By Zhong-he Shen, W.K. Scholz, S.K.W. Nanda, K.O. Whittlinger, S. Chen & B.M. Sullivan Reprinted with permission of Nalge Nunc International
Introduction
The 384-well plate is designed and manufactured with same footprint as a 96-well plate. The plate is designed for high throughput screening applications.
Fast, uneven evaporation in MicroWell® plates can contribute to aberrant results in biological and chemical applications. This is one of the most frequently encountered problems in MicroWell plate applications. To convert an assay from 96-well format to a high density 384-well format, it is necessary to consider evaporation phenomena and compare the results to 96-well plates.
Questions regarding how well size, uniformity, and geometry affect the performance of the 384-well plate are answered by variance analysis. Furthermore, a comparison of sealing mechanisms, including a loose lid, a breathable membrane and solid adhesive tape with regard to evaporation rate, cross contamination and edge effect, demonstrates the advantages of utilizing a membrane type sealing material.
The rounded square well design allows maximum volume and maximum area for dispenser and reader access. The following calculations demonstrate that:
Round Well
Rounded Square
% increase
Access area
0.107 cm2
0.136 cm2
27
Volume
101.5 µL
125 µL
23
The unique rounded square well design greatly increases dispenser access area and well volume (at same liquid height) as compared to a completely round well design.
Surface Area to Volume Ratio
The 384-well plate well surface area to volume ratio is nearly double that of the 96-well plate. Therefore, the 384-well plate gives enhanced signal in solid phase reactions in which surface plays an important role in the assay.
"Wicking" Test
"Wicking" test results
Scintillation cocktail was pipetted into the wells of a 384-well plate and the plate was sealed with solid adhesive tape. After sitting overnight at room temperature, the 384-well plate was illuminated with a UV lamp (366 or 254 nm).
The plate on the left side has the traditional square well design. Scintillation cocktail has accumulated on the top of wells. This phenomenon is referred to as wicking. The plate on the right side has the unique rounded squared well design; no wicking is observed.
Evaporation
Fast and uneven evaporation in MicroWell plates leads to aberrant results in biological and chemical applications, often seen as higher or lower signal and increased % CV. Evaporation rates were measured at different temperatures and humidities in 96- and 384-well plates with different sealing materials.
TABLE I Evaporation rates (mg/cm2hr) at different temperatures for lid covered 96- and 384-well plates
Temperature °C
384-well Plate
96-well Plate
4
0.092
0.133
21.5
0.257
0.5-0.63
37
0.33-0.36
0.76
60
5.44
8.58
Temperature strongly affects evaporation rate.
384-well plates have significant lower evaporation rates than 96- well plates.
The total well opening area is 52.22 cm2 for 384-well plates and 37.475 cm2 for 96-well plates.
TABLE II Evaporation rates (mg/cm2hr) at different humidities and with different sealing systems for 384-well plates and 96-well plates.
384-well Plate
96-well Plate
78% RH
92% RH
78% RH
92% RH
Lid
0.36
0.10
0.85
0.20
Membrane
0.94
0.28
0.99
0.22
Membrane and lid
0.21
0.07
0.27
0.16
Solid tape
0.04
0.01
0.05
0.01
Higher humidity greatly reduces evaporation.
Solid adhesive tape provides the most effective barrier against evaporation.
A breathable adhesive membrane with a lid significantly reduces evaporation when gas exchange is desirable.
Both the adhesive membrane and solid tape can be pierced with a pipette tip for access into the well.
Conclusions
The 384-well plate is useful for high throughput screening applications
surface area to volume ratio, approximately double that of a 96-well plate
enhances solid-phase assay signals, allowing a reduction in reagent utilization without loss of signal
The unique rounded square well design
provides maximum volume compared to a traditional round well design
provides maximum area for dispenser and reader access
and prevents wicking phenomenon
Evaporation studies demonstrate
the evaporation from the 384-well plate is slower than from the 96-well plate
increased humidity reduces evaporation, a positive effect for some applications
Advantages of an adhesive membrane- or tape- covered plate include
decrease evaporation rate
prevent edge effect
seal individual wells, prevent environmental and well-to-well contamination
Variance analyses indicate
the dispensing technique dominates the total variance
variance due to well bottom thickness, well geometry is negligible
the 384-well plate is symmetrical in well arrangement
