selectively bind with the antigens, changing the impedance of the electrical circuit which is detected via impedance spectroscopy. Antibodies to SARS-CoV-2 spike S1 protein and its receptor-binding-domain (RBD) are detected at a limit-of-detection of 2.8 × 10^-15 and 16.9 × 10^-15 m, respectively, and read by a smartphone-based user interface. The sensor can be regenerated within a minute by introducing a low-pH chemistry that elutes the antibodies from the antigens, allowing successive sensing of test samples using the same sensor. Sensing of S1 and RBD antibodies is specific, which cross-reacts neither with other antibodies such as RBD, S1, and nucleocapsid antibody nor with proteins such as interleukin-6. The proposed sensing platform could also be useful to detect biomarkers for other infectious agents such as Ebola, HIV, and Zika.

Keywords: 3D printing; 3D sensors; COVID-19; antibody tests; gold nanoparticles; micropillars; pandemics; reduced graphene oxide.

Figure 1

Schematic of the manufacturing process of the 3D‐printed COVID‐19 test chip (3DcC) by Aerosol Jet nanoparticle 3D printing. a) Glass substrate with patterned gold film forming the base for working electrode (WE), counter electrode (CE), and reference electrode (RE) of the electrochemical cell of 3DcC. b) Construction of Aerosol Jet machine where gold ink is converted into an aerosol consisting of micro‐droplets using ultrasonic energy and transported to a nozzle by nitrogen (N₂) gas where it is focused on the gold film of the WE using a sheath gas (also N₂) to form the micropillars. The entire process is digitized with the CAD program controlling the printing process. c) An AJ‐printed 10 × 10 gold micropillar array where pillar‐to‐pillar gap is indicated. d) Details of AJ printing of a single micropillar where rapid layer‐by‐layer stacking of the micro‐rings of the nanoparticle ink are achieved using surface tension (γ) of the printed ink. The entire process is achieved without the use of any support structure. Once a layer was printed, the ink loses solvents due to the heat from the platen (which was heated to 150 °C using a custom heater). The dried ink provides a base to receive the next micro‐ring; and the process is repeated. e) Process of fabrication of the PDMS housing of the 3DcC device. A PDMS structure is created using a PMMA master mold using replica molding. The dimensions of sections A and A1 are 1 × 1 × 5 mm³ and 2 × 1 × 10 mm³, respectively. This structure then acts as a reverse‐mold for the PDMS housing that contains a cavity for microfluidic channel as shown. f) The 3DcC device formed by placing the PDMS housing with microfluidic channel on the glass substrate with the micropillar electrodes. Prior to this step, the micropillar electrodes were functionalized with reduced graphene oxide (rGO) and viral antigens as described in Figure 2.

Figure 2

Functionalization of 3D‐printed micropillar electrode and 3DcC sensor operation. a) AJ‐printed gold micropillars prior to the surface treatment (step 1). b) Coating of the electrodes by carboxylated (—COOH) rGO sheets by a simple drop‐casting process (step 2). An SEM image shows the decoration of rGO sheets on the gold pillar. The electrostatic or van der Waals interactions allow the rGO sheets to be connected to the micropillar. The surface porosity of the 3D‐printed micropillar shown in the SEM images in Figure 3c likely aid in this process. Molecular structure of a rGO sheet is shown where —COOH and —OH groups are indicated. c) Coupling of the viral antigens with the rGO sheets using EDC:NHS chemistry (step 3). The EDC and NHS molecules activate the —COOH group of rGO sheets. Recombinant antigens of the SARS‐CoV‐2 are immobilized and bound to rGO sheets by establishing strong covalent bonds between the —COOH group of rGO and —NH₂ group of antigens via an amidation reaction. Two antigens, namely, spike S1 and RBD, were separately immobilized in this manner. Bovine serum albumin (BSA) treatment on the pillar surface blocked the non‐specific sites of the sensor. d) Antibodies selectively attached to the specific antigens upon introduction to the sensor via an antibody–antigen interaction (step 4). e,f) Schematics showing the sensing principle of the 3DcC device. The electrode/electrolyte interface of the WE was expected to form an electrical double layer (C _dl), inner Helmholtz plane or IHP, outer Helmholtz plane or OHP, and a diffusion layer during the redox reaction. An equivalent electrical circuit is shown. When antibodies are introduced (f), they rapidly bind with the antigens on the electrode surface, altering the Nyquist plot (schematics in (e) and (f)) which is captured by electrical impedance spectroscopy (EIS).

Figure 3

Physical and chemical characterization of the 3DcC device. a) An optical image of the 3DcC device made by the fabrication process described in Figures 1 and 2. b) Optical and SEM images of the AJ‐printed gold micropillar array (10 × 10) at different magnifications. c) SEM images showing the morphology of a single gold micropillar at different magnifications. The tips of the micropillars are pointed and have a small circular dip at the top (diameter 20 µm) which is a result of the printing process described in Figure 1d. Surface morphology of the micropillars consisted of gold clusters that created a characteristic surface porosity. This morphology helps attach rGO flakes to the electrode surface. d) SEM images showing the morphology of a gold micropillar after rGO decoration. The rGO sheets have formed secondary 3D networks adjacent to the micropillars. The rGO has also formed wrinkles likely due to π–π interactions amongst graphene sheets. e) Raman spectra of an AJ‐printed gold micropillars without coating, after coating with rGO, and after immobilization of antigens on rGO–Au. The spectra show both defect and graphitic peaks for the coated samples. The graphitic peak is shifted to higher frequency upon S1 antigen immobilization. f) EDX studies showing composition (in atomic %) before coating, after coating with rGO, and after immobilization of antigens on rGO–Au. g) The 3DcC device interfaced with a portable potentiostat which was connected to a smartphone via a USB‐C connection to record the signal using PStouch software.

Figure 4

Sensing of antibodies to SARS‐CoV‐2 spike S1 antigen at different molar concentrations with regeneration. a) Nyquist plots of the 3DcC sensor measured by EIS method without and with the spike S1 antibodies at concentrations of 0.01 × 10 ⁻¹⁵ m, 1 × 10 ⁻¹⁵ m, 1 × 10⁻¹² m, 100 × 10⁻¹² m, 1 × 10⁻⁹ m, 10 × 10⁻⁹ m, and 30 × 10⁻⁹ m in pbs solution. b,c) Nyquist plots similar to that in (a) after two successive sensor regenerations by a low‐pH chemistry consisting of 1.0 m (pH 2.5) formic acid solution. The regeneration was achieved within 60 s. For all concentrations, the signal in (b,c) was within 95% of that in (a). d) The charge transfer resistance (R _ct) for the 3DcC sensor for each concentration of antibodies and control serum before and after each regeneration for the data in (a–c). The fetal bovine serum (fbs) and rabbit serum (rs) in (a–d) were utilized as control biofluids. For all measurements, a 50 × 10 ⁻³ m pbs (pH 7.4) solution containing an equimolar concentration (5 × 10 ⁻³ m) of a ferro/ferricyanide mediator was used. Three successive readings were obtained at each concentration of the antibodies for the three data sets. Detection frequencies from 1 to 10 000 Hz were applied to obtain this data. There was no incubation time for all the measurements in this figure. The R _ct values in (d) were calculated by fitting the data in (a–c) to a Randles equivalent circuit shown in Figure 2e.

Figure 5

Sensing of antibodies to SARS‐CoV‐2 receptor binding domain (RBD) antigens at different molar concentrations with regeneration. a) Nyquist plots of the 3DcC sensor measured by EIS method without and with the RBD antibodies at concentrations of 1 × 10 ⁻¹⁵ m, 1 × 10 ⁻¹⁵ m, 1 × 10⁻¹² m, 100 × 10⁻¹² m, 1 × 10⁻⁹ m, 10 × 10⁻⁹ m, and 20 × 10⁻⁹ m in pbs solution. b,c) Nyquist plots similar to that in (a) after two successive sensor regenerations by a low pH chemistry consisting of 1.0 m (pH 2.5) formic acid solution. The regeneration was achieved within 60 s. For all concentrations, the signal in (b,c) was within 95% of that in (a). d) The charge transfer resistance (R _ct) for the 3DcC sensor for each concentration of antibodies and control serum before and after each regeneration for the data in (a–c). The fetal bovine serum (fbs) and rabbit serum (rs) in (a–d) were utilized as control biofluids. For all measurements, a 50 × 10 ⁻³ m pbs (pH 7.4) solution containing an equimolar concentration (5 × 10 ⁻³ m) of a ferro/ferricyanide mediator was used. Three successive readings were obtained at each concentration of the antibodies for the three data sets. Detection frequencies from 1 to 10 000 Hz were applied to obtain this data. There was no incubation time for all the measurements in this figure. The R _ct values in (d) were calculated by fitting the data in (a–c) to a Randles equivalent circuit shown in Figure 2e.

Figure 6

Detection time and regeneration studies of the 3DcC device. a,b) The detection time for spike S1 and RBD antibodies during the EIS measurements, respectively, for twelve different 3DcC devices. The device impedance is plotted against the detection time. The spike S1 and RBD sensors reached 93.2% and 92% of their saturation impedance at 11.5 s, respectively, allowing the signal detection in seconds. The concentration of antibodies was set to 1 × 10⁻⁹ m for this measurement. A frequency range of 1–10 000 Hz was used to obtain this data. The (Z _im) data is recorded from 3 s after the introduction of antibodies as the EIS measurement had to overcome the solution resistance, R _s, prior to obtaining the charge transfer resistance (see schematic in Figure 2e). c,d) Regeneration study showing Nyquist plots and charge transfer resistance for the detection of spike S1 antibodies for the 3DcC device. The regeneration is carried out 9 times. For each measurement, the sensor was first exposed to pbs, then to 1 × 10⁻⁹ m concentration of S1 antibodies in pbs, and finally to formic acid (1 m) with an incubation time of 60 s. The charge transfer resistance as a function of the number of regenerations is plotted in (d). e,f) Regeneration data for sensing of RBD antibodies by the 3DcC device. For both the sensors, a minimal loss in sensor performance is observed after nine regenerations.

Figure 7

Cross‐reactivity and reproducibility studies of the 3DcC device. a) Cross‐reactivity test for the 3DcC device designed to detect spike S1 antibodies. Nyquist plots for the device are plotted for multiple antigens and antibodies in absence and presence of spike S1 antibodies. IL‐6 or IL‐6 antigen (0.05 × 10⁻⁹ m), nucleocapsid (N) antibody (1 × 10⁻⁹ m), and RBD antibody (0.1 × 10⁻⁹ m) are used for this measurement. b) The R _ct values were calculated from the Nyquist plots shown in (a). The 3DcC device provided minimal interference with other related proteins. c) Cross‐reactivity test for the 3DcC device designed to detect RBD antibodies. Nyquist plots for the device are plotted for multiple antigens and antibodies in absence and presence of RBD antibodies. IL‐6 antigen (0.05 × 10⁻⁹ m), N‐antibody (1 × 10⁻⁹ m), and spike S1 antibody (0.1 × 10⁻⁹ m) are used for this measurement. d) The R _ct values were calculated from the Nyquist plots shown in (c). The 3DcC device provided minimal interference with other related proteins. e,f) The sensor reproducibility test on six different 3DcC sensors in presence of S1 antibodies (1 × 10⁻⁹ m in pbs). The sensor‐to‐sensor variation is evaluated by calculating the R _ct values for each sensor. This variation is within about 7%. The error bar is from at least three repeated measurements of the sensor. g,h) Reproducibility test data for sensing of RBD antibodies (1 × 10⁻⁹ m in pbs) from six different sensors. The sensor‐to‐sensor variation in this case is about 5%.

Figure 8

Study of the kinetics of antigen–antibody interactions. a) Nyquist plots for spike S1 antibodies at concentrations of 10 × 10⁻⁹ m (a) and 30 × 10⁻⁹ m (b) at the micropillar surface showing their association, equilibrium, and regeneration phases. c) Variation in the R _ct values during antigen–antibody interaction for spike S1 antibodies at 10 × 10⁻⁹ and 30 × 10⁻⁹ m concentrations. Repeat 1, 2, 3, and 4 are the replicate plots in the equilibrium phase. d) Schematic of the association, equilibrium, and regeneration of the kinetics shown in (a), (b), and (c). In association phase, the target antibodies (10 × 10⁻⁹ and 30 × 10⁻⁹ m) with buffer solution are loaded into the 3DcC device. The antibodies attach to the antigens. However, some unbound antibodies may be present in the solution near the sensor surface and contribute to the R _ct. In equilibrium phase, fresh pbs (without antibodies) is used to wash the 3DcC device, thus removing the unbound antibodies in the solution, and reducing the R _ct slightly. In the regeneration phase, a solution of formic acid (1 m; pH 2.5) is used to regenerate the sensor surface, thus eluting the antibodies from the antigens and lowering R _ct significantly.