An Advanced Healthcare Sensing Platform for Direct Detection of Viral Proteins in Seconds at Femtomolar Concentrations via Aerosol Jet 3D-Printed Nano and Biomaterials

Abstract

Sensing of viral antigens has become a critical tool in combating infectious diseases. Current sensing techniques have a tradeoff between sensitivity and time of detection; with 10-30 min of detection time at a relatively low sensitivity and 6-12 h of detection at a high (picomolar) sensitivity. In this research, uniquely nanoengineered interfaces are demonstrated on 3D electrodes that enable the detection of spike antigens of SARS-CoV-2 and their variants in seconds at femtomolar concentrations with excellent specificity, thus, overcoming this tradeoff. The 3D electrodes, manufactured using a high-resolution aerosol jet 3D nanoprinter, consist of a microelectrode array of sintered gold nanoparticles coated with graphene and antibodies specific to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike antigens. An impedance-based sensing modality is employed to sense several pseudoviruses of SARS-CoV-2 variants of concern (VOCs). This device is sensitive to most of the pseudoviruses of SARS-CoV-2 VOCs. A high sensitivity of 100 fm, along with a low limit-of-detection of 9.2 fm within a test range of 0.1-1000 pm, and a detection time of 43 s are shown. This work illustrates that effective nano-bioengineering of interfaces can be used to create an ultrafast and ultrasensitive healthcare diagnostic tool for combating emerging infections.

Keywords: 3D biosensor; nano-bio-interfaces; nanoprinting; pseudovirus; ultrarapid diagnosis; variants of concern.

Figure 1.

Packaging of pseudoviruses SARS-CoV-2 VOCs. A) Structure of SARS-CoV-2 and spike. B) The expression of SARS-CoV-2 spike proteins was confirmed by Western-blotting.

Figure 2.

3D nano-printed sensor for rapid detection of SARS-CoV-2 VOCs. A) Schematic presentation of a 3D nano-printed sensor. The device consists of three electrodes such as working, counter, and reference wherein the working electrode was made of an array of micropillars of gold nanoparticles. This array was manufactured by an Aerosol Jet 3D nanoparticle printer. B) SEM image of the nano-printed micropillar array. C,D) SEM images of graphene coating on the gold microelectrode array (graphene-Au). The zoomed-in SEM image (D) shows the assembly of wrinkled graphene on the tip of a micropillar. E) Stepwise representation of the surface functionalization to coat the SARS-CoV-2 spike S1 antibodies on rGO-Au via a unique EDC-NHS conjugation chemistry. In reaction chemistry, the EDC activates the -COOH groups of graphene, while NHS acts as a stabilizer. The amide bonds were formed between spike S1 antibodies and the graphene-Au surface. Captured antigens by antibodies specific to SARS-CoV-2 spike proteins on the rGO-Au microelectrode hinder the electron transfer due to oxidation, thus enhancing the electrode impedance.

Figure 3.

Sensor characterization. A) Electrochemical impedance spectroscopy (EIS) measurements show the Nyquist plots of the sensor. The sensor’s electrode (rGO-Au) was functionalized with SARS-CoV-2 spike S1 antibodies before and after measurements. B) Schematic illustration of an equivalent circuit between the counter and working electrodes. R_ct is the charge transfer resistance, R_s is the solution resistance, C_dl is double-layer capacitance and Z_w is the Warburg resistance. C) The diameter of the semicircle shown in a typical Nyquist plot is the R_ct. An AC signal with an amplitude of 1.0 mV and a fixed frequency range (10 000–1 Hz) was applied to conduct the EIS experiments. D) The cyclic voltammetry (CV) of the sensor was conducted with and without spike S1 antibodies. With antibody coating, the sensor current is decreased due to blocking of electrons by antibodies on the electrode surface. Both EIS and CV measurements were performed in presence of PBS (50 mm) mixed with a ferro/ferricyanide (5 mm) redox mediator.

Figure 4.

SARS-CoV-2 sensor calibration studies. A) Sensing results with different concentrations of SARS-CoV-2 spike S1 antigens. Sensing of SARS-CoV-2 antigens was conducted using EIS measurements. The Nyquist plots of the SARS-CoV-2 antigen sensor (A) were recorded as a function of S1 antigen at 0.1–1000 pm. B) Their corresponding R_ct values are plotted with respect to the different S1 antigen concentrations. In Nyquist plots, the deviation of fitted (shown in Figure 3C) and raw semicircles was less than ±3.0%. A p-value of <0.0001 indicates a statistically significant difference (B). C) Imaginary impedances (Z_im) of the sensors at different antigen concentrations were plotted against detection time in seconds. D) Modulus of impedance (|Z|) is plotted against frequency at different concentrations. Three repeated measurements were recorded for each antigen concentration. All EIS experiments for sensing antigens were recorded by applying an AC signal with an amplitude of 1 mV and a frequency range of 10 000–1 Hz. The PBS (50 mm) solution mixed with a ferro/ferricyanide (5 mm) mediator was used to prepare the titrate concentration of spike S1 antigens.

Figure 5.

Sensing of SARS-CoV-2 VOCs. Based on titration measurements in Figure 4, the sensor was used to measure SARS-CoV-2 VOCs at identical conditions. A) The preparation of different SARS-CoV-2 pseudoviruses is demonstrated in Figure 1 and tested with the sensor using EIS measurement. For this study, VSV and Mock are taken as controls. WT, Alpha, Beta, Delta and Gamma are the five VOCs used in this study. B) Corresponding R_ct values are calculated for both controls and VOCs and plotted to evaluate p-value (<0.0001). For each virus and control, the sensor was run at least three repeated times. C) The detection phase and modulus of impedance are plotted with frequency for each EIS measurement. D) The Zim of the sensors for detecting different VOCs were plotted against detection time in seconds. EIS measurements for sensing antigens were recorded by applying an AC signal with an amplitude of 1 mV and a frequency range of 10 000–1 Hz. The PBS (50 mm) solution mixed with a ferro/ferricyanide (5 mm) was used to prepare different concentrations of recombinant spike S1 antigens.

Figure 6.

Sensor selectivity and reproducibility. The selectivity studies of the sensor is in presence of different antigens such as IFNs, IL-6, and NC with and without SARS-CoV-2 spike antigen (0.1 pm). The concentration of all interfering antigens is set to 10 pm. The baseline of the sensor is set at 0.1 pm concentration of SARS-CoV-2 S1 antigens. Individual interfering antigens are then added to this solution at a 1:1 ratio. A) Nyquist plots of the sensor with different interfering antigens, and B) their corresponding Rct values calculated by fitting the impedance graphs in (A). C) Nyquist plots of four identical sensors tested in the same buffer solution mixed with SARS-CoV-2 antigens (0.1 pm), and D) their corresponding R_ct values. p-values were obtained as < 0.0001 when the sensors were compared with the baseline response. The PBS (50 mm) solution mixed with a ferro/ferricyanide (5 mm) mediator was used for the studies shown in this figure.