In situ Synthesis of Oligonucleotides on Plasma Treated Polypropylene Microporous Membrane

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【刊名】Chinese Chemical Letters


Nowadays much attention has been focused on highly parallel DNA hybridization assayson miniaturized gene-chips'. The substrates for in situ synthesis of DNA microarrayare usually glass slides developed by Southern et al.2, or silicon wafers. However,when fabricating a gene-chip by means of in situ synthesis3, there is limitation to theprobe density and coupling efficiency, so better substrate materials remain to bediscovered2. Recently, plasma treatment has been used to alter the surface properties ofpolymers without changing their bulk properties4-8. We here report the successfulgrafting of -NH2 on a polypropylene microprous membrane by simple plasma-treatmentin a mixture of nitrogen(N2) and hydrogen(H2). The modified microporous polypro-pylene membrane serves well as a substrate for the in situ synthesis of oligonucleotidesand an average coupling yield was more than 98%. The polypropylene microporous membrane (diameter=47 mm, average aperture=0.45 μm, named as membrane A in this paper) and the reference porous polypropylenemembrane (AP01-81, designated as membrane B in this paper) were obtained fromGermany Pall Corporation. The latter was polyacrylamide modified with PEG (poly(ethylene glycol)) spacer, loading with free primary amino functions (0.6 μmol/cm2).The commercially available ultra-high pure gases (purity of N2 99.9%; H2 99.99%)were used without further purification. Generally, by means of the interaction ofplasma on the polymer surfaces in mixture of N2 and H2, the structures of the membraneswere changed and grafted with amino groups. Our typical treatment condition is:discharge power of 80 W, treatment time of 120s and vacuum of 2 Pa. The fluxes of N2and H2 were 30 mL/min and 60 mL/min, respectively. The gases were introducedthrough flow controllers and the pressure was about 150 Pa. As shown in Figure 1a for the attenuated total reflectance Fourier transforminfrared spectroscopy (ATR-FTIR) on the Nicolet Nexus 870 FTIR instrument with aDTGS detector at a resolution of 4 cm-1 and 64 scans., it is found that the untreatedmembrane A exhibits no absorbance at 3299.9, 1727.3, 1638.3 and 1544.5 cm-1.However, after the plasma treatment all the above four bands are present in Figure 1band c (b: face, c: inverse). The band 1638.3 cm-1 is usually assigned to the N-Hbending vibration in primary amine and amide (R-NH2, R-CONHR), and the band1544.5 cm-1 to the N-H deformation, the N-H bending and the C-N stretching vibrationin secondary amine (R-NHR). The weak absorbance band at 1727.3 cm-1 is related tothe polar carbonyls and the broad band with a maximum at ~3299.9 cm-1 to amine andamide (R-NH2, R-CONH). The above observation consists with that reportedpreviously8-9, demonstrating the plasma treatment leads to the grafting of amino groupsand others amides. Moreover, it seems that the intensity of the absorbance for theinverse side (Figure 1c) is slightly weaker than that for the face (Figure 1b), meaning apossible difference in bombarding strength of plasma action for both surfaces. Thiswas taken into consideration of performing characterization and in situ oligonucleotidesynthesis in our investigation. The above plasma grafting was further verified by XPS analysis of the treatedmembrane A. Three peaks having binding energies at about 285 eV, 400 eV and 530eV were observed10, where are ascribed to C1s, N1s and O1s, respectively, clearlyindicating the existence of N atoms in the treated surface. The N1s photoelectron peak Figure 1 ATR-FTIR spectra of the membrane A surfaceIn situ Synthesis of Oligonucleotides 399of the modified membrane A (not shown here) was deconvoluted into two componentsby peak fitting. The first at 398.57 eV is attributed to -N-C nitrogen (80.82%) and thesecond at 400.25 eV can be attributed to imine (C=N) or amide (-N-C=O) (19.18%)11-13,verifying the success of plasma grafting of groups associated with N for membrane A. The plasma modified membrane A was then submitted to automatic oligonucleotidesynthesis (5'-NH2-AAC CAC CAA ACA CAC-3') as previously described according tothe standard phosphoramidites chemistry protocol3, except that the modified membraneA (1 cm2) was placed into the closed sandwich-like fluidic reactor system jointed to theModel 391 DNA synthesizer. It is known that if a mole of nucleoside monomers (dA,dT, dC and dG) were coupled to the treated membrane or the previously synthesizedoligonucleotide, a mole of dimethoxytrity (DMT), which is used to protect the 5'-hydroxide (5'-OH) group of the nucleoside monomers, will be eliminated at the nextdeprotection step. The thin solution of DMT displays a highest absorption at 498 nmaccording with the Beer's Law. Checking the absorbance of DMT of four parallel tests(Shimadzu UV-2201 spectrophotometer), an average coupling yield was more than 98%for each step and it is very true for in situ synthesis of DNA. Our previous studyindicated that the coupling efficiency of the prime 2-6 bases was in the range of 88% ~93% on a modi

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