PMC:4996384 / 4779-11045 JSONTXT

{"project":"2_test","denotations":[{"id":"27600212-19562201-69481637","span":{"begin":1094,"end":1096},"obj":"19562201"},{"id":"27600212-22863116-69481638","span":{"begin":1443,"end":1445},"obj":"22863116"},{"id":"27600212-24069412-69481639","span":{"begin":1446,"end":1448},"obj":"24069412"},{"id":"27600212-24069412-69481640","span":{"begin":2293,"end":2295},"obj":"24069412"},{"id":"27600212-18331002-69481641","span":{"begin":2672,"end":2674},"obj":"18331002"},{"id":"27600212-22863116-69481642","span":{"begin":4669,"end":4671},"obj":"22863116"},{"id":"27600212-24069412-69481643","span":{"begin":4672,"end":4674},"obj":"24069412"}],"text":"2. Experimental Section\n\n2.1. Reagents and Chemicals\nGold nanoparticles (AuNPs) with a diameter of 20 nm were purchased from BBI Solutions (Cardiff, UK). Adenosine, guanosine, HO-(CH2)11-PEG-SH (MW 336.5Da) named PEG300, Bis-(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium (BSPP) and all buffer reagents were purchased from Sigma‑Aldrich (Saint Quentin Fallavier, France). CH3O-PEG-SH (MW 2000 Da) named PEG2000, was purchased from Rapp Polymere GmbH (Tübingen, Germany). Different concentrations of adenosine and 1 µM guanosine were prepared in the SPR running buffer (HEPES 10 mM, MgCl2 5 mM, NaCl 150 mM and 0.005% Tween20, pH 7.4). Ultrapure water (18.2 MΩ·cm) was used throughout.\nThe oligonucleotides were purchased from Eurogentec (Angers, France) with a thiol modification at their 5' position and a thymine spacer (five or ten thymine nucleotide) before the sequence of interest. Sequences are listed in Table 1. APT4 and APT8 correspond to the full adenosine aptamers and Split‑APT4, Split‑APT8 and Split-APT are their split sequences, designed according to the literature [17]. CN8 sequence was used as a negative control.\nmicroarrays-04-00041-t001_Table 1 Table 1 Oligonucleotide sequences.\n\n2.2. DNA Microarray Fabrication\nThiolated oligonucleotides were grafted on gold-coated prisms (Horiba Scientific-GenOptics, Orsay, France) as Self-Assembled Monolayers (SAM) according to the protocol described in previous work [21,22]. Briefly, the gold surface lying on the prism was first cleaned by plasma treatment (0.6 mbar, 75% Oxygen, 25% Argon, power 40 W, 6 min) in a plasma generator (Femto, Diener Electronic, Ebhausen, Germany). Then, droplets of approximately 4 nL of HK2PO4 buffer (1 M, pH 9.25), containing a mixture of 20 µM of thiol-modified DNA probes and 10 µM of PEG2000 or PEG300 solution, were deposited on the surface using a piezoelectric dispensing system (Siliflow, Valence, France). The deposition was done under a controlled atmosphere of 85% humidity and left at room temperature for 60 min. After overnight drying, prisms were thoroughly rinsed with deionized water and dried under argon stream for few seconds. The size of the spots is approximately 500 µm in diameter, with a 1.3 mm pitch. The probes coverage surface is estimated to 8 pmol/cm² [22]. Prior to the SPR measurements, the functionalized prism surface is blocked with a PEG2000 or PEG300 solution (150 µM) for 60 min incubation at room temperature. Then, surfaces were rinsed with deionized water and dried under argon stream.\n\n2.3. AuNPs-ssDNA Conjugation\nGold nanoparticles modified by ss-DNA were prepared according to the literature with some modifications [29]. In a typical experiment, to 1mL of citrate coated AuNPs solution (1.16 nM), 3 mg of BSPP was added, and the mixture was stirred at room temperature in order to allow phosphine ligands to replace the citrate ligands. Following overnight incubation, 2 mg of NaCl were added to the stirring mixture until the color change from red to cloudy purple. The solution was centrifuged at room temperature for 10 min at 16750 G to collect the precipitated AuNPs. The supernatant was removed and the pellet re-suspended in BSPP buffer (1 mg/mL of BSPP). Nanoparticle purification and concentration steps were carried out in a Sigma Laborzentrifugen 3K15 centrifuge.\nTo functionalize AuNPs with ssDNA conjugates, 10 µL of 5'-thiolated DNA at 10 µM (PBS, pH 7.4) was mixed with 70 µL of the concentrated gold colloid prepared in the first step, 10 µL of PBS (pH 7.4) and 10 µL of BSPP buffer (1 mg/20 µL of BSPP). The mixture was incubated under gentle agitation at room temperature for 24 h, then 7 µL of PEG solution at 150 µM (PBS, pH 7.4) was added and the solution was incubated with gentle agitation for a further 2 h. The solution was then centrifuged (10 min, 16750 G) to remove the excess of reagents. The supernatant was discarded and the precipitate was washed with the SPR running buffer. After a second centrifugation, the final precipitate was re‑suspended in 1 mL of SPR running buffer. UV-visible absorption spectroscopy was used for optical characterization of the AuNPs-ssDNA conjugates. The measurements were taken using a ND-1000 Nanodrop (Labtech International, Uckfield, UK) spectrometer. AuNPs and AuNPs-ssDNA conjugates were quantified by measuring the absorbance at λ = 520 nm and calculating the concentration using Beer’s law based on their extinction coefficients: ε520 (20 nm) = 8.8 × 108 M−1·cm−1. The maximum UV–Vis absorption peak of unmodified AuNPs and AuNPs-ssDNA conjugates in solution were 520 nm and 528 nm, respectively.\n\n2.4. SPRi Measurements\nAs for previous work [21,22], the SPRi experiments were carried out using a SPR imager apparatus (SPRi-Lab, Horiba Scientific-GenOptics) equipped with an incoherent light source (λ = 635 nm). The reaction chamber consists of a hexagonal reactor PEEK flow cell (~15 µL of volume). The flow cell was connected to PEEK tubing coupled with a degassing system (Alltech, Carquefou, France) and a syringe Cavro pump (Tecan, San Jose, CA, USA). The experiments were performed at 25 °C. All the injections were dispensed using a 500 µL injection loop. The SPR data were acquired using the software furnished by Horiba Scientific-GenOptics. Acquisition of the reflectivity signal, registered with a 12-bit camera, was launched upon stabilization of the baseline. The reflectivity values were averaged over the replicates of each spot series and plotted upon time.\nFor the hybridization experiments, each ssDNA injection (at 1 µM) or AuNPs-ssDNA conjugates injection (at 200 pM) was dispensed via a flow rate of 50µL/mL or an alternating back-and-forth flow mode (15 µL dispensed volume, 10 µL aspirated volume, 50 µL/min flow rate). For biochip recycling, 50 mM NaOH was injected for 8min in order to denature the hybridized complementary strands of DNA and regenerate single stranded DNA on control spots. Concerning the adenosine detection assays, a series of adenosine solutions (from 1 nM to 1 µM) were co-injected with AuNPs-SplitAPT conjugates (200 pM) under the alternating flow. To assess the target-specificity of the sensing system, 1 µM guanosine solution was also co-injected with AuNPs-SplitAPT conjugates (200 pM).\n"}