A vast majority of research is dependent on the
transfer of a measured volume of liquid from one
vessel to another, such as from a bulk container to a
microplate. Because this technique is often employed
up to several times per hour, the product used to perform
the task must be user friendly and comfortably
suited to the application. General-purpose and specialized pipets and peristaltic pumps dominate the liquid handling market for virtually every volume
range, viscosity, and container size, although each
may exhibit distinct disadvantages for niche applications
such as assay development, where bulk dispensing
is required in microplate format.
Pipets, especially multichannel pipets, accurately
and precisely deliver a predetermined fluid volume to
a microplate well, or a single column/row of wells in
a microplate. Pipets are especially useful for dispensing
liquid in milliliter, microliter, or smaller volume
ranges since most are highly precise and accurate.
Although this method has been the overwhelmingly
popular liquid handling choice for several decades,
inconsistent and poor pipetting techniques are common
issues that result in assay performance variability,
even from a single operator. These problems can
be compounded by repetitive strain injuries such as
carpal tunnel syndrome.
Aside from operator error, dry pipet tips must be
preconditioned by repeatedly drawing up and dispensing
a chosen volume of liquid to reduce surface
tension within the tip walls, which is of concern if
using expensive reagents. Viscous liquids with high
surface tension can dramatically affect assay results
because the true volume of liquid dispensed through
the small pipet tip opening can be distorted. Additionally,
since pipets contain moving parts, they
must be regularly cleaned and calibrated to ensure
optimal dispense performance without damage from
aerosols or deposits created from the liquids used.
Finally, with advances in microplate technology
and miniaturization, laboratories have transitioned
from 96-well microplates to higher-density microplate
geometries such as 384- and 1536-well formats.
These higher densities can be a daunting task for manual pipetting, thus requiring other methods of
liquid dispensing.
Peristaltic pumps are commonly used for continuous
or semicontinuous liquid dispensing, and since the
liquid is entirely contained within flexible tubing,
they are ideal for sterile applications without risk of
cross-contamination. They are also well suited for
use when conservation or recycling of reagents is
desired because the liquid can be pumped back to the
source reservoir. Peristaltic pumps incorporate rollers
fixed to a rotating rotor which, when encircled
by flexible tubing, compress the tubing at regular
intervals between two consecutive rollers, forcing
a volume of liquid to pass through the tube.
This volume is defined as a slug. Volume adjustments
are determined by the distance between
the rollers, rotor position, tubing tension, inner
diameter of the tubing, and tip geometry. This
dispensing style is suited for automated systems
with a high degree of precision and accuracy.
Since there are no moving parts in contact with
the liquid, these pumps are relatively inexpensive
to maintain, requiring only periodic replacement
of the tubing.
The disadvantage of many peristaltic pumps is
that, by design, their volume ranges are often not
suitable for microdispensing since it is increasingly
difficult to effectively overcome cohesive
forces of the liquid for accurate dispensing at very
low volume levels.
Accuracy is also compromised if the pump dispenses
volumes of liquid (slugs) in fractions
rather than full volumes, because the variation
between the fractions is significant when compared
to full volumes. This is especially problematic at
microvolumes because the liquid is no longer forced
out of the tubing by the force of the roller, but rather
the speed of the rotor itself. Cohesive forces in the
liquid cause uneven distribution of the liquid. In
addition, cassettes, which contain the tubing, are
rated for a limited lifetime because the tubing will
stretch to a certain degree due to prolonged contact
with the rollers. This stretching can adversely affect
dispense performance, or worse, rupture the tubing.
An improved dispensing
method
Figure 1 - MicroFlo Select Dispenser.
In order to provide accurate bulk dispensing that is
both precise throughout a range of volumes and easy
to use with low maintenance, BioTek Instruments, Inc. (Winooski, VT) designed the MicroFlo™ Select Dispenser (Figure 1). The flexible design of the
device allows for dispensing into microplates from 6-
to 1536-well formats with a variety of plate heights,
and also dispenses into microtubes and other tubes
up to 4 inches in height. Dispense height is automatically
adjusted by the instrument’s software, and
the dispenser can be automated for high-throughput
applications. Stable-performing, long-lasting peroxide-cured tubing in each of eight channels eliminates
the cumbersome process of preconditioning,
and the low dead volume reduces reagent waste. The
MicroFlo Select uses a specialized peristaltic design
for a wide volume range from 1 μL to 9999 μL, with
precision and accuracy comparable to, or better than,
multichannel pipets.
Three selectable cassette sizes (1 μL, 5 μL, and 10 μL)
each incorporate the appropriate dimension tubing
to produce full volumes of liquid instead of fractions,
and also use optimized tip geometries, thus reinforcing
the precision and accuracy of each volume dispensed.
These cassettes can be autoclaved without
the expense and time associated with recalibration,
and both the cassettes and cassette tubing may be
easily and quickly replaced as needed.
Experimental protocol
In order to determine precision and accuracy
ranges of the MicroFlo Select using each of the
three cassette sizes, a standard blue dye (FD&C
#1) was dispensed per the following protocols: A
10-μL cassette with 0.022-in.-i.d. molded polypropylene
tips was inserted into the MicroFlo Select,
the variable dispense rate was set to the highest
setting, and the dispense height was set to 15.362
mm. The concentrated dye solution was undiluted
for the 10-μL dispenses, diluted by a ratio of
1:5 with distilled water for dispenses between 15
μL and 75 μL, and diluted again by a ratio of 1:2
before the 100-μL dispenses so that the absorbance readings were in the range of 0.5 and 2.5 o.d. The
prepared dye solutions were then dispensed into
individual 96-well microplates (Corning Life Sciences,
Lowell, MA) in quantities ranging from
10 μL to 100 μL, and distilled water was added
to each microplate well with a calibrated μFill™ microplate dispenser (BioTek Instruments,Inc.)
for a total volume of 250 μL per microplate well.
A 5-μL cassette with 0.014-in.-i.d. molded polypropylene
tips was inserted into the MicroFlo Select,
the variable dispense rate was set to the lowest setting
for dispenses between 1 μL and 4 μL, and the
medium setting was used for the remaining dispenses.
The concentrated dye was undiluted for dispenses between 1 μL and 10 μL, then diluted per the above
ratios for dispenses between 15 μL and 50 μL, and 75
μL and 100 μL, respectively. The prepared dye solutions
were then dispensed into microplates in quantities
ranging from 1 μL to 100 μL, and distilled water
was added to each microplate well as described above
for a total volume of 250 μL per microplate well.
A 1-μL cassette with 0.010-in.-i.d. molded polypropylene
tips was inserted into the MicroFlo
Select, and the variable dispense rate was set to
the highest setting. The concentrated dye was
undiluted for dispenses between 1 μL and 10 μL,
and diluted per the above ratio of 1:5 for dispenses
between 15 μL and 50 μL. The prepared dye solutions
were then dispensed into microplates in
quantities ranging from 1 μL to 50 μL, and distilled
water was added to each microplate well as
described above for a total volume of 250 μL per
microplate well. All microplates were read using a Power - Wave™ microplate spectrophotometer (BioTek Instruments, Inc.) at dual wavelengths (450 and
630 nm) to reduce background noise.
Results
The data in Table 1 emphasize two important points.
First, there is wide variability inherent to fractional
slugs, especially in low volumes. For example, using
the 10-μL cassette to dispense 15 μL, or one full slug
and one-half slug of liquid results in a high degree of
variability due to the effects of cohesive forces, while
using a 5-μL cassette or 1-μL cassette to dispense 15
μL with multiple full slugs of liquid (two and ten
slugs, respectively) results in a high degree of precision.
Second, all precision and accuracy data in the
table, when used with the appropriate cassette, are
comparable to, or better than, those expected with
use of a multichannel pipet.
Summary
When dispensing liquids in microplate or even
tube format, pipets and peristaltic pumps each offer
advantages and disadvantages in ease of use, precision
and accuracy, and flexibility. The MicroFlo
Select Dispenser is the first instrument of its kind to
combine the advantages of both technologies, with a
wide range of choices in dynamic dispense volumes,
microplate types and styles, tube sizes, and cassette
selection, in one compact and user-friendly unit
to accommodate bulk dispensing requirements and
optimal assay integrity.
Mr. Greene is Product Manager, BioTek Instruments,
Inc., Highland Park, P.O. Box 998, Winooski, VT 05404-0998, U.S.A.; tel.: 888-451-5171; fax: 802-655-7941;
e-mail: [email protected].