Immobilization of oriented protein molecules on poly(ethylene glycol)-coated Si(111).
A high-density poly(ethylene glycol) (PEG)-coated Si(111) surface is used for the immobilization of polyhistidine-tagged protein molecules. This process features a number of properties that are highly desirable for protein microarray technology: (i) minimal nonspecific protein adsorption; (ii) highly uniform surface functionality; (iii) controlled protein orientation; and (iv) highly specific immobilization reaction without the need of protein purification. The high-density PEG-coated silicon surface is obtained from the reaction of a multi-arm PEG (mPEG) molecule with a chlorine terminated Si(111) surface to give a mPEG film with thickness of 5.2 nm. Four out of the eight arms on each immobilized mPEG molecule are accessible for linking to the chelating iminodiacetic acid (IDA) groups for the binding of Cu(2+) ions. The resulting Cu(2+)-IDA-mPEG-Si(111) surface is shown to specifically bind 6x histidine-tagged protein molecules, including green fluorescent protein (GFP) and sulfotransferase (ST), but otherwise retains its inertness towards nonspecific protein adsorption. We demonstrate a particular advantage of this strategy: the possibility of protein immobilization without the need of prepurification. Surface concentrations of relevant chemical species are quantitatively characterized at each reaction step by X-ray photoelectron spectroscopy (XPS). This kind of quantitative analysis is essential in tuning surface concentration and chemical environment for optimal sensitivity in probe-target interaction.
Cha
Taewoon
T
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Guo
Athena
A
Jun
Yongseok
Y
Pei
Duanqing
D
Pei
Duanquing
D
Zhu
Xiao-Yang
XY
D002713:Chlorine / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004577:Electron Probe Microanalysis; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007098:Imino Acids / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D015233:Models, Statistical; D011092:Polyethylene Glycols / Q000737:chemistry*; D040081:Protein Array Analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D012825:Silicon / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D007098:Imino Acids; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D002713:Chlorine; D003300:Copper; C008109:iminodiacetic acid; D012825:Silicon