Biomedical Engineering Reference
In-Depth Information
Image Analysis
Digital images of ArrayPlates
TM
were analyzed with software (ArrayPlate
TM
Fit v.3.31a, HTG, Tucson, AZ) that extracted luminescence intensity data
for each array element in a plate. The resulting data were exported as
comma-separated value (CSV) files that were processed further with soft-
ware (ArrayPlate
TM
Crunch, HTG, Tucson, AZ) that allowed manipulation
of the intensity data, for instance, to normalize signals within arrays to any
combination of array elements. Intensity data CSV files were also imported
into Excel spreadsheets (Microsoft, Redmond, WA) for further analysis.
1.5.2 Results and Discussion
Reagent Programming of Universal Arrays
The 96-well ArrayPlates
TM
contain the same universal array of 16 distinct el-
ements printed at the bottom of each well. Each element consists of a position-
specific, covalently bound 'anchor' species that incorporates an oligonucleotide
25-mer recognition feature. Since identical arrays are printed across all wells
of all plates, the manufacture of ArrayPlates
TM
is standardized and subject
to rigorous quality control procedures.
In spite of this standardized production, ArrayPlates
TM
provide an open
architecture to allow customized assays: A 'reagent programming' hybridiza-
tion immobilizes specific capture reagents at preselected positions in the uni-
versal array. This is achieved using a cocktail that contains 16 bifunctional
'programming linker' species. Each programming linker contains both an
oligonucleotide complementary to a specific anchor and an analyte-specific re-
gion. Thus, the hybridization of linkers to anchors immobilizes analyte-specific
reagents at predetermined positions within the array (Fig. 1.1, top left panel).
Reagent programming provides versatility. The analyte-specific region of a
programming linker can be an oligonucleotide, a peptide, a protein or a chem-
ical compound, depending upon the type of assay that is to be performed:
Programming linkers that consist of antibody conjugated to anchor-binding
oligonucleotide are suited for multiplexed ELISAs or for setting up arrays of
antigens. Programming linkers that have two oligonucleotide regions serve to
capture target RNA, DNA or oligonucleotides. Conjugates of anchor-binding
oligonucleotide and substrate peptides can be used for instance, for multi-
plexed kinase and phosphatase assays. With reagent programming, different
combinations of assay capacity versus content become possible. For example,
the user can program all the wells in a plate identically to measure 16 targets
per sample across 96 samples. Alternatively, by programming arrays in pairs
and splitting samples across two wells, 32 targets (16
×
2) can be measured in
48 samples (96
÷
2).
