The phosphorous amide approach is of the highest coupling efficiency among the DNA in situ synthesis approaches at present. The strict anhydrous condition is required in the process of DNA in situ synthesis, and the DNA synthesis in the molecular stamp method must be performed in the anhydrous glove box. Because various volatile reagents remain in the glove box, the reagent gases invade into the coupling reaction system so that the coupling efficiency is decreased. This decrease is caused by the pyridine contained in the oxidizing agent. An improved oxidizing agent system of I 2/Ac 2O/AcOH/THF for the synthesis of oligodeoxy-nucleotides was reported. Using this oxidizing agent, 16-mer oligodeoxynucleotide probes were successfully synthesized on the CPG(controlled pore size glass) or the modified glass slide and a single step coupling efficiency of 98.2% was achieved. The synthesized DNA probes were hybridized with fluorescence and gold-labeled target oligodeoxynucleotides respectively. The fluorescence intensity and gray level for the synthesized probes in the I 2/Ac 2O/AcOH/THF oxidizing agent were similar to those in the traditional phosphorous amide approach possessing a water- and pyridine-containing oxidizing agent I 2/H 2O/Pyridine/THF. The reported oxidizing agent will make the contact printing approach to in situ synthesized DNA microarrays more feasible by eliminating the decrease of coupling efficiency which is because the cross reagent interfering arises from pyridine and water.
The factors that influence the colorimetric gene detection of gold label silver stain and improve the detection signals were studied. The influence of amino DNA probes and thiol DNA modified gold nanoparticles was investigated based on a sandwich hybridization system. An increase in amino probe concentration brought about an increase in hybridization signal which reached a threshold corresponding to the saturated concentration of amino probes bounded onto a glass slide surface. Since the steric hindrance effect of nanoparticles was dominant over the influence of a surface area, the bigger gold nanoparticles led to weaker hybridization signals. The hybridization efficiency enhanced significantly with the increase of the thiol DNA modified nanoparticle concentrations. Experimental results showed that 125 μmol/L of the amino DNA probe concentration, 15 nm of the gold nanoparticle diameter, and 4.07 nmol/L of the thiol DNA modified gold nanoparticle concentration were optimal for the detection system. The hybridization signals can be improved remarkably by choosing optimal hybridization conditions.
A sensitive method is described for the determination of trace antimony based on the antimony-bromopyrogallol red (BPR) adsorption at a carbon paste electrode (CPE). Three steps were involved in the overall analysis: preconcentration,reduction and stripping. Optimal conditions were found to be an electrode containing 25% paraffin oil and 75% high purity graphite powder as working electrode;a 0.10 mol/L HCl solution containing 40 μmol/L BPR as accumulation medium;a 0.20 mol/L HCl solution as reduction and stripping electrolyte;accumulation time,150 s;reduction potential and time,-0.50 V,60 s;scan range from -0.50 to 0.20 V. Interferences by other ions were studied as well. The detection limit was found to be 0.5 nmol/L for 150 s preconcentration. The linear range was from 1.0 nmol/L to 0.50 μmol/L. Application of the proposed method to the determination of antimony in water and human hair samples gave good results.
Microporous polyamide-6 membranes were hydrolyzed in 0.01 mol/L NaOH/CH 3OH medium by refluxing for about 36 h. By investigating the UV spectra, the hydrolyzing conditions were optimized. A pH value of 12 and reaction time of 36 h are the preferred conditions for hydrolysis. The treated membrane can be applied for in site synthesis of oligonucleotide and, for example, the oligonucleotide probes of 5′-AACCACCAAACACAC-3′ were successfully synthesized on the hydrolyzed membrane,which was proved by the high resolution X-ray photoelectron spectroscopy(XPS) of P 2p. The single step coupling efficiency was determined by UV spectra to be in excess of 98 %.
