A biotin-hydrogel-coated quartz crystal microbalance biosensor and applications in immunoassay and peptide-displaying cell detection.
A biotin-coated quartz crystal microbalance (QCM) chip was prepared by dip-coating a long-chain alkanethiol-modified crystal with precoupled dextran-biotin hydrogels. The resulting biotin chip was used to affinity-immobilize streptavidin (SAv) and was then further employed for various biosensor assays. First, the SAv chip allowed efficient on-line binding of biotinylated bovine serum albumin (bBSA), followed by a sensitive and specific response toward anti-bovine serum albumin (BSA) antibodies. Three consecutive immunoassays were reproducibly demonstrated with a single chip. The apparent binding kinetics with k(on)=5.9 microM(-1) h(-1), k(off)=10.1 h(-1), and K(D)=1.71 microM was readily resolved by fitting the real-time sensorgrams. Second, the capability of the SAv chip to selectively recognize recombinant Escherichia coli with flagella displaying an artificial SAv binding peptide, Strep-tag II, was demonstrated by QCM analysis and verified by scanning transmission electron microscope (STEM) image analysis with biotin-coated gold nanoparticles as the label. Finally, the affinity of the cell-displayed Strep-tag II peptide to surface-coated SAv, K(D)=6.8 x 10(8) CFU/ml, was resolved on-line using equilibrium binding kinetics by QCM. This study presents an easy, economical, and reliable method of preparing high-performance SAv-coated biotin chips with potential for application in real-time repetitive immunoassays, on-line binding kinetics studies, and high-affinity peptide screening.
Chen
Hsiu-Mei
HM
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan. hsiumei@mail.ntust.edu.tw
Huang
Tsun-Han
TH
Tsai
Ruey-Ming
RM
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001710:Biotin / Q000032:analysis* / Q000737:chemistry; D002417:Cattle; D003460:Crystallization; D004926:Escherichia coli / Q000737:chemistry; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D046208:Microchip Analytical Procedures / Q000379:methods; D046529:Microscopy, Electron, Transmission; D015394:Molecular Structure; D019151:Peptide Library; D011791:Quartz / Q000737:chemistry*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000276:immunology
D019151:Peptide Library; D011791:Quartz; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D012710:Serum Albumin, Bovine; D001710:Biotin