Development of a label-free electrochemical aptasensor for Rift Valley fever virus detection

Materials and reagents

RVFV Nucleoprotein, Rubella virus-like particles, Zika Virus Envelope Protein and Dengue Virus NS1 Protein Serotypes 1–4 was obtained from The Native Antigen Company (Kidlington, United Kingdom), Pure Cube Ni-NTA (Nickel-Nitrilotriacetic acid) Agarose beads were purchased from Cube Biotech (Monheim, Germany). Magnesium chloride, sodium carbonate anhydrous, sodium azide, acetic acid, boric acid, Ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate, ethanol, Ethidium bromide solution, Tetramethyl ethylenediamine (TEMED), hydrochloric acid, acrylamide/bis-acrylamide, Bromophenol Blue, sodium acetate, ammonium persulfate (APS), Potassium ferrocyanide (K₄Fe(CN)₆), potassium ferricyanide (K₃Fe(CN)₆), sodium chloride, mercapto-1-hexanol (MCH), phosphate buffer saline (PBS: pH 7.4), Human serum and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Ontario, Canada). Tris-base and urea were purchased from Bioshop Inc. (Ontario, Canada). The DNA library (5’-TCCCTACGGCGCTAAC-N40-GCCACCGTGC-TACAAC-3’), labelled forward primer (5’-FAM-TCCCTACGGCGCTAAC-3’), unlabelled reverse primer 5′ -poly dA20-HEG-GTTGTAGCACGGTGG − 3′, M13 primers, and the aptamer sequences were synthesized by Metabion International AG (Planegg, Germany). PCR reagents (10x buffer, dNTPs, MgCl₂, Taq polymerase, 6X loading dye, and 100 bp ladder) were obtained from ACE Biotech (Riyadh, Saudi Arabia). DNase/RNase Free Distilled Water, PCR™8/ GW/TOPO™ TA Cloning Kit with One Shot TOP10 and the DH5α T1R chemically competent E. coli cells were obtained from Invitrogen Inc. (New York, USA). Amicon Ultra (0.5 mL) centrifugal filters 3 KDa were purchased from EMD Millipore (Alberta, Canada). SpinX cellulose acetate centrifuge filter tubes with pore size of 0.22 μm were purchased from Corning life sciences (Massachusetts, USA).10X TE (Tris-EDTA) Buffer Solution was purchased from Teknova (USA). Agarose powder and ampicillin were obtained from Bio-Rad (California, USA). X-Gal was obtained from Bio Basic Inc. (Toronto, Ontario, Canada). Luria Bertani Agar was purchased from watin bio-life Advanced Diagnostics mfr (Riyadh, Saudi Arabia).

The binding buffer composed of 20 mM Tris, 1 mM MgCl₂ and 150 mM NaCl, pH 8. The washing solution used in the beads coupling step was 30 mM of Imidazole prepared in binding buffer. Elution buffer consists in 300 mM Imidazole prepared in binding buffer. Tris-EDTA buffer is composed of 1 mM EDTA with 10 mM Tris (pH 7.4). The storage solution was 0.05% sodium azide in binding buffer.

Instrumentation

Bio-Rad T100 Thermal cycler (Hercules, California, USA) was used for the PCR amplification. The concentration of DNA and protein was determined using Nanodrop 2000 UV–Vis spectrophotometer (Thermo Scientific, Ottawa, Canada). The fluorescence intensity of the DNA was measured by using Nanodrop ND3300 fluorospectrometric (Thermo Scientific, Ottawa, Canada). The electrophoresis was run through Powerpack-current Power supply and supplied by Bio-Rad (California, United States). UVP BioDoc-It Imaging system (UK) was used in the DNA band analyzing. All electrochemical measurements, including cyclic voltammetry (CV) and square wave voltammetry (SWV), were conducted using a Metrohm (Switzerland) Autolab potentiostat, specifically, the PGSTAT302N model. The experimental setup was managed by Nova 1.11 software. Screen-Printed Gold Electrodes were purchased from Metrohm DropSens, Inc (Asturias, Spain). They are composed of Ceramic substrate (3.4 × 1.0 × 0.05 cm) comprising a gold auxilliary electrode and silver as reference. The working electrode diameter is 4 mm.

SELEX process

Immobilization of RVFV Nucleoprotein (N) on Ni-NTA Agarose beads

The mechanism of binding was based on the interaction between Histidine tag of the nucleoprotein and the Ni-NTA on the agarose beads (Figure S.1). The process of conjugating the RVFV nucleoprotein to the Ni-NTA Agarose beads started by applying the beads to many washes using a binding buffer to remove any residual preservative solution. Then, 100 µL of RVFV were added to tube containing 2 mL of the binding buffer. The washed beads were mixed with the protein solution at a 1:1 ratio and incubated at 4 ^◦C overnight with end-over-end rotation. Subsequently, the protein-conjugated beads underwent a washing process using 2 mL of washing solution. Following that, the beads were subjected to incubation in the washing buffer for 1 h, during which rotation was performed to block the activity of unbound sites present on the beads surface. Following the blocking of the conjugated beads, a total of five washes were performed using binding buffer. Then, the conjugated beads were stored at 4 ^◦C in TE buffer mixed with 0.05% sodium azide for further use in the SELEX process.

In vitro selection of DNA aptamers against RVFV protein

First, the DNA library and primers were designed as it was previously described by our group²³. In the first cycle of selection 100 µL of the RVFV protein-conjugated beads are incubated with 3 nmol of DNA library while the amount of 150 pmol is used in the subsequent SELEX cycles. Prior to the incubation of the beads, these latter were washed five times with 300 µL of binding buffer to remove the unbound protein and eliminate the sodium azide. In parallel, the ssDNA was subjected to a heating process at a temperature of 90 ^◦C for 5 min. Subsequently, it was cooled down to 4 °C for a period of 10 min, followed by a further 10 min incubation at room temperature. The ssDNA mixture was poured into the washed beads into the Spin-X filter tube. Following that, the filter tube was incubated at room temperature for a duration of 2 h with continuous rotation in an end-over-end method. After that, the beads were washed five times using binding buffer until the total absence of any observable fluorescence. This step was conducted to confirm the elimination of the non-bound DNA. In parallel, the bound DNA was separated using 300 µL of elution buffer and treated with heating process at approximately 90 °C for 10 min. This step was repeated until the last elution showed no fluorescence signal. Next, a centrifugal filter with a molecular weight cutoff of 3 kDa was used to concentrate and desalt the DNA that had been collected. Following the end of the seventh cycle, a negative-selection step was performed, involving the incubation of a DNA pool obtained from the previous positive cycle with blank beads; non conjugated to the RVFV nucleoprotein. After each cycle DNA was amplified by PCR generating double-stranded DNA (dsDNA) with around 72 bp (bp). Then, the dsDNA was separated into ssDNA using denaturing Urea PAGE (Polyacrylamide Gel Electrophoresis). The SELEX cycles were stopped at the 12th cycle, then unlabelled primers were used to amplify the eluted ssDNA after purification, and the final product was used in the cloning step. Forty reactions were used for the PCR amplification step, each containing 50 µL of the master mixture: 10x buffer, 10 mM dNTP, 25 mM MgCl₂, 0.2 µM primers (forward and reverse), and 2 units of Taq polymerase. The PCR thermal cycle started at 95 °C for 5 min, then 15 cycles of 95 °C for 30 s/54°C for 30 s/72°C for 45 s, and a 10 min extension at 72 °C as a final step. The amplified product was confirmed by running 2% agarose gel electrophoresis with ethidium bromide at 110 V for 30 min. Subsequently, the amplified dsDNA underwent ethanol precipitation, wherein three volumes of pure cold ethanol and 0.1 volume of 3 M sodium acetate (pH 5.5) were added. The solution was incubated for 1 h at -80 °C and then centrifuged at 4 °C for 30 min. Next, the supernatant was removed, and the pellet washed by 75% ethanol and dried at 37 °C to eliminate any residual ethanol. The pellet was reconstituted in a water and formamide loading dye mixture with a volume ratio of 1:2 and this mixture was incubated at 90 °C. Then, the fluorescence-labelled ssDNA was separated by running the mixture through a 12% denaturing urea polyacrylamide gel at a voltage of 300 V for 1 h. The fluorescence-labelled ssDNA band was visualized using a gel imager. The band was subsequently cut from the gel and the DNA was extracted by adding TE buffer, freeze/thaw cycle, followed by incubating the DNA mixture in the rotator at 37 °C overnight. Finally, the eluted ssDNA solution was desalted and concentrated by a 3 KDa filter, then used to start a new SELEX cycle.

Cloning and sequencing of the aptamers selected against RVFV nucleoprotein

After the last round of SELEX, symmetric PCR was run, then the bands were checked using 2% agarose gel. The ligation was carried out by incubating the ligation mixture: 2 µL of PCR product, 1 µL of salt solution, 2 µL of H₂O, and 1 µL of the vector at room temperature for 30 min. After that, E. coli competent cells were transferred into ice and 2 µL of the ligated product were immediately added. Then, the cells were incubated at the ice for 30 min and transferred at 42℃ for 30 s and the tube was placed on ice for 1–2 min. 250 µl of Super Optimal broth with Catabolite repression (SOC) media was subsequently added to the cells and rotated at 37 ℃ for 1 h. Next, 50 µl of ampicillin and 40 µl of X-gal were added to the plates and spread. After 1 h rotation, the cells were added on three different plates with three different volumes (25 µl, 50 µl, and 75 µl) and kept upside down at 37 ℃ for 24 h. The plates were examined to identify colonies with visible growth, and those displaying a positive white or light blue appearance were selected. The ssDNA aptamer inserts were subjected to amplification through colony PCR, using M13 forward and reverse primers. Verification of the successful incorporation of the aptamer sequences was achieved through the detection of a distinct 300 bp band via 2% agarose gel electrophoresis. Ultimately, the PCR products from the colonies were subjected to sequencing and alignment using the PRALINE program, accessible at https://www.ibi.vu.nl/programs/pralinewww/.

Binding affinity study of the aptamers to RVFV and determination of dissociation constants (Kd)

A series of fluorescein-labelled aptamers were diluted using the binding buffer (0-400 nM). Each solution was then treated with the heating/cooling cycle. Following that, the sample was incubated with 50 µL of beads and transferred into spin X filter tubes. The solution was subjected to incubation at ambient temperature for 60 min with continuous rotation. The beads underwent a washing process using 300 µL of binding buffer. This process was repeated five times. For the elution step, 300 µL of elution buffer was added to the sample. The tubes were then incubated at 90 °C for 10 min, after which centrifugation was performed at 13,000 rpm for 1 min. The eluates were collected, and the fluorescence of each sample was determined using a nanodrop fluorometer at an emission wavelength of 515 nm. Subsequently, the concentration of ssDNA input (from 0 to 100 nM) was plotted against the measured fluorescence intensity. Saturation curves have been created for each aptamer sequence. The Kd values were determined through nonlinear regression analysis using Prism software.

Fabrication of the electrochemical aptasensor

The selected aptamer was first conjugated to a thiol group to enable its immobilization on the gold electrodes. Then, 10 µL of the RVFV-aptamer (1 µM) was dropped onto the surface resulting in the spontaneous formation of a self-assembled monolayer (SAM). The incubation of the Au electrode with the aptamer was carried out overnight in a water-saturated environment at 4 °C. Subsequently, the electrode underwent a washing process using a PBS buffer solution with a pH of 7.4. The blocking of the remaining sites was accomplished by incubating the electrodes with 1 mM MCH prepared in PBS buffer (pH 7.4) for 30 min at room temperature. After washing with PBS, the electrode was ready to use for the electrochemical experiments. The label-free detection of the nucleoprotein was performed by SWV conducted in a solution containing 5 mM ferro/ferricyanide prepared in a 10 mM PBS buffer with a pH of 7.4. The SWV scan potential range was from 0.4 to -0.4 V with a step potential of -5 mV, an amplitude of 20 mV, and a frequency of 25 Hz.

The selectivity of the RVFV aptasensor toward its the targeted nucleoprotein was studied by incubating the functionalized gold surface with Rubella virus-like particles, Zika Virus Envelope Protein, Dengue Virus NS1 Protein, BSA, and human globulin. The test was performed by incubating 10 µL of the potential interferents (50 pg/mL) on the RVFV electrode. Then, the sensor response was evaluated as described above. To demonstrate the clinical applicability of the aptasensor, human serum was diluted 100 folds in 10 mM PBS buffer. Then, it was spiked with increasing concentrations of RVFV protein (50 pg/ml to 1000 pg/ml). 10 µL of each spiked serum was incubated with the RVFV aptasensor. Then, the corresponding electrochemical response was recorded and compared to that obtained in the buffer solution.