Biomedical Engineering Reference
In-Depth Information
staining of arrays has been shown as a genuine alternative to fluorescent detection
with the advantage to use less sophisticated and therefore less expensive detection
devices, as costs can represent a real entry barrier for diagnostics markets. Ana-
lysing protein microarrays using a colorimetric nanogold probe coupled with silver
enhancement (gold-silver detection) has been described Liang and co-workers
[
19
]. In this method, the gold nanoparticles are introduced to the microarray by the
specific binding of gold-conjugated antibodies or streptavidins and then coupled
with silver enhancement to produce a black image of microarray spots, which can
be easily detected with a conventional CCD camera. The method showed good
detection sensitivity (1 pg of IgG immobilised on slides or 2.75 ng/ml IgG in
solution) and a good linear correlation between the signal intensity and the log-
arithm of the sample concentration. The examination of this method in analysing a
demonstrational ToRCH antigen microarray developed showed identical results
when compared to fluorescent detection schemes. These results suggested that the
colorimetric gold-silver detection method had potential applications in proteomics
research and clinical diagnosis. Nevertheless it seems that detection systems based
on silver reduction are very sensitive to environmental changes (e.g. temperature)
and timing, making it somewhat difficult to use in routine analysis.
The majority of colorimetric (also called chromogenic) detection for minia-
turised ELISAs is based on colorimetric substrates for ELISA development with
alkaline phosphatase (AP) or for horseradish peroxidase enzyme (HRP). Com-
pared to the Gold/Silver detection they are typically less expensive and have been
used for conventional ELISAs for years. Typical reagents commercially available
1FA2CA04F788
)
do include:
p-Nitrophenyl Phosphate, Disodium Salt (PNPP) is a widely used substrate for
detecting AP in ELISA applications. PNPP produces a yellow water-soluble
reaction product that absorbs light at 405 nm. PNPP is available either as a
crystalline powder, 5 mg tablets or as a ready-to-use formulation.
2,2
0
-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt
(ABTS) is used to detect HRP and yields a water-soluble green end reaction
product. The green product has two major absorbance peaks, 410 nm and 650 nm.
ABTS is less sensitive than the OPD and TMB substrates (see below) for HRP
detection. Colour development is slow (approximately 20 min) which may be
advantageous if unacceptable background results from the use of the OPD or TMB
substrates due to higher sensitivities. ABTS is available in either tablet or a ready-
to-use for formulation.
o-phenylenediamine dihydrochloride (OPD) is used to detect HRP and yields a
water soluble yellow-orange reaction product. The reaction product has an
absorbance maximum of 492 nm. OPD is available in either powder or tablet
forms and easily prepared by dissolving in Stable Peroxide Substrate Buffer or
buffered hydrogen peroxide solution.
3,3
0
,5,5
0
-tetramethylbenzidine (TMB) soluble substrates yield a blue colour
when detecting HRP. The major absorbance maxima or the reaction product are
370 nm and 652 nm. The colour then changes to yellow with the addition of
