Biomedical Engineering Reference
In-Depth Information
the protein mixture. Note that conventional, protein based blocking mixtures
are not necessary. Once the protein mixture is applied to the array the mix-
ture is allowed to incubate at 30
◦
C for at least 2 hours. For high sensitivity
measurements longer incubation times may improve results. The protein in-
cubation can be performed in either a static mode, in which the solution is
allowed to interact with the array without dynamic movement, or in a flow
mode, where solution is circulated over the array in either a continuous loop
or a reciprocating fashion. For the static mode, simple reaction vessels are
fashioned over the arrays by application of adhesive-backed wells or other
similar devices, while for circulation mode more technically evolved solutions
are required. Both methods yield equivalent results; the advantage of mixing
is a reduction in the incubation time needed to reach maximal binding levels.
Pre-Crosslink Wash, Crosslinking, and Post-Crosslink Wash
At the end of the incubation period the protein solution is replaced with PIB,
allowing removal of unbound protein while retaining cognate protein binding
to aptamers through a
nity interaction. The arrays are then exposed to UV
irradiation, causing covalent crosslink formation between BrdU residues on
the aptamers and proximal electron-rich amino acids of the cognate proteins.
Optimal wavelength for the crosslink is 308 nm, which can be introduced by
excimer laser excitation or broad spectrum UV which is filtered to eliminate
sub-300 nm wavelengths. Optimal energy levels have been calibrated on the
laser and empirically determined to be 3 J/cm
2
, which gives the highest levels
of specific crosslinking.
The specificity imparted to the microarray assay in the crosslinking step
is a key feature of photoaptamer technology. The photoSELEX process se-
lects those aptamers that e
ciently crosslink their target protein. Because the
photoactivated complex is short-lived, e
cient crosslinking requires close and
stable contacts between BrdU and the target amino-acid, a requirement for
π
-bond orbital overlap has been proposed [50]. Although polyanion-binding
proteins may bind to aptamer DNA, the probability that this binding will
result in productive geometry for photocrosslinking is low. We have shown
that the photocrosslinking step can improve aptamer specificity by an order
of magnitude or more over the specificity due to a
nity interactions alone [48].
Although these measurements were made in solution, they are consistent with
results obtained on microarrays, both with simple protein mixtures and with
target proteins spiked in to serum [48].
Since the crosslinked aptamer-protein complex is covalently linked to the
substrate it is possible to use extremely rigorous denaturing conditions to
fully remove any remaining proteins from the substrate or non-cognate array
features. Examples of denaturing components of washing solutions include
0.02 M NaOH, 0.1 M AcOH, 1% SDS, 2% TritonX-100, 0.5 M NaCl, 0.02 M
DTT, 8 M Urea, 4 M GuHCl, 50
◦
C, and sonication. However, since the bind-
ing interaction of aptamers to cognate proteins is extremely specific there is
