Chemicals and materials

Hydrogen tetrachloroaurate trihydrate, silver nitrate, dithiobis (succinimidyl propionate), 11-mercaptoundecanoic acid (MUA) and MMC were purchased from Sigma-Aldrich. Analytical-grade ascorbic acid was obtained from MP Biomedicals. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide were obtained from Thermo Fisher Scientific. A Milli-Q water system was used to generate ultrapure water (18.2 MΩ cm) to synthesize the nanoparticles. Two commercially available LFA products purchased from Abbott (ARTG #345192) and SD Biosensor (ARTG #345219) were used to test the clinical samples by following the instructions from the providers.

Production of SpyCatcher, SpyTag and SCV2 RBD–mNeonGreen fusion proteins

To produce SpyCatcher and SpyTag, SpyCatcher–MatterTag and EGFP-SpyTag fusions were designed via a rigid linker (AEAAAKEAAAKEAAAKA) and flexible linker (GGGS), respectively. The chimeric genes were commercially synthesized and cloned into the pCDNA3.1 vector. The fusion proteins were expressed in ExpiCHO-S cells according to the manufacturer’s instructions (Thermo Fisher Scientific). For purification, the filtered supernatant was loaded on a Strep-Tactin 4Flow column (IBA Lifesciences) equilibrated with a purification buffer (100 mM Tris–HCl (pH 8.0), 150 mM NaCl). The column was then washed with 50 column volumes of purification buffer. The fusion protein was eluted by a purification buffer containing 50 mM biotin (IBA Lifesciences). The purities of the purified proteins were analysed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The expression and purification of SCV2–mNeonGreen fusion protein is described elsewhere².

Engineering yeast cells and production of multifunctional SynBioNFs

EBY100 yeast cells were employed to display eight different functional fusion proteins on the cell walls, including (1) W25–MatterTag, (2) Sb68–SpyTag, (3) Sb68–MatterTag, (4) Nb21–StrepTag, (5) Nb21–SpyTag, (6) Nb21–MatterTag, (7) DD7–MatterTag and (8) DD5–SpyTag. These fusion gene sequences were commercially synthesized and cloned (Gene Universal; Supplementary Data 1 and 2) into the pCTCON2 expression plasmid for yeast surface display by following previous work². In our design, the target-binding nanobodies (Sb68, W25 and Nb21) and functional peptide tags (MatterTag, SpyTag and StrepTag) were fused into the N and C termini of Aga2p, respectively. A flexible linker (GGGGS) with 15 and 30 amino acids was used at the N and C termini of Aga2p to avoid steric hindrances between the fused proteins/peptides. Furthermore, HA and c-Myc peptide tags that allow the quantification of the displayed fusion proteins were included at the N and C termini of each gene construct, respectively. The fusion proteins carrying Aga2p were able to immobilize on the yeast cell walls by interacting with the Aga1p anchor protein.

To achieve the fusion protein display, EBY100 yeast cells were incubated with recombinant DNA (10 µl, 1,000 ng) under the stimulation of a square wave using electroporation. The produced yeast cells were cultured in the SDCAA medium and monitored until the optical density at 600 nm (OD600) reached 5–10. The yeast cells were then transferred into galactose containing the SGCAA medium and diluted to OD600 of 1.0 to induce fusion protein expression. Following the culture for 48 h, the yeast cells were collected and confirmed the fusion protein expression by performing a flow cytometry analysis, as described below.

For the preparation of SynBioNFs, the yeast cells with the display of fusion proteins were collected from the SGCAA medium (50 ml, OD600 of 6–10) and washed with PBS through centrifugation (2,000×g, 10 min), followed by resuspending into PBS supplemented with a protease inhibitor cocktail with EDTA (10 ml per tablet). The mechanical fragmentation of yeast cells was conducted using a sonicator (Sonics ultrasonic processor VC-505) with a 3 mm tip diameter and 171 mm length at an ultrahigh intensity by repeating the following conditions for five times: 40% amplitude; 1 s ON and 1 s OFF pulse for 2 min. Ultimately, SynBioNFs were obtained using centrifugation (2,500×g, 15 min) to collect the supernatant products and purified through a filter unit (100 nm, Millipore).

Flow cytometry profiling of fusion proteins

The yeast cells with fusion protein expression (10⁷ cells ml^–1) were collected and washed using PBS containing 0.1% bovine serum albumin (BSA) (500 µl) through centrifugation (1,500×g, 4 min) at 4 °C. To enable the labelling of the yeast surface protein, the yeast cells were incubated with anti-Myc antibody labelled with DyLight 650 (1:100 dilution) and RBD–mNeonGreen, or anti-dengue NS1 protein followed by anti-His antibody labelled with PE (1:100 dilution) in PBS containing 0.1% BSA (100 µl) with rotation and away from light at 4 °C for 1 h. The labelled yeast cells were centrifuged (1,500×g, 4 min) and washed with PBS containing 0.1% BSA (500 µl), followed by resuspending them into PBS containing 0.1% BSA (500 µl) for testing. The yeast cell controls without the use of anti-Myc or anti-His, anti-dengue NS1 antibody and RBD–mNeonGreen were prepared with the same protocol. The obtained yeast cells were subject to flow cytometry profiling (CytoFLEX, Beckman Coulter) using two lasers (488 and 633 nm) and two band-pass filters (525/40 and 660/20 nm). The data were acquired using CytExpert (2.4.0.28) and analysed with FlowJo software (10.8.1).

SCV2 culturing using cell line

SCV2 was cultured in Vero E6 cells. The Vero E6 cells were first cultured in Dulbecco’s modified Eagle’s medium supplemented with 2% heat-inactivated foetal bovine serum. When the cells were 70–90% confluent, the viral inoculum was inoculated into the Vero E6 cells and incubated at 37 °C (5% CO₂); the cytopathic effect was observed. SCV2 was harvested in the supernatant via centrifugation at 4,500×g for 10 min. Virus was gamma irradiated at a dose of 50 kGy to inactivate it.

RT-qPCR quantification of cultured SCV2

To quantify the cultured SCV2 stock, RT-qPCR was performed. MagMAX-96 viral RNA isolation kit was used to extract the SCV2 RNA. The gBlock synthetic E gene standards were utilized to establish the copy-number-related calibration curve. The test employed the AgPath-ID One-Step RT-PCR master mix with the following primers: CoV-E-fwd (5’-AGT ACG AAC TTA TGT ACT CAT TCG TT-3’), CoV-E-R2 (5’-ATA TTG CAG CAG TAC GCA CAC A-3’) and TaqMan probe (CoV E probe 5’-6-FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-MGB-3’). The detection of SCV2 was conducted in duplicates by using the mean for calibration on an Applied Biosystems instrument. The cycling conditions were 45 °C for 10 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 45 s.

Conjugating detection SynBioNFs with gold–silver alloy nanoboxes

The conjugation of SynBioNFs with gold–silver alloy nanoboxes was performed via the SpyCatcher-/SpyTag-mediated self-assembly. SpyCatcher-coated gold–silver alloy nanoboxes were first prepared as follows: gold–silver alloy nanoboxes were synthesized following our previous work³⁰. One millilitre of nanoboxes were centrifuged at 800×g for 15 min. Then, 10 µl Raman reporter (MMC) and 2 µl linker molecule (MUA) were incubated with the above nanoboxes for 5 h. After removing the free MMC and MUA by centrifuging at 800×g for 15 min, 10 µl of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (10 mM) and 20 µl of N-hydroxysulfosuccinimide (10 mM) were added to activate the carboxyl group on MUA. Then, 0.5 µg SpyCatcher was incubated with the nanoboxes for 30 min at room temperature. The SpyCatcher-coated nanoboxes were purified with centrifuging at 800×g for 15 min and resuspended into 200 µl of 0.1% BSA.

Next, 30 µl of the SpyCatcher-coated nanoboxes were incubated with 4 µl of SynBioNFs (Sb68–SpyTag) (8 µg µl^–1) in 60 µl of 10 mM PB, 1 mM PBS and 1% BSA buffer for 10 min. The final products were collected by centrifuging at 600×g for 10 min and washing with 0.1% BSA.

NanoFCM characterization of SynBioNFs and nanobox bioconjugates

NanoFCM measurements were performed on a nanoFCM flow NanoAnalyser (NanoFCM). The NanoAnalyser was first calibrated for concentration and size using the standard nanoparticles provided by the company. The size distribution of SynBioNFs was obtained by comparing with the cocktail size standard (that is, premixed silica nanoparticles with different diameters). To profile the fluorescence profiling of gold–silver-alloy-nanobox-conjugated SynBioNFs against RBD–mNeonGreen, 30.0 µl of the conjugates were incubated with 0.5 µl of RBD–mNeonGreen (350 µM) at room temperature for 30 min and the products were washed three times with 0.1% BSA via centrifugation at 600×g for 10 min, followed by recording the events for 1 min. The same amount of gold–silver alloy nanobox and SynBioNFs without reacting with RBD–mNeonGreen were used as negative controls to set the threshold.

SynBioNF-enabled SERS detection of RBD, SCV2, simulated patient samples and clinical COVID-19 samples

The gold microelectrodes were prepared in-house by a photolithography approach with 4-inch borosilicate glass wafers and following a previously established protocol³¹. After photolithography, the wafer consisted of an array of 28 circular gold microelectrodes with inner working electrodes (1.00 mm in diameter) and outer counter electrode (0.12 mm in diameter). The working and counter electrodes were separated by 1 mm. To contain the sample on the gold microelectrodes, a well structure made of polydimethylsiloxane was attached to the wafer. Before functionalization, the gold microelectrodes were washed with 1× PBS. Subsequently, SynBioNFs (Sb68–MatterTag) were pipetted on the gold microelectrodes and incubated for 30 min at room temperature. Excess SynBioNFs (Sb68–MatterTag) were removed by washing three times with 1× PBS. Finally, the gold microelectrodes were blocked with 5% BSA in 1× PBS for 1 h at room temperature. Before use, the gold microelectrodes were washed with 1× PBS. A 30 µl mixture of the patient sample (20 µl sample + 10 µl PBS/1% Tween-80) was then pipetted on the gold microelectrode and incubated for 45 min under stimulation of nanomixing by alternating-current electrohydrodynamics (frequency, 500 Hz; amplitude, 800 mV). In particular, the inclusion of PBS/1% Tween-80 buffer in the patient samples was aimed to inactivate the virus. For clinical samples that are not contagious after treatment (for example, gamma irradiation), the samples can be directly applied on the platform without the use of PBS/1% Tween-80 buffer. Subsequently, after washing the gold microelectrodes with 1× PBS, the bioconjugates of SynBioNFs (Sb68–MatterTag) and nanoboxes were incubated for 20 min under the same nanomixing conditions as above. Finally, the excess bioconjugates were removed by washing with 1× PBS. The gold microelectrodes were then subject to confocal Raman mapping (WITec alpha300 R spectrometer) and collecting/analysing the data using WITec Suite FIVE software. Specifically, a He–Ne laser with an excitation wavelength of 632.8 nm, ×20 objective, electron-multiplying charge-coupled device camera, 0.05 s integration time and 1 µm step size was used for scanning the images with a size of 60 µm × 60 µm.

Clinical sample details

SCV2-positive clinical patient samples were supplied by the Molecular Diagnostics Unit at Pathology Queensland, and consisted of nasopharyngeal swabs resuspended in PBS. These samples were tested using in vitro diagnostics RT-qPCR at the Molecular Diagnostics Unit very early in the pandemic, whereas diagnostic assays were still being completely validated. Negative and positive samples were provided by the Infectious Diseases Laboratory, Microbiology Prevention Division, Pathology Queensland, and tested using the validated BGI platform. Patient samples were collected under the following ethics approval: HREC ref. no. HREC/2020/QRBW/70461; project title, optimizing clinical diagnostics for SCV2. A waiver of consent was approved by this ethics committee and compensation was not applicable for this study.

Fluorescent platform detection of SCV2

Sixty microlitres of SynBioNFs (Sb68–MatterTag) were incubated on the gold surface at room temperature for 2 h, followed by washing three times with PBS to remove free SynBioNFs. Then, 60 µl of the sample solution (that is, SCV2 or medium control) was loaded and incubated on the sensing area at room temperature for 1 h. The gold chips were washed with PBS three times to remove the uncaptured targets. Next, 50 µl of the bioconjugates were applied on the chips and incubated for 1 h. To prepare the bioconjugates, 500 µl SynBioNFs (Nb21–StrepTag) and 10 µl fluorescence beads (coated with streptavidin) were incubated in an Eppendorf tube at room temperature for 1 h and purified through centrifugation. After getting rid of the free bioconjugates, the gold chips were imaged under a fluorescence microscope. The acquired images were then analysed with ImageJ software (1.53).

Electrochemical detection of SCV2

Sixty microlitres of SynBioNFs (Sb68–MatterTag) were incubated on the screen-printed electrodes at room temperature for 2 h. After washing away the free SynBioNFs (Sb68–MatterTag), 60 µl of the sample solution (that is, SCV2 or medium control) was applied on the inner circular working electrodes for an incubation of 1 h and subsequently washed three times with PBS for electrochemical detection. For DPV measurement, 40 µl of 2.5 mM [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ redox couple in 1× PBS (pH 7.4) containing 0.1 M KCl was added onto the screen-printed electrodes to record the current. The DPV scan was conducted on an electrochemical analyser CHI 650D (CH Instruments) using a scan voltage from –0.2 to 0.4 V, pulse amplitude of 50 mV, pulse width of 50 ms, potential step of 5 mV and pulse period of 10 ms. The CV measurements were performed in 10 mM PBS in the presence of the [Fe(CN)₆]^3−/4− redox system (pH 7.4, 2.5 mM [Fe(CN)₆]^3−/4−). The data were recorded between –0.6 and 0.6 V at a scan rate of 100 mV s^–1.

LFA for SCV2 detection

Lateral flow test strips (width, 7 mm; length, 80 mm) were prepared using nitrocellulose HP-80 FF strips with laminate backing (Cytiva) and medium-sized absorbent pads attached at the top of the strips. Multiple strips were prepared using a programmable high-speed strip cutter (KinBio). Each strip was spotted with 0.2 µl (200 ng) of capture CR3022 monoclonal antibodies and dried in a 37 °C incubator for 15 min. The CR3022 antibodies were prepared in-house using Chinese hamster ovary cell culture.

Samples for the lateral flow strips were set up in 0.2 ml thin-walled tubes and incubated at 37 °C for 10 min. Next, 10 µl of bioconjugates of SynBioNFs (Nb21–SpyTag) and spherical gold nanoparticles (coated with SpyCatcher) in PBS was mixed with 10 µl SCV2 or medium control. Then, 1% BSA and 1% Tween-80 were included in each reaction. Each reaction was incubated at 37 °C for 15 min. The whole reaction (20 µl) was aliquoted into wells of a 96-well plate, and the tests strips were dipped into the wells to allow the samples to run vertically up the strips towards the absorbent pad for 1–2 min. Then, 50 µl PBST (1× PBS + 0.05% Tween-20) was added to the wells and incubated for further 5 min to move all the bioconjugates up the strip. Visual colorimetric reactions at the capture line were imaged using a digital camera.

ELISA-based assay for stability and avidity test

For the ELISA assay, 10 µg ml^–1 of recombinant SCV2 RBD protein diluted in 1× TBS (20 mM Tris (pH 8.0), 300 mM NaCl) was coated on MaxiSorp ELISA plate wells for 1 h at room temperature. The wells were then incubated with the blocking buffer (3.00% BSA in TBS with 0.05% Tween-20) for 1 h at room temperature. Then, 100 µl of anti-SCV2 RBD SynBioNFs (Nb21–SpyTag) (1:5) or rabbit polyclonal antibody (1:10,000) were added to each well and incubated for 1 h at room temperature. For a thermal stability analysis, SynBioNFs or rabbit polyclonal antibody aliquots were incubated at the indicated temperature for 1 h and subsequently added into the respective wells. For avidity assay, urea, guanidine hydrochloride, Triton X-100, sodium dodecyl sulfate or NaCl were added at the indicated concentrations for 1 h at room temperature. To test the acidic pH conditions, a mix of citric acid and sodium phosphate buffers (pH, 2.6–7.6) were used. For alkaline pH conditions, a mix of sodium carbonate and sodium bicarbonate buffers (pH, 9.2–10.8) were used. After washing the wells with TBST five times, HRP-conjugated anti-Myc tag antibody (1:5,000 dilution in 3% BSA–TBST) or HRP-conjugated goat anti-rabbit IgG secondary antibody (1:10,000) was added to the wells for 1 h. The wells were washed with TBST and finally 100 µl of TMB substrate was added to each well, and the reaction was stopped by 100 µl of 1 M sulfuric acid.

Octet assay for measurement of protein interaction

Streptavidin sensors (ForteBio) were pretreated in 200 µl of 10 mM PBS for 10 min. Each well was loaded with 200 µl of the solution. The assay was performed by setting a program: the sensors were dipped in PBS for 120 s in the initial baseline step, loaded with 75 µg of strep RBD per well in the loading sample, dipped in PBS for 120 s in the second baseline step, interacted with SynBioNFs (Nb21–SpyTag) or soluble Nb21 nanobody for 300 s in the association step and ended with disassociation in PBS for 600 s.

Statistical analysis

The diagnostic sensitivity, specificity and accuracy of the SynBioNF-based screening of clinical samples on the SERS platform were determined based on the confusion matrix (Fig. 6c) with the following formulas:

Sensitivity = Number of true positive assessments / Number of all positive assessments = 81/(81 + 3) = 96.43%;

Specificity = Number of true negative assessments / Number of all negative assessments = 50/(0 + 50) = 100 %;

Accuracy = Number of correct assessments / Number of all assessments = (81 + 50)/(81 + 3 + 0 + 50) = 97.76%.

Two-tailed t-tests, receiver operating characteristic curve and Bland–Altman analysis were performed in GraphPad Prism (v. 9.2).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41565-023-01415-1