Biomedical Engineering Reference
In-Depth Information
purposes. After UV illumination, the photolabile linker could be cleaved to allow
postsynthetic biological screening such as agar overlay assay. Different building
blocks containing carboxylic acid, aldehyde, and isocyanide were applied onto a
cellulose support. The Ugi MCR could occur efficiently with the addition of water.
The compounds produced through in situ MCR displayed high purity, with most of
them showing a purity level above 90%. This approach may hold substantial promise
for creating small-molecule libraries in the future.
13.3 FABRICATION OF SMMs
Following the completion of library synthesis, small molecules from 384-well plates
can be deposited automatically onto the glass slides by high-precision robotic printers.
Depending on the type of microarray spotter used, small molecules could be deposited
onto glass slides by contact printing or piezoelectric mode. The typical spot size for
microarray is around 100
m in diameter, and the typical spot volume is around
1 nL. This miniaturized assay saves a substantial amount of expensive bioreagent
compared with other assay platforms, such as microplate-based assays.
Immobilization technology sits at the heart of array fabrication. It is of paramount
importance to immobilize biomolecules stably onto a solid support during microarray
fabrication. Three approaches are generally used for the fabrication process: (1) a
noncovalent immobilization approach, (2) a covalent immobilization approach, and
(3) an in situ synthesis approach.
13.3.1 Noncovalent Immobilization Approach
Noncovalent interactions have commonly been employed for SMM immobilization.
The strong interaction between two complementary DNA stands is frequently utilized
for decoding purposes in combinatorial chemistry. In 2007, Schroeder et al. intro-
duced an elegant DNA-directed strategy to immobilize cell-specific peptide ligands
onto an array [29]. In this approach, various biotinylated peptide ligands were first
coupled to streptavidin-DNA conjugates. The resulting peptide-DNA conjugates
were then hybridized to a DNA microarray containing complementary DNA strands.
The peptide array obtained could be used for the cell adhesion study of NIH-3T3
fibroblasts. Experiments showed that cells could grow properly on the spot in the
presence of suitable ligands. This method not only proved the feasibility of fabricat-
ing a live cell array in a DNA-directed manner, but also created additional functions,
such as controlled release of cells on an array. Melkko et al. realized another creative
design [30]. The team generated self-assembling chemical libraries by linking two
different small molecules together through a specially designed DNA tag. The tag
consists of two parts: a unique oligonucleotide sequence for decoding purpose that
can be hybridized onto a DNAmicroarray, and an assembly oligonucleotide sequence
for mediating the self-assembly of two molecules. Using this approach they were able
to identify tight binders against proteins with multiple pockets, such as human serum
albumin and bovine carbonic anhydrase (see also Chapter 11).
