Biomedical Engineering Reference
In-Depth Information
the well bottom very irregularly and show significant changes in the spot to spot
distances. An elegant solution for this initial drawback is beside de-ionisation of the
polymer microplates the usage of shielding plates. These conductive plates inserts,
either provided as a part of the microplate or as a separate metal plates, are acting as
effective faraday cages and allow a field free deposition of the protein droplets onto
the surface, when ejected from above the microplate. When printing is finished the
shielding plates are just taken of the spotted microplate and can be re-used [ 17 ]
Fig. 4 shows a metal shielding plate providing 96 discrete chimneys (top) can be
inserted into a standard 96 well plate (middle) and non contact spotting can be done
from above the plate (bottom), thereby increasing print speed significantly.
As mentioned before, a movement along the z-axis with the spotting head.
It can be used for highly accurate deposition of capture probes to the bottom of
wells, too. Typical spotting heights vary from 200 to 500 lm. Although time
consuming, this mode of direct printing provides a robust method to array flexible
protein capture array patterns on a given substrate Fig. 5 shows Direct printing
into microplate wells with two non contact dispensers. Each dispenser creates a
separate array in separate wells. For visualisation purposes a blue dye was used to
illustrate a precise sample deposition.
3.2 Microarrays with Up to 1400 Spots Per Well
The number of spots that can be printed into a single well of a 96 well plate
depends on the shape of the well (round or squared) and varies from single spots
having a printing volume between 100 pL and 100 nL to print patterns that
accommodate more than 1400 spots per well (Fig. 6 ). Using non-contact printing
technology more than 1000 spots per well can be generated. The picture shows
[1400 spots deposited with a sciFLEXARRAYER S11 (Scienion AG, Berlin,
Germany).
Nevertheless, most applications developed for diagnostic purposes have
between 30 and 300 spots, where 10-100 analytes are printed as triplicates (three
replicates). Replicates give some level of security if one spot isn't displayed
adequately or shows an effect that is not foreseeable. In addition replicates are a
very good QC tool for analysing array results, since principally they should show
identical behaviour.
Microplate formats have been used for ELISA purposes for many years [ 18 ]
and proven that the polymer materials work very nicely for well coating purposes.
When array spotting is applied, surface defects in the plate's bottom become
clearly visible immediately, when a spot is located in such an area. Thus, using a
defect free high quality microplate is essential before starting any array based
diagnostic developments. Figure 7 shows a microarray printed into a well of a
microplate with an unwanted result. Very poor protein binding, combined with non
uniform spots and unwanted particles in a microplate do not allow for proper
analysis of an experiment .
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