Biochips – fundamentals and applications - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 334

Most materials developed for covalent binding of proteins cannot be directly used
unless their surfaces are functionalized. Normally, inorganic oxides, particularly silica,
porous glass, and oxidized metals, are derivatized with organosilanes [55]. The most
popular examples are aminopropyltrimethoxysilane (APTES) treated glasses, silica,
and quartz. A typical procedure is to treat the surface with 10% APTES in chloroform,
toluene or in aqueous silane at PH 3-4. The alkoxy groups in APTES are hydrolyzed
by the surface-free water and then connected to the surface silanol groups (Si ß OH)
via Si ß O ß Si bonds. Post-thermal curing is sometimes necessary to cross-link the
residual free silanol groups and thus form a uniform saline fi lm. If the matrix of silane
is absolutely anhydrous, a monolayer can be obtained. Otherwise, a multiple layer
would exist on the surface (Fig. 11.24).
Gold-based substrates are also widely investigated for covalent binding of probes.
Instead of using polycrystalline gold surfaces, thin gold fi lms are now preferable
since they can be easily deposited on solid supports like glass and silicon wafers by
evaporation or sputtering in an ultra-high vacuum. A thin layer of Cr or Ti is gener-
ally required to deposit prior to gold coating. After careful cleaning, a self-assembly
monolayer (SAM), namely, gold-thiol monolayer can be prepared by exposing gold
surface to the vapor or solution of thiols. The affi nity of thiols to gold is so strong that
they can replace most of other adsorbates on the gold surface in virtually any solvent,
which make this preparation very simple. Depending on the end group located on the
monolayer surface, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or glutar-
aldehyde (GA) can be served as cross-linker to couple protein to the gold-thiol surface.
Because of the excellent electronic properties of gold, these surfaces can be applied
in more advanced and label-free detection technologies such as surface plasmon reso-
nance (SPR) and mass spectrometry.
Except inorganic substrates, functionalized polymer membranes are another choice
to immobilize proteins. For instance, commercial SPR instrument producer Biacore
adopts epoxy-terminated thiol to couple carboxy-methyl-modifi ed dextran polymers,
which contains extensive reactive sites for activation and covalent attachment of pro-
teins of interest. Another new approach is to use the copolymerization of methacrylate,
styrene or vinyl alcohol monomers on glass surface followed by subsequent function-
alization using photografting. The optical properties of the surface can be tailored to
the refl ectance of light or scattering effects. This technology has been used to make
highly refl ective microarray surfaces at applied wavelength for potential applications
in highly sensitive luminescent-based assay systems.
Although functionalized surface technology has achieved much success in the past
few decades, there are still challenges in building and using functionalized protein
microarrays. First, the notorious liability of proteins raises concerns about their stabil-
ity and integrity on the functionalized surface. Second, it is time consuming and costly
to produce proteins of good purity and yield, and not many proteins can be purifi ed
at all. Third, functionalized surface lacks the ability to prevent non-specifi c adsorp-
tion. Finally, the methods used to attach proteins to the substrate surface may affect the
behavior of the proteins. Improvements in these aspects are crucial to enhance sensitiv-
ity and to avoid false positives.