7. Chemical Modification for Nonspecific Adsorption
Nonspecific adsorption of biomolecules is simple and moderate. For WGMB
applications, microspheres acts as the solid support. Plasma cleaned spheres introduce
surface hydroxyl groups which interact with methoxy/ethoxy components of the silanes.
For our nonspecific adsorption experiments, we use 3-aminopropyltriethoxysilane1
(APTES) leaving a rug of amines as reactive groups (Fig.9). We also use a modified
technique for vapor phase deposition in a vacuum chamber2. Briefly, 1 mL of fresh
APTES is placed into a vacuum chamber, refluxed briefly, slowly pressurized, and cured
in a hot oven at 120° C. NH2 carries a pKa of about 8, meaning at our pH buffer (PBS
7.4), it takes on a proton becoming NH3+, highly attractive to negatively charged
molecules.
Fig. 9. Amino-silanization using APTES on cleaned microsphere support.
This type of modification was used for all nonspecific binding experiments which will be
discussed next.
8. Molecular and Artificial Examples: BSA, Antibody IgG, Polystyrene Spheres
BSA is an abundant protein found in the serum of bovine having a molecular
weight of 66,000 g/mol. A BSA layer on a counter-charged glass surface has a thickness
of about 3 nm3,4. Binding experiments were first conducted to demonstrate the ability of
the instrument to sense small-molecule protein adsorption5 and to correlate the
measurements with thickness estimation based on theory mentioned earlier. With a pI of
4.7, BSA has a net negative charge in pH 7.4 buffer. Therefore, our amino-silanized
spheres are attractive to these biomolecules. 20 μl of a 50 μM solution of BSA in 10 mM
phosphate buffered saline was mixed with 980 ul of PBS buffer bringing the final
concentration of protein to 1 M. Sample was continuously stirred and resonant dip
traces were tracked (Fig. 10). Complete saturation was observed in ~35 seconds.
Fig. 10 BSA adsorption. Resonance dip trace from 1310-nm laser recorded over 5
minutes.
In equation (25) we relate the wavelength shift of binding events to thickness of a
monolayer. We measure a thickness of ~2.1 nm indicating a surface coverage of xxx.
(discuss calculation with Dr. Arnold).
Antibody IgG too is a protein with a molecular weight of 155,000 g/mol, about
2.5 times the size of BSA. It has a pI of around 6.8 so similar buffer conditions and
surface modification methods were used. At a 62 nM concentration, the resultant dip
trace is given in Fig.11. Normalized shift gives us a thickness of 4.6 nm, approximately
2.5 times the shift we observed with our BSA experiments.
Fig.11 Dip trace of antibody IgG nonspecific adsorption onto amino-silanized sphere.
Simulation of Viral Binding with Polystyrene Spheres
We then moved onto larger molecules which would more closely simulate large
virion detection, such as HIV. We looked at polystyrene spheres of diameter 100 nm.
The polystyrene beads were carboxylated, thus amino-silanization methods were again
employed. Polystyrene beads were injected into a 1cm3 cuvette with constant stirring.
Complete saturation was observed at ~3 hours after injection. Compared to the shift
resulting from BSA adsorption, we estimated a shift of ~60 nm for a 100 nm particle.
We measure a thickness of approximately 45 nm (Fig.12) which agrees with theory
predicted (Dr. Teraoka’s analysis). (answer, does it fit theory, what did you expect?)
Fig. 12. Binding of polystyrene nanoparticles to sphere surface and resonant shift
associated with binding events. Inset, comparison of shift between BSA and IgG.
We will next discuss virus sensing using WGM and present results on nonspecific
virus adsorption experiments. We follow with a modified surface treatment protocol and
incorporation of microfluidic capabilities to our system for specificity detection purposes.
1
N. Zammatteo, S. Hamels, F. De Longueville, I. Alexandre, J. L. Gala, F. Brasseur, and
J. Remacle, "New chips for molecular biology and diagnostics," Biotechnol Annu Rev 8,
85-101 (2002).
K. H. Choi, J. P. Bouroin, S. Auvray, D. Esteve, G. S. Duesberg, S. Roth, M. Burghard,
“Controlled deposition of carbon nanotubes on a patterned substrate,” Surf Sci 462, 1952002 (2000).
2
3
T. J. Su, J. R. Lu, R. K. Thomas, Z. F. Cui, J. Penfold, "The conformational Structure of
Bovine Serum Albumin Layers Adsorbed at the Silica-Water Interface," J Phys Chem B
102, 8100-8108 (1998).
4
T. J. Su, J. R. Lu, R. K. Thomas, Z. F. Cui, "Effect of pH on the Adsorption of Bovine
Serum Albumin at the Silica/Water Interface Studied by Neutron Reflection," J Phys
Chem B 103, 3727-3736 (1999).
5
F. Vollmer, D. Braun, and A. Libchaber, "Protein detection by optical shift of a resonant
microcavity," Appl. Phys. Lett 80, 4057-4059 (2002).