Zepto-molar electrochemical detection of Brucella genome based on gold nanoribbons covered by gold nanoblooms

Gold nanoribbons covered by gold nanoblooms were sonoelectrodeposited on a polycrystalline gold surface at −1800 mV (vs. AgCl) with the assistance of ultrasound and co-occurrence of the hydrogen evolution reaction. The nanostructure, as a transducer, was utilized to immobilize a Brucella-specific probe and fabrication of a genosensor, and the process of immobilization and hybridization was detected by electrochemical methods, using methylene blue as a redox marker. The proposed method for detection of the complementary sequence, sequences with base-mismatched (one-, two- and three-base mismatches), and the sequence of non-complementary sequence was assayed. The fabricated genosensor was evaluated for the assay of the bacteria in the cultured and human samples without polymerase chain reactions (PCR). The genosensor could detect the complementary sequence with a calibration sensitivity of 0.40 μA dm3 mol−1, a linear concentration range of 10 zmol dm−3 to 10 pmol dm−3, and a detection limit of 1.71 zmol dm−3.

Os-C1 Au Amp, CV 600 fM 1.0-300 pM -S11 5'-HS(CH2)6GCGTTCCAAAGGGCAGGA TCATTGA-3' Os-C2 Au CV, DPV 6 pmol 5-20 pmol Helicobacter pylori S12 5'-GGGGCAGAGCCTCACAACCT-(CH2)3-SH-3' AQMS Au CV, OSWV 0.5 nM --S13   Legends of supplementary materials: S1: Variation of OCP of the Au/nAu electrode during the self-assembling process of p-ssDNA on its surface. S2: Cyclic voltammograms MB obtained using the genosensor (intercalated MB) and the Au/nAu electrode ( M MB present in the solution phase). The potential sweep rate was 50 mV s -1 . S3: A comparison of the figure of merit of some genosensors. S4: DPVs of the genosensor recorded for the repeating fabrication. S5: DPVs recorded for the regeneration of the genosensor. Blue curves show DPVs for the genosensor before hybridization, and red curves show DPVs for the genosensor before re-hybridization. S6: Variation of the peak current in DPVs for 1.0×10 -10 mol dm -3 t-ssDNA over 39 days to check the stability of the genosensor. S7: DPVs for MB recorded using the genosensor at different concentrations of the Brucella DNA genome (A), and the dependency of the peak current on concentration (B). S8: DPVs for MB recorded using the genosensor at two concentrations of bacterial genomes of Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella and Acinetobacter (negative controls). S9: DPVs for MB recorded using the genosensor for the healthy and patient samples.

Materials
All chemicals were of analytical grade from Merck (Germany) or Sigma (USA) and were used without further purification. All solutions were prepared by redistilled water. A 24 base thiolated oligonucleotide probe (probe oligonucleotide, p-ssDNA) was designed based on the common genomic sequence in all the Brucella species. p-ssDNA was The oligonucleotide stock solutions were prepared with 20 mmol dm -3 Tris-HCl buffer, pH 7.4 solution (Tris) and were kept frozen.

Apparatus
Electrochemical measurements were carried out in a conventional three-electrode cell powered by a -Autolab potentiostat/galvanostat (the Netherlands). An Ag/AgCl, 3 mol dm -3 KCl, a platinum wire, and a bare (Au, 2 mm of diameter) or modified gold disk electrode with gold nanoribbons covered by gold nanoblooms (Au/nAu) were used as the reference, counter and working electrodes, respectively. The system was run on a PC by In order to obtain information about the morphology and size of gold nanostructures, field emission scanning electron microscopy (FESEM) was performed using a Zeiss, Sigma-IGMA/VP (Germany). The samples were coated by a 2-5-nm thin film of gold through sputtering.

Preparation of the modified electrode
Before sonoelectrodeposition of gold nanoribbons was covered by gold nanoblooms, the Au electrode was polished on a sand papers and then on a polishing pad with 50 nm-alumina powder lubricated by glycerin. Polishing was continued to attain a mirror-like Au electrode surface. The electrode was then cleaned by immersion in a 1:3 water/ethanol mixture and ultrasonication for 8 min in an ultrasound bath. The electrode was further electropolished by immersion in a 500 mmol dm -3 H 2 SO 4 solution and applying potential in the range of cathodic to anodic edges of the electrolyte stability in a regime of cyclic voltammetry for 20 consecutive cycles. Upon this pretreatment, clean and stable Au electrode surface was attained. The Au electrode was then placed in the cell containing the synthesis solutions comprising 5 mmol dm -3 HAuCl 4 + 0.5 mol dm -3 KCl. Sonoelectrodeposition of gold nanoribbons covered by gold nanoblooms was performed at 1800 mV for 300 s, while the synthesis solution and also the Au electrode surface were irradiated by ultrasound wave of 45 W power. The modified electrode was then rinsed thoroughly with distilled water. For preparation of the Au/nAu electrode for open circuit potential (OCP) measurements, an Au screen-printed electrode was directly transferred to the synthesis solutions and the sonoelectrodedeposition process was done similarly.

Immobilization of p-ssDNA
Immobilization of p-ssDNA probe was performed by dropping 10.0 L of 10.0 mol dm -3 p-ssDNA solution dissolved in Tris on the Au/nAu electrode surface and kept refrigerated at 4 C for 8 h. Then the electrode was rinsed with Tris. The DNA-modified Au/nAu electrode was further treated with 1.0 mmol dm -3 6-Mercapto-1-hexanol at room temperature for 30 min to obtain a well aligned p-ssDNA monolayer. Then the electrode was washed again with Tris and double distilled water, respectively, to remove unspecific absorbed p-ssDNA. The obtained electrode was denoted as the genosensor.

Hybridization
The hybridization process was performed by immersing the genosensor into Tris containing various concentrations of t-ssDNA for 1 h at 37 C. Then, the electrode was rinsed with Tris to remove the un-hybridized t-ssDNA, and then it was incubated in a solution containing Tris + NaCl (20 mmol dm -3 ) + methylene blue (MB, 20 mol dm -3 ) for 5 min at 37 C. Finally, the electrode was rinsed with Tris to remove the physically absorbed MB.

Electrochemical measurements
The real surface area of the Au/nAu electrode was measured electrochemically. The Au/nAu electrode was transferred to a solution of KCl (0.5 mol dm -3 ) containing Electrochemical detection of the DNA hybridization was performed in an electrochemical cell containing 10 mL Tris by recording cyclic voltammograms (CVs) and differential pulse voltammograms (DPVs) for the reduction peak of intercalated MB.
The parameters of DPV recording were pulse width of 25 mV, a pulse time of 50 ms, and a scan rate of 10 mV s -1 . The concentration of t-ssDNA was quantified as the MB reduction peak current.
Bacteria culture, human samples, and genomic DNA extraction Brucella agar culture medium with the addition of 5% horse blood was used for the cultivation of Brucella organisms by incubation at 35±2 °C for 48-72 h. 7 human serum samples with Brucellosis (Wright and 2ME tests were positive in all samples) were provided from veterinary administration of Fars province. It should be noted that a healthy blood sample was evaluated with suspicious samples. From 7 human serum samples with Brucellosis, 6 were measured after amplification by PCR and one sample was measured without amplification. Before performing the required investigations, the genomes were placed for 5 min at 90 ° C.