Surface Modification Of COC Substrate By UV-Induced Graft Polymerization For Preparing Biomolecular Micro Array Chips

High density immobilization of biomacromolecules has advantages of high throughput,miniaturization,fast handling,which has been broadly used in the bio-related field including cell analysis,disease diagnostics,drug screening and gene sequencings.Substrate materials are the basis for the preparation of microarray chip,and developing new substrates and exploring matched surface chemical modification methods were significant for the development of microarray chips technology.Cyclic olefin copolymer(COC)was an ideal substrate for the preparation of polymer-based microarray chips because of its high glass transition temperature,excellent optical properties,low autofluorescence,good resistance to chemical solvent and processability.However,the low surface energy and reaction inertness of COC surface greatly restrict its application as substrate of microarray chips.UV-induced graft polymerization has many advantages such as short modification time,easy operation,no damage to the bulk properties of substrate and precisely temporal/spatial controllability,which was suitable for the surface modification of polymer substrates.Here three strategies aiming to surface modification of COC surface were developed based on the UV-induced graft polymerization.The inert surface C-H bonds of COC were converted into functional reactive groups or reactive micro-/nano-structures by these strategies for the preparation of biomolecular microarray chips.And the main research contents and results were listed as follows.1.A simple but effective method was developed by grafting anhydride groups on the COC surface to prepare protein microarray with a high immobilization density.In this work,the copolymer brush of maleic anhydride(MAH)and vinyl acetate(VAC)was introduced on the COC surface by UVinitiated graft polymerization.The introduction of poly(MAH-co-VAc)brushes on COC substrate was confirmed by the attenuated total reflection Flourier transformed infrared(ATR-FTIR)spectroscopy,X-ray photoelectron spectroscopy(XPS)and the water contact angle measurement.Grafting poly(MAH-co-VAc)brushes resulted in a decrease in transmittance at wavelengths below 350 nm,but hardly influenced the transparency of the COC substrate in the visible light range,suggesting that this surface modification strategy will not impair the transparency of COC.The influence of monomer concentration and irradiation time on the graft density of anhydride group was investigated and the highest graft density of 3.2 ?mol/cm2 could be obtained with the monomer concentration of 7.5 M and irradiation time of 4 min.The graft density of the anhydride groups on the modified COC was ranged from 0.46 ?mol/cm2 to 3.2 ?mol/cm2.Different concentrations of rabbit anti-rat IgG were immobilized on the COC surface through the ring-opening reaction between the anhydride group and the amino group.When the concentration of immobilized IgG was 17 ?g/mL,the highest immobilization efficiency and density were 88%and 0.168 ?g/cm2,respectively.The immunoassay experiment demonstrated that the microarray exhibited a satisfactory response to target goat-anti-rabbit IgG with a wide dynamic range of 3.91-2000 ng/mL.And the limit of detection(LOD)was 7.81 ng/mL.2.A strategy to construct three-dimensional(3D)nano-structure on COC for the high density immobilization of DNA probes was developed by using UV-induced emulsion graft polymerization technique.Firstly,grafting polymerization of methyl methacrylate(MMA),glycidyl methacrylate(GMA)and ethylene glycol dimethacrylate(EGDMA)on the COC surface was achieved under the UV irradiation.The effect of monomer concentration on surface morphology of modified COC was investigated.It was observed by the scanning electron microscopy(SEM)and atomic force microscopy(AFM)that a uniform 3D graft layer composed of loosely stacked nanoparticles with a diameter of about 50 nm could be formed when the monomer concentration was 18 wt%.The optical transparency of the modified COC substrate was characterized by UV-Vis spectrophotometry.The introduction of 3D structure on the surface substrate has little influence on the optical transparency,and it was only decreased by 5%in the wavelength around 550 nm and 650 nm.DNA probes with different concentrations were immobilized on the 3D structure modified COC by reaction of end-capped amine groups of DNA probe and surface epoxy groups.The influence of monomer and DNA probe concentration on the immobilization efficiency of DNA probes was investigated.When grafting with 18wt%monomer and incubating with 5 ?M Probe A,the immobilization efficiency of DNA microarray could reach the maximum of 93%,corresponding to the immobilization density of 1.8 pmol/cm2.Compared with the one-dimensional(1D)and two-dimensional(2D)structure modified COC microarray chips,the immobilization efficiency of probes on the 3D structure was significantly increased by 2-4 times.Finally,the sensitivity of the 3D DNA microarray chip was studied by the hybridization experiment with the target DNA.The highest hybridized density and the LOD was 0.43 pmol/cm2 and 75 pM,respectively.The microarray was able to discriminate the single,two and three base mismatched sequence in single nucleotide polymorphism(SNP) discrimination test.3.A method of high density immobilization of peptide probes on COC surface was developed by combining UV-induced graft polymerization and enzyme-catalyzed reaction.Firstly,the 3D hydrogel column microarray was introduced on the COC surface by the UV-induced graft copolymerization of acrylic acid(AA)and polyethylene glycol diacrylate(PEGDA).Secondly,the Poly-L-lysine(PLL)molecules were coupled to the hydrogel microarray through the condensation reaction between the carboxyl group and the ?-amino group in the PLL.Peptide probes was conjugated to the PLL-modified hydrogel microarray via acyl transfer reaction between the y-amide groups in glutamine groups of peptide probes and ?-amino groups in PLL catalyzed by glutamine transaminase(MTG).The influence of pH value,ionic strength,immobilization temperature and reaction time on the reaction efficiency were explored.The optimum enzyme-catalyzed reaction conditions were at 40? for 2 h and in acetic acid-sodium acetate buffer solution of pH=5.According to the measurement of SEM results,the height of the hydrogel microarray could be adjusted by the concentration of PEGDA.When the concentration of PEGDA increased from 0 to 30 wt%,the height of the hydrogel column increased from 0.95 ?m to 8.1?m.The maximum immobilization density could reach 0.14?g/cm2 when the height of the hydrogel column was 7.23 ?m.The PLGLA sequence in the peptides could be specifically cut off by the matrix metalloproteinase 2(MMP-2).Thus,the 3D peptides microarray chip could be used to detect MMP-2 and the LOD was 25.8 ng/mL.