Sequence-selective recognition of nucleic acids using nanoparticle probes
A technology of nucleotide sequences and metal nanoparticles, applied in nanotechnology for sensing, nanotechnology, nanotechnology, etc., can solve the problems of difficult preparation and low solubility of PNA/nanoparticle conjugates
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Embodiment 1
[0119] Example 1: Materials
[0120] Chemical reagent: tetrachloroauric(III) acid trihydrate (HAuCl 4 ·3H 2 O), trisodium citrate, Triton X-100, sodium dodecyl sulfate (SDS), Zonyl FSN-100 (F(CF 2 CF 2 ) 3-8 CH 2 CH 2 O(CH 2 CH 2 O) 0-15 H), tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St Louis, MO). 40-nm silver nanoparticles were purchased from Ted Pella (Redding, CA). All other reagents certified as analytically pure were used as received. Morpholino oligomers were purchased from Gene Tools LLC (Philomath, OR). oligonucleotides from 1 st Acquired by Base Pte Ltd (Singapore).
[0121] Instrument: with NanoDrop TM A 1000 spectrophotometer (Thermo Scientific) was used for quantification of morpholino oligonucleotides and ssDNA samples. The absorption spectrum of the gold nanoparticle colloid was collected with an Agilent G1103A UV-Vis spectrophotometer. This spectrophotometer was also used for melting analysi...
Embodiment 2
[0122] Example 2: Synthesis of gold nanoparticles
[0123] By reducing HAuCl with citrate as described by Grabar, K.C. et al., Anal.Chem., 1995, 67, 735-743 4to prepare gold nanoparticles with average diameters of ~40 nm and ~13 nm, respectively. All glassware used to prepare gold nanoparticles was sequentially filled with freshly prepared aqua regia (HNO 3 :HCl=1:3), rinse thoroughly with ultrapure water, and then dry in an oven at 100°C for 2-3 hours. 60mL 0.01% (w / v) HAuCl 4 Boil in a round bottom flask with reflux condenser and stir vigorously. For gold nanoparticles with an average diameter of ~40 nm, add 0.6 mL of 1.0% (w / v) sodium citrate to HAuCl 4 solution. For gold nanoparticles with an average diameter of ~13 nm, add 4.5 mL of 1.0 wt% sodium citrate to HAuCl 4 in solution. The reaction mixture was maintained at boiling point and stirring was continued for about 15 minutes. The suspension was stored at 4°C until subsequent use. Assuming spherical particles a...
Embodiment 3
[0124] Example 3: Preparation of metal nanoparticles modified with mercapto-morpholino oligonucleotides
[0125] By activating the sulfhydryl groups, the 3'-disulfamide-modified MO was treated with 0.1 MDTT in 0.2M phosphate-buffered saline (PBS, pH 8.0) for 1 hour; the thiolated MO was then purified using a NAP-5 column (GE Healthcare). The purified thiolated MO samples were stored at 4°C until subsequent use. To avoid interchain disulfide formation of the thiolated MO chains, the purified MO was dissolved in 5 mM tris(2-carboxyethyl)phosphine (TCEP) (pH 7.5) before mixing with gold or silver nanoparticles. Incubate for 10 minutes. Unless otherwise stated, the mixture solution containing ˜2 μM thiolated MO, ˜2 nM or 4 nM FSN-capped nanoparticles, ˜0.1 wt % SDS, and 10 mM phosphate buffer (pH 7.5) was incubated at room temperature for 1 h or 2 h. Hour. Then, excess ssDNA was removed by centrifugation at 7.0K rpm for 10 minutes. Unreacted MO was removed by centrifugation at...
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