A SARS-Cov-2 sensor based on upconversion nanoparticles and graphene oxide

Since the beginning of the COVID-19 pandemic, there has been an increased need for the development of novel diagnostic solutions that can accurately and rapidly detect SARS-CoV-2 infection. In this work, we demonstrate the targeting of viral oligonucleotide markers within minutes without the requirement of a polymerase chain reaction (PCR) amplification step via the use of oligonucleotide-coated upconversion nanoparticles (UCNPs) and graphene oxide (GO).


S-II Characterization of PAA-UCNPs and oligonucleotide UCNPs
Following the solvothermal synthesis, core-shell UCNPs had oleic acid (OA) ligands on their surface. Initially, a ligand exchange procedure was performed where OA ligands were replaced with poly-acrylic acid (PAA). This enabled the transfer of UCNPs to water and facilitated their subsequent functionalization with amino-modified oligonucleotides. The chemical coupling was performed utilizing an EDC coupling reaction to create an amide bond between the amino groups of oligonucleotides and the carboxylic groups of the PAA on the UCNP surface.
Successful coupling was assessed via zeta potential (see Figure S2) and Fourier transform infrared spectroscopy (FTIR) (see Figure S3). A clear decrease in the net charge was observed upon oligonucleotide functionalization, which was attributed to the increase in negative charge arising from the oligonucleotide backbone. FTIR analysis showed that upon oligonucleotide coupling the characteristic peak corresponding to the carboxyl group of PAA disappeared and two new peaks at1650 cm -1 and 1560 cm -1 corresponded to the vibrations of the amide group (C=O and N-H) appeared. Figure S2. Zeta potential measurements of PAA coated core-shell UCNPs and oligonucleotide coated core-shell UCNPs (0.5 mg/mL). A clear change in the zeta potential charge of the nanoparticles was observed indicating successful oligonucleotide coupling. Figure S3. Fourier-transform infrared spectroscopic measurements of oleate-capped core-shell UCNPs, PAA coated core-shell UCNPs and oligonucleotide coated core-shell UCNPs. The presence of PAA is confirmed by S6 the appearance of a strong COOH peak at 1700 cm -1 . The appearance of the peaks at 1650 cm -1 and 1560 cm -1 is related to the creation of amide bonds due to the conjugation of the oligonucleotides via EDC coupling.

S-III Oligonucleotide synthesis and purification
Oligonucleotides were synthesized on an Applied Biosystems 394 automated DNA/RNA synthesizer using a standard 1.0 μmole phosphoramidite cycle of acid-catalyzed detritylation, coupling, capping, and iodine oxidation. Stepwise coupling efficiencies and overall yields were determined by the automated trityl cation conductivity monitoring facility and were >98.0%.
All -cyanoethyl phosphoramidite monomers were dissolved in anhydrous acetonitrile to a concentration of 0.1 M immediately prior to use with coupling time of 50 s for normal A, G, C, and T monomers and was extended to 600 s for 5'-Amine monomer (5'-TFA-Amino-Modifier C6-CE Phosphoramidite was purchased from Link Technologies Ltd.).
5'-Amino modified oligonucleotides on resin were treated with a solution of diethylamine (10% in acetonitryl) for 20 min in order to selectively remove the cyanoethyl protecting groups.
Cleavage and deprotection of oligonucleotides were achieved by exposure to concentrated aqueous ammonia solution for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C.

S7
Purification was carried out by reversed-phase HPLC on a Gilson system using a Brownlee Aquapore column (C8, 8 mm x 250 mm, 300Å pore) with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) increasing from 0% to 50% buffer B over 20 min with a flow rate of 4 mL/min (buffer A: 0.1 M triethylammonium bicarbonate, pH 7.0, buffer B: 0.1 M triethylammonium bicarbonate, pH 7.0 with 50% acetonitrile). Elution was monitored by ultraviolet absorption at 298 nm. After HPLC purification, oligonucleotides were freeze dried then dissolved in water without the need for desalting.
All Purified oligonucleotides were characterised by electrospray mass spectrometry. Mass spectra of oligonucleotides were recorded using a XEVO G2-QTOF MS instrument in ESmode. Data were processed using MaxEnt and in all cases confirmed the integrity of the sequences. Figure S4. UV-Vis spectrum of GO demonstrating the increased absorbance and 540 nm compared to a wavelength of 655 nm.

S-V Target recognition in biological fluid
After demonstrating the successful response of our sensor to varying concentrations of cDNA we also investigated whether its response would be affected by the presence of biological fluids. Figure S5 shows the fluorescence signal obtained following incubation of UCNPs, hybridized to cDNA, in saliva, which confirms the function of the sensor.

S-VI RNA target recognition
The efficiency of detection of a SARS-CoV-2 oligonucleotide target was tested upon the use of a synthetic cRNA sequence, which was designed to represent the same RdRP/Hel target used through our study (see Table S1). Figure S6 shows that following hybridization of UCNPs with an increasing concentration of cRNA and upon incubation with GO, a gradual increase in fluorescence intensity was observed. was then cooled to room temperature and a mixture of NaOH (100 mg, 2.5 mmol) and NH4F (148.16 mg, 4 mmol) dissolved in 10 mL dry methanol was injected dropwise and left to stir at room temperature for a further 45 min. After that, the methanol was evaporated from the mixture for 30 min under Ar at 100 °C followed by another 30 min under vacuum. The reaction mixture was then heated to 310 °C, at a rate of 15°C/min under Ar, for 1h 20 min to form the nanoparticles. Finally, the reaction was left to cool down to room temperature. The purification of the core UCNPs was done by a series of three centrifugations (8000 rpm, 15 min). In between each centrifugation, the pellet was resuspended in EtOH (15mL) via a sonication bath. After S13 the final round of purification, the white UNCP pellet was left to dry at 80 °C before their use further experiments.

Synthesis of Core-Shell UCNPs
Core-shell UCNPs were prepared following a modified version of a previously published protocol. 28 By using a Schlenk line, YCl3·6H2O (151.68 mg, 0.5 mmol) was dissolved in a mixture of 1-octadecene (15 mL) and OA (6 mL min for the formation of core-shell UCNPs. After this, the reaction was left to cool down to room temperature before purification (three times centrifugation at 8000 rpm for 15 min and washing with EtOH). The core-shell UCNPs' pellet was collected and re-dispersed in tetrahydrofuran.

Ligand exchange with (Poly)acrylic acid (PAA)
The UCNPs surface was further functionalized in a homogeneous ligand exchange step with PAA to bring the UCNPs in water. Briefly, a solution of PAA (250 mg, MW ≈ 1.8 kDa) dissolved in tetrahydrofuran (3 mL) was added to the core-shell UCNPs coated with OA (21 mg in 7 mL tetrahydrofuran). To allow for ligand exchange, the mixture was stirred for 96 h at S14 room temperature. After the reaction, the nanoparticles were collected via centrifugation and washed with ethanol (20 mL) twice. The particles' pellet was dried and re-suspended in phosphate buffered saline and stored at 4 °C.

Synthesis and characterisations of oligonucleotide coated UCNPs
The PAA coated core-shell UCNPs were incubated with the amino-modified oligonucleotides and functionalized via the carboxylic groups on the PAA ligand using EDC amino-coupling

Sensor calibration
A stock solution of 5 mg/mL GO is prepared by dissolving 50 mg of GO in 10 mL phosphate buffered saline. The calibration was carried out by adding increasing concentrations of GO, in 0.1 mg/mL increments to a dispersion of a fixed concentration of oligonucleotide coated UCNPs (0.5 mg/mL). The corresponding fluorescence spectra of the UCNPs were monitored to determine the concentration of the GO that would result in optimum fluorescence quenching.

Targeted DNA/RNA detection using oligonucleotide coated UCNPs
The oligonucleotides on the UCNP surface were hybridized with their complementary sequence before incubating with the GO to prevent the interaction between the single-stranded DNA coated UCNPs and the GO. 0.5 mg/mL of oligonucleotide coated UCNPs were incubated in phosphate buffered saline with various concentrations of the cDNA strand (ranging from 5 S15 fM to 5 nM) while shaking. Then, a solution of GO dispersed in phosphate buffered saline was added and left incubating for 10 min before performing the fluorescence measurements.