U.S. patent application number 11/107880 was filed with the patent office on 2005-08-18 for liquid dispensing apparatus and method.
This patent application is currently assigned to Genetix Limited. Invention is credited to Davies, Douglas, Haslam, James Keith, Stephens, Sarah Katharine.
Application Number | 20050180892 11/107880 |
Document ID | / |
Family ID | 24294267 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050180892 |
Kind Code |
A1 |
Davies, Douglas ; et
al. |
August 18, 2005 |
Liquid dispensing apparatus and method
Abstract
A multiwell plate for microarraying comprising a self-sealing
lower membrane and optionally also a self-sealing upper membrane.
Spotting is performed by pushing a pin down through the liquid and
then on to pierce the self-sealing lower membrane. A slide having
an upper spotting surface is arranged under the well plate. The
liquid sample forced through the lower membrane by the pin tip can
thus be deposited directly onto the spotting surface. The pin is
then withdrawn upwards through the lower membrane, which
automatically reseals preventing further loss of liquid. The
optional resealable upper membrane also prevents loss of sample
liquid by evaporation and spillage. By contrast to the prior art,
there is the major advantage that the pin head does not have to
traverse between the well plate and slide to collect sample liquid,
thus dramatically increasing operational speed for an automated
microarraying apparatus employing the multiwell plate. Excellent
spot reproducibility is also observed.
Inventors: |
Davies, Douglas; (Dorset,
GB) ; Haslam, James Keith; (Dorset, GB) ;
Stephens, Sarah Katharine; (Hampshire, GB) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Genetix Limited
|
Family ID: |
24294267 |
Appl. No.: |
11/107880 |
Filed: |
April 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11107880 |
Apr 18, 2005 |
|
|
|
09573998 |
May 19, 2000 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
Y10T 436/25625 20150115;
G01N 35/1065 20130101; B01J 2219/00612 20130101; Y10T 436/2575
20150115; B01J 2219/00605 20130101; G01N 2035/1037 20130101; B01J
19/0046 20130101; Y10T 436/25 20150115; B01J 2219/00387 20130101;
B01J 2219/00596 20130101; B01J 2219/00659 20130101; C40B 60/14
20130101; B01L 3/0244 20130101; Y10T 436/25375 20150115; B01J
2219/00585 20130101; B01J 2219/00527 20130101 |
Class at
Publication: |
422/100 |
International
Class: |
B32B 005/02 |
Claims
1-4. (canceled)
5. A well plate comprising: an upper surface; a lower surface; an
array of wells extending between the upper surface and the lower
surface; a lower membrane extending over the lower surface to form
a self-sealing, liquid-tight base for the wells.
6. A well plate according to claim 5, wherein the lower membrane is
formed of a single sheet of material.
7. A well plate according to claim 5, wherein the lower membrane is
formed of multiple sheets.
8. A well plate according to claim 7, wherein there is one sheet
for each of the wells.
9. A well plate according to claim 5, further comprising an upper
membrane extending over the upper surface to form a top for the
wells.
10. A well plate according to claim 9, wherein the upper membrane
is made of self-sealing material.
11. A well plate according to claim 5, further comprising a
removable cover extending over the upper surface to form a top for
the well plate.
12. A well plate according to claim 5, wherein the array of wells
conforms to a square grid having a grid spacing of one of: 2.25 and
4.5 millimeters.
13. A well plate according to claim 5, wherein the array of wells
has one of: 96, 384 and 1536 wells.
14-20. (canceled)
Description
BACKGROUND ART
[0001] The invention relates to an apparatus for and method of
dispensing liquid. More especially, but not exclusively, the
invention relates to dispensing liquid from well plates as widely
used in the field of chemistry and biotechnology for microarraying
and other applications.
[0002] Microarraying is a technique in widespread use. Conventional
microarraying is based on standard multi-well plates having a 4.5
mm grid and 384 wells. However, larger array sizes of 1536 wells
are becoming more widely used, these larger arrays conform to a
2.25 mm grid. Liquid samples are stored in the wells of a well
plate. The liquid may be assays or any other biological or chemical
sample of interest. To spot the liquid from a well, a pin is dipped
in the well to retrieve an amount of the liquid. The pin carrying
an amount of the sample liquid is then moved across to a spotting
surface of a microscope slide or other suitable surface. A spot of
liquid is deposited on the slide by bringing the pin into close
proximity, or by physically contacting the tip of the pin, with the
slide surface.
[0003] FIG. 1A of the accompanying drawings shows schematically a
pin 110 conventionally used for spotting. The pin is in the form of
a split pin, with liquid 111 being attracted to and carried on the
pin by capillary action. The liquid is discharged from the pin onto
the spotting surface by lowering or tapping the pin on the spotting
surface so that the liquid transfers from the pin tip onto the
spotting surface.
[0004] FIG. 1B of the accompanying drawings shows schematically a
modified split pin design, also used for spotting in the prior art.
Split pin 110 incorporates a reservoir 112 and has a blunt end so
that liquid 111 extends beyond the pin tip. Liquid can be deposited
onto a spotting surface by pressing the blunt end of the pin in
contact with the spotting surface or by bringing the pin into very
close proximity with the surface such that surface tension causes a
drop to be transferred from pin to surface.
[0005] These pin designs have in common that they rely on capillary
action to gain a reservoir of sample liquid sufficient for many
spot depositions. This avoids having to dip into the well for each
spot.
[0006] Most microarray pins in the prior art float vertically in a
common head. They rest in the lowest position by gravity or spring
biasing. The head tends to over travel by a small amount and the
pins will lift in the head by the over travel.
[0007] Regardless of the pin design, spotting is carried out with
the following basic steps. The pin is moved to above the well
plate. The pin is dipped in a well of the well plate to retrieve
some liquid. The pin carrying the liquid is moved over to above the
spotting surface. The retrieved liquid is deposited from the pin
onto the spotting surface, either with only one spot, or with
several spots for a pin that carries a reservoir of sample liquid.
The pin is moved back to the well plate to retrieve more liquid for
further spotting.
[0008] FIGS. 2A to 2C of the accompanying drawings show the basic
spotting process. In FIG. 2A, a pin 110 from a pin head 120 is
lowered downwards, for example mechanically, to dip it into a well
112 of a well plate 114 and thereby retrieve an amount of liquid
sample. In FIG. 2B, the liquid sample is deposited onto a spotting
surface. In FIG. 2C, the pin is cleaned in a washing stage 118,
typically after many spotting actions (i.e. FIG. 2A to FIG. 2B
repeats) prior to commencing spotting with a different liquid
sample.
[0009] Design effort has been concentrated on speeding up the head
transit times so that the time taken between dipping in the well
plate and spot deposition on the slide is reduced. As mentioned
further above, the pins are also sometimes designed to use
capillary action for storing a charge of liquid in the pin
sufficient for depositing a number of spots. This also speeds up
the spotting procedure by reducing the number of times the head
needs to be traversed between the well plate and the slide.
SUMMARY OF THE INVENTION
[0010] According to a first aspect of the invention, there is
provided a well plate with a self-sealing lower membrane. Spotting
is performed by pushing a pin down through the liquid and then on
to pierce the self-sealing lower membrane. A spotting surface is
arranged under the well plate. The liquid sample forced through the
lower membrane by the pin tip can thus be deposited directly onto
the spotting surface. The pin is then withdrawn upwards through the
lower membrane, which automatically reseals preventing further loss
of liquid.
[0011] By contrast to the prior art, the pin head does not have to
traverse between the well plate and spotting surface to collect
sample liquid, thus dramatically increasing operational speed.
Moreover, experiments have proved that the spot size obtained by
deposition through the self-sealing membrane is highly consistent.
In prior art systems which rely on one dip of the pin into the well
to put down many spots, larger variances in the spot size tend to
occur as the pin becomes dry through evaporation and
deposition.
[0012] The well plate may also be provided with a self-sealing
upper membrane, thereby fully enclosing the liquid in the well. The
pin then pierces first through the upper membrane and then on
through the liquid and the lower membrane. When the pin is
withdrawn, the upper membrane self seals in the same manner as the
lower membrane. Consequently, loss of sample from the wells by
evaporation is prevented. This is especially useful for valuable or
toxic samples and has the further advantage of greatly reducing the
risk of sample contamination. Additionally, the upper membrane
wipes the shank of the pin as it is withdrawn. This cleaning action
cleans the pin while at the same time reducing loss of sample
liquid. Moreover, the upper membrane ensures that accidental
dropping of the well plate will not result in spillage.
[0013] Well plates may be provided in a variety of sizes and
configurations. Standard 96, 384 or 1536 geometries may be
provided. Specially sized well plates may also be developed to suit
specific applications, or as a proprietary measure.
[0014] According to a second aspect of the invention, there is
provided a head apparatus for operation with the multi-well plate
of the first aspect of the invention. The head apparatus comprises
a pin head and a mounting frame adapted to hold a multiwell plate
beneath the pin head. A motor stage is operable to drive the pins
of the pin head down through the multiwell plate. The pins can thus
be actuated through the wells and through the self-sealing membrane
to deposit a sample directly onto a spotting surface held below the
head apparatus. The pin head and a well plate held thereto can thus
be moved around together by a robotic guidance system, instead of
moving the head independently of the well plate as in the prior
art.
[0015] The pins are preferably fixed in the body portion so that
their tips lie in a common plane distal the body portion. Slidably
mounted pins are not necessary, resulting in a considerable cost
saving.
[0016] Alternatively the pins may be individually actuatable and
addressable, for example using conventional pin array addressing
mechanisms.
[0017] Advantageously, the head may further comprise an abutment
arranged to stop the pin tips being advanced beyond a plane defined
by the abutment. The abutment is designed to contact the spotting
surface simultaneously with the pins for spotting. The abutment
thus defines the maximum travel of the pins during spot deposition.
The pins are preferably constrained so that they either stand off
slightly from the spotting surface or only just contact it at their
points of maximum travel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] For a better understanding of the invention and to show how
the same may be carried into effect reference is now made by way of
example to the accompanying drawings in which:
[0019] FIG. 1A is a schematic section of a split pin of the prior
art;
[0020] FIG. 1B is a schematic section of another split pin of the
prior art;
[0021] FIG. 2A shows sample collection by a pin from a well of a
multi-well plate according to the prior art;
[0022] FIG. 2B shows sample deposition from a pin onto a microscope
slide according to the prior art;
[0023] FIG. 2C shows pin washing according to the prior art;
[0024] FIG. 3 is a schematic section through a part of a multi-well
plate according to an embodiment of the invention with two pins and
a slide also being shown;
[0025] FIG. 4 is a schematic elevation of one example of a
multi-well plate according to the embodiment of FIG. 3;
[0026] FIG. 5 shows a multi-well plate with pin head above and
spotting surface below, in an operational configuration;
[0027] FIG. 6 shows a multi-well plate with an alternative pin head
above and microscope slide below, in an operational
configuration;
[0028] FIG. 7 shows in elevation a pin head with spotting surface
and multi-well plate; and
[0029] FIG. 8 is a schematic diagram of an automated spotting
apparatus according to an embodiment of the invention.
DETAILED DESCRIPTION
[0030] FIG. 3 is a schematic drawing showing a section of part of a
multi-well plate 30 according to an embodiment of the invention.
Two pins 10 and 10', in two different operating positions, and a
spotting surface 16 are also shown. The multi-well plate 30
comprises a plurality of wells, two of which are shown in FIG. 3.
Each well is partially defined by a side wall 12 of generally
frusto-conical shape defined in a main body of the well plate. The
shape of the sidewall is not critical. The main body is made of
conventional material. The main body has an underside to which is
secured a lower membrane 14, and an upper side to which is secured
an upper membrane 16. The lower membrane 14 defines a base to the
wells. The upper membrane 16 defines a lid to the wells. The
meniscus of a quantity of sample is shown with reference numeral
18. In a concrete example, the meniscus position shown may
represent about 5 microliters of sample liquid in each well, each
well having a total volume of about 30 microliters. As well as
sealing the well against spillage, the upper membrane 16 has the
function of preventing sample loss through evaporation, which is
important for precious, volatile or hazardous samples. The upper
and lower membranes are made of self-sealing material, in other
words material that automatically reseals after piercing with a pin
or needle. The material used was obtained from USA/Scientific
Plastics (Europe) Limited. This material has an adhesive backing,
the adhesive being inert medical grade. Materials such as rubber,
silicone or PTFE may also be suitable for the membranes. Other
flexible, resilient materials may also be suitable.
[0031] In operation, to dispense an amount of sample, a pin is
driven down from the position shown by pin 10'. The pin travels
down through the upper membrane 16, then through the sample liquid
and on through the lower membrane 14 until in close proximity with
an upper surface of the spotting surface 16. This position is shown
by pin 10 in FIG. 3. The liquid sample collected on the tip during
its passage through the well is then deposited onto the spotting
surface.
[0032] Experiments have shown that the quantity of sample carried
through the membrane is highly consistent, providing spots of 80
micrometer diameter on a glass spotting surface, with very low
variance. Spot size can be varied by using pins with varying tip
diameters. In the experiments, the tips were not pressed against
the spotting surface, but rather brought into a nominally zero
stand-off or offset with the spotting surface using an abutment
arrangement described further below. Liquid deposition is thus
driven by surface tension and fluid flow effects, or by throwing
the liquid off the tip by deceleration. In the experiments, the
pins were traditional surgical needles made of 316 grade stainless
steel modified by flattening the needle tips to a diameter of 50
micrometers. In the experiments, the lower membrane resealed,
apparently perfectly, with no compromise to the sealing properties
over a test with 1000 piercing actions in a single piercing
position. No loss of sample (other than through the spot
deposition) or damage to the membrane was detectable.
[0033] Other needle tip shapes and dimensions may be used to
provide dosage control of the amount of deposited liquid. For
example, fluted needle tips may be used.
[0034] FIG. 4 shows an elevation of a whole multi-well plate 30, a
corner of the upper membrane 16 being peeled away for ease of
viewing. The lower membrane 16 is also visible. The well plate
conforms to the industry standard dimensions for a 384 well plate.
Accordingly, the wells are arranged in a 4.5 mm square grid and the
well plate is generally rectangular with outer dimensions of 124.5
mm by 82 mm to accommodate an array of 16 by 24 wells.
[0035] Although the membranes are shown as single sheets adhesively
bonded to the well plate main body, it will be understood that the
upper or lower membranes may be segmented. For example, individual
membrane sections could be provided for each well or for groups of
wells. Further, the membranes could be mechanically clamped onto
the surfaces of the well plate main body, for example by a plate
perforated with a grid of holes. Moreover, it will be understood
that the upper membrane is optional. The upper membrane could be
dispensed with altogether. Alternatively, the upper membrane could
be fitted initially, after filling the well plate with sample
liquid, to prevent evaporation and spillage during transport and
storage. The upper membrane could then be removed immediately prior
to microarraying. It will thus be understood that the upper
membrane need not be a self-sealing membrane in all cases.
[0036] FIG. 5 shows another elevation view. The multi-well plate of
FIG. 4 is shown again, together with a head 32 arranged thereabove
and a non-standard slide 16 thereunder. The head 32 has 384 pins
(actually needles as described above) arranged in conformity with
the well array of the multi-well plate, i.e. in a 16 by 24 array
having a 4.5 mm grid.
[0037] Contrary to conventional designs, the pins 10 are fixed in a
body part 31 of the head in order to allow them to be pushed
through one or more membranes, as is required with the multiwell
plates described herein. Fixing the pins is a great advantage since
it would be very costly to build a head with 384 gravity located
pins for a conventional multiwell plate. The small mass of the pins
makes them sensitive to variations in the fit of the pins in their
guide holes. As the number of pins is increased, it becomes
increasingly difficult to avoid some pins falling freely and other
pins sticking in their holes. Adding pin biasing springs has been
proposed-to overcome this problem, but this is not ideal since it
increases impact forces on the pin tips, thereby increasing pin
damage and wear rates. In any case, the biasing would have to be
heavy if the pins were to be able to penetrate one or
membranes.
[0038] FIG. 6 is comparable to FIG. 5, but shows a smaller 48-pin
head (reference numeral 32) with the pins arranged in a 4 by 12
array, again in a 4.5 mm square grid. Arranged under the 384 well
plate 30 there is an industry standard microscope slide 16.
[0039] FIG. 7 is an illustration of a pin head 32 with ancillary
mounting frame 33 and motor stage 37. The head 32 comprises a body
portion 31 and an array of pins 10 extending down. The mounting
frame 33 has a lower lip 39 for receiving and holding a multiwell
plate 30 from an autofeed system (described further below). Any
suitable guide, slot or retaining means could be used. The head 32
is held in the mounting frame 33 by the motor stage 37 which
comprises a linear motor that is drivable to move the head up and
down in the z-axis as illustrated. The body portion 31 of the head
32 has four pillars 44 serving collectively as an abutment. The
pillars 44 are secured to the body portion 31 and extend down to
terminate in a plane arranged approximately at or slightly below
the common plane of the tips. The abutment extensions 44 from the
head body portion 31 stop the pins at a fixed position just above
or at the upper surface of the slide 16 so that liquid can pass
from the pins to the spotting surface by fluid flow.
[0040] In use, the z-axis linear motor drives the body 31 down
within the mounting frame 33 so that the pins 10 fixed in the body
31 pass through the wells of well plate 30 until the abutment 44
touches the spotting surface 16 whereupon the liquid is
transferred.
[0041] FIG. 8 shows an automated microarraying apparatus according
to an embodiment of the invention. A pin head 32 is held by a
robotic xyz guidance system 40. An autofeed stacking system 35 is
operable to load well plates 30 into the head 32 from a stack 34 of
well plates. The autofeed stacking system 35 is also operable to
restack well plates after use in another stack 36. The stack and
restack processes may automatically remove and replace lids from
the well plates, as is known from the prior art. Lids may be
provided instead of or in addition to an upper membrane. A number
of non-standard slides 16 are also shown. The slides are
non-standard in that they are over-dimensioned by the provision of
a margin region extending beyond the dimensions of a conventional
slide. The xyz guidance system 40 is operable to position the head
and well plate above any of the slides 16 for spotting. A wash
station 42 is also shown which is provided for cleaning the head 32
after unloading a well plate into the restack stack 36.
[0042] It will be appreciated that although particular embodiments
of the invention have been described, many modifications/additions
and/or substitutions may be made within the spirit and scope of the
present invention.
* * * * *