Biochips – fundamentals and applications - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 332

acids were incubated with patient serum to detect and characterize autoantigens in the
serum. Bacarese-Hamilton and coworkers used antigen arrays to detect serum antibod-
ies against allergens and infectious antigens [41,42].
Functional arrays are concerned with elucidating novel protein interactions, and are
thus more analogous to the yeast two-hybrid system [43] and co-immunoprecipitation
studies [44, 45]. Compared with antigen - antibody array, functional arrays show great
potential in the mapping of interacting proteins on a system-wide or genome-wide
scale. Major advantages of this in-vitro technique are that we can control the condi-
tions of the experiment, modify the state of the proteins under investigation, and study
the interaction of proteins with non-proteinaceous molecules. Several pioneer inves-
tigators have conducted a number of studies in this fi eld. Ge [46] arrayed 48 purifi ed
human proteins onto a nitrocellulose membrane and probed with different proteins,
nucleic acids, and small organic compounds. Results show that a double-stranded DNA
probe bound more tightly to a phosphorylated form of protein PC4 than to the unmodi-
fi ed form. Zhu et al. [35] used functional arrays to identify 150 putative phospholipid-
binding proteins. MacBeath and Schreiber [34] proved that the activities of purifi ed
proteins can be retained when spotted onto chemically derivatized glass slides which
simplifi es the mass production of functional microarrays. Indeed, functional arrays
are not limited to whole proteins. Espejo et al. [47] used immobilized glutathione
S-transferase-fused protein interaction domains to fi sh out proteins from total cell lysates.
Capture array involves the immobilization of non-protein molecules onto the sur-
face which can interact with proteins in the solute phase. Generally, capture molecules
may be broad capture agents based on chromatography type surface chemistries such
as ion exchange, hydrophobic and metal affi nity functionality, or they may be highly
specifi c such as molecular imprinted polymers or oligonucleotide aptamers.
11.3.2.2 Surface functionalization for protein arrays
No matter what kind of biochip is chosen for use, the fi rst and sometimes the most
important step to construct a biochip is to effectively immobilize proteins or biomol-
ecules on a solid surface. The ideal properties of protein biochip surface are:
● High immobilization density
● No or little infl uence on the activity of immobilized biomolecules
● Effective inhibition of non-specifi c adsorption
● Strong suppression of matrix effect of complex biological solution on the detec-
tion of the one component of interest
● Orientation of immobilized protein with binding site towards the solution.
At present, a wide range of solid substrates are available for protein immobilization.
According to the protein attachment strategies, namely, adsorption, affi nity bind-
ing, and covalent binding, all these substrates can be separated into three main parts.
Surfaces like ploy(vinylidene fl uoride) (PVDF), poly(dimethylsiloxane) (PDMS),
nitrocellulose, polystyrene, and poly-1-lysine coated glass can adsorb proteins by elec-
trostatic or hydrophobic forces. A potential drawback of such substrates is the diffi culty