U.S. patent application number 11/784157 was filed with the patent office on 2007-08-09 for crystallization methods for laboratory cap and well.
Invention is credited to Christian Houde, Jean-Pascal Viola.
Application Number | 20070184551 11/784157 |
Document ID | / |
Family ID | 4163275 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070184551 |
Kind Code |
A1 |
Viola; Jean-Pascal ; et
al. |
August 9, 2007 |
Crystallization Methods for laboratory cap and well
Abstract
The invention relates to a device for molecular and
macromolecular crystallization. More particularly, the device
comprises a well and a transparent cap for growing
diffraction-quality protein crystals by conventional vapor
diffusion techniques. The present device is particularly
advantageous in that it allows the pre-filling of the well with a
solution for transport and handling.
Inventors: |
Viola; Jean-Pascal; (Ville
d'Anjou, CA) ; Houde; Christian; (Montreal,
CA) |
Correspondence
Address: |
Roger H. Stein;Wallenstein & Wagner, Ltd.
53rd Floor
311 South Wacker Drive
Chicago
IL
60606-6630
US
|
Family ID: |
4163275 |
Appl. No.: |
11/784157 |
Filed: |
April 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09913088 |
Jan 23, 2002 |
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PCT/CA00/00119 |
Sep 2, 2000 |
|
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11784157 |
Apr 5, 2007 |
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Current U.S.
Class: |
436/4 ;
422/400 |
Current CPC
Class: |
C30B 7/00 20130101; B01L
2200/0689 20130101; B01L 3/50825 20130101; B01L 3/50853 20130101;
B01L 2200/02 20130101; B01L 3/06 20130101; C30B 29/58 20130101;
B01L 2300/042 20130101 |
Class at
Publication: |
436/004 ;
422/102 |
International
Class: |
G01N 31/00 20060101
G01N031/00; B01L 3/00 20060101 B01L003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 1999 |
CA |
CA 2,261,326 |
Claims
1. A method for forming molecular or macromolecular crystals
comprising the steps of: dispensing a precipitating solution in a
well having an open upper end; providing a droplet of a solution
containing a molecule or macromolecule on a crystallization support
formed on an underside of a cap; inverting the cap so the support
is positioned over the open upper end of the well and the droplet
is suspended over the precipitating solution contained in the well;
and, engaging a first locking means associated with the cap and a
second locking means associated with the well to simultaneously
interlock the cap onto the well and to close and seal the open
upper end of the well.
2. The method of claim 1 wherein the support is transparent, the
well has a light transmitting bottom and a resilient member is
disposed between each cap and each well to ensure the sealing
thereof.
3. The method of claim 2 further including the step of observing
the activities within the sealed well through the transparent
support with the aid of light passing through the light
transmitting bottom of the well.
4. The method of claim 1 wherein the cap and the open upper end of
the well are tubular and the step of engaging includes rotating the
first and second locking means relative to one another.
5. A method of claim 1 further comprising the step of pre-filling
the well with the precipitating solution and sealing the well
before shipment to a purchaser.
6. A method of forming crystals by vapor diffusion comprising the
steps of: providing a tray having a base with a plurality of wells
supported on the base with each well having an open upper end and a
light transmitting bottom; providing a plurality of caps, each with
a transparent crystallization support formed on an underside
thereof; placing a precipitating solution within each well; placing
a droplet of solution containing a molecule or macromolecule to be
crystallized and a precipitating agent on each of the
crystallization supports; inverting each cap so each
crystallization support is positioned over each open upper end of
each well and the molecule or macromolecule is suspended over the
precipitating solution contained in each well; and, engaging a
first locking means associated with each cap and a second locking
means associated with each well to simultaneously interlock each
cap onto a well and to close and seal the open upper ends of the
wells.
7. The method of claim 6 further including the step of positioning
a resilient sealing member on each cap so as to enhance the seal
between each cap and each well without the need or use of a
contaminating sealing material between each cap and well.
8. A method of claim 6 further including the step of examining and
monitoring crystal growth under a microscope through the
crystallization support with the aid of light passing through the
light transmitting bottom of the well.
9. The method of claim 6, wherein the caps and the upper end of
each well are tubular and the step of engaging includes rotating
the first and second locking means relative to one another.
10. A method according to claim 6, further comprising the step of
pre-filling each well with the precipitating solution and sealing
each well before shipment to a purchaser.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a device for handling
molecular and macromolecular crystallization. More particularly,
the device comprises a well and a cap assembly for growing protein
crystals by conventional vapor diffusion techniques. The present
device is particularly advantageous in that it facilitates the
pre-filling of the well with a solution for transport and handling
prior to utilization by a technician.
BACKGROUND OF THE INVENTION
[0002] Crystallography is an extremely useful tool for scientists,
and is therefore a field of research attracting a lot of interest.
It is a powerful means that provides precise and detailed
description of the three-dimensional structure of the molecules,
and is of great help in the understanding of their functions.
Crystallography of macromolecules like proteins is extensively used
today, academically as well as industrially.
[0003] Although three-dimensional structures of simple proteins
have been obtained through crystallization methods, it is not
always easy to obtain crystals from macromolecules. For example,
the preferred conditions for the crystallization of a given
molecule can take several hundreds if not thousands of trials. As a
result, means and methods have been developed to perform a great
number of trials relatively quickly, including hanging-drop and
sitting-drop methods. All such methods use the benefit of vapor
diffusion to obtain the crystals.
[0004] The hanging-drop method is currently the most commonly used
technique for scanning various crystallization conditions of
macromolecules, such as proteins. It comprises suspending a droplet
of approximately 2 to 20 .mu.L of solution containing the
macromolecule to be crystallized and a precipitating agent, over a
precipitating solution, such as conventional polyethylene glycol
20% or ammonium sulfate 40%, contained in a reservoir or well. The
system is then sealed hermetically. After a while, vapor diffusion
of the solvent or solvent mixtures between the droplet and the
solution in the reservoir reaches equilibrium. The end result is a
decrease of water in the droplet, and an increase of the
macromolecule and precipitating agent concentration therein, thus
causing crystallization of the macromolecule in optimized
conditions. The actual technique for the set up of the hanging-drop
or sitting-drop experiments is a long and arduous work and has to
be performed by qualified and skilled technical personnel.
[0005] Conventionally, a commercially available tray made of an
inert thermoplastic material comprising a plurality of reservoirs
or wells is prepared, and the precipitating solution is placed in
each reservoir or well manually. The macromolecule solution is then
mixed with a precipitating agent on a glass plate (coverglass) and
the whole is inverted over the wells, thus making the macromolecule
solution overhanging the well. Prior to placing a glass plate over
a well, the rim of each well is greased to ensure a proper seal.
Care must be taken when placing the plate over each well, since the
grease can easily contaminate the macromolecule solution. The
crystallization process is followed with the help of a microscope.
After the crystal is obtained, the glass plate is removed. Again,
this must be done with great care to prevent contamination of the
crystallized macromolecule with grease, and/or breaking of the
glass plate. On top of that, the plates are hardly reusable for any
experiment because the grease is hard to remove, and some of it
remains on the plates.
[0006] An advantage of the hanging-drop and sitting-drop methods is
that they provide screening conditions for crystallization, and
truly represent a microcrystallization technique. The vapor
diffusion in the hanging or sitting drop allows screening of a
large range of conditions and necessitates a relatively small
amount of macromolecules. Further, it allows a relatively clear
visualization of the results, and the eventual crystals are free,
i.e., they do not adhere or are stuck to any surface.
[0007] Typically, several hundreds of experiments are required to
find appropriate crystallizing conditions for the production of
high quality crystals. Accordingly, hanging-drop and sitting-drop
experiments are a very labor-intensive process demanding skilled
and experienced technical personnel. For example, multiple
aspirating and dispensing is steps of components, multiple greasing
steps etc. must be performed in the experimental set up. Further,
for each well, a separate coverglass must be manually inverted. The
number and complexity of steps can therefore produce an undesirable
wide variation in experimental results.
[0008] As stated above, grease is conventionally used to provide a
seal between the well and the coverglass. Other ways for sealing
the system have been proposed. For example, grease can be replaced
with immersion oil or an adhesive tape. As with grease, these
sealing means have serious drawbacks. Grease is not always easy to
dispense around the upper rim of the well, and is a time consuming
operation. Technicians repeating the operation thousands of times
occasionally suffer physical pain to their hands. Other significant
problems and risks are present when manipulating the crystal on a
greasy cover slide. The cover slide sometimes breaks during the
process, which may cause injury to the technician, in addition to
loosing the crystals. The immersion oil is also problematic. One
has to use a determined volume of oil. Too much oil leads to
contamination within the well, while not enough will lead to
non-hermetic seal that may result in the evaporation of the
precipitating solution. An adhesive tape allows quicker and simpler
manipulations, but all experiments are sealed at the end of the
set-up, thus introducing experimental variations between the
1.sup.st and the 24.sup.th drop. Further, crystals often stick to
the tape, rendering impossible the recovery of the crystals, and
the operations for the recovery of the drop are also
problematic.
[0009] These conditions promoted the robotization of the procedure.
Some automated crystallization devices already exist. The
well-known Cyberlab-200.TM. apparatus dispenses solutions in wells,
greases the upper rim of each well, pours droplets on cover is
slides held by a vacuum arm, and places the cover slides over the
wells. However, such apparatus still has some drawbacks, namely a
complicated experimental set-up, and the notable use of grease.
Further such apparatus is extremely expensive.
[0010] Relevant references in the field include U.S. Pat. No.
2,366,886; U.S. Pat. No. 3,107,204; U.S. Pat. No. 3,297,184; U.S.
Pat. No. 3,537,956; U.S. Pat. No. 3,597,326; U.S. Pat. No.
3,649,464; U.S. Pat. No. 3,692,498; U.S. Pat. No. 3,729,382; U.S.
Pat. No. 3,745,091; U.S. Pat. No. 3,907,505; U.S. Pat. No.
4,038,149; U.S. Pat. No. 4,154,795; U.S. Pat. No. 4,495,289; U.S.
Pat. No. 4,917,707; U.S. Pat. No. 5,271,795;
[0011] It would therefore be highly desirable to develop a device
for crystallizing macromolecules that would overcome the above
deficiencies. Such device would eliminate the requirement of
external means like grease, oil or an adhesive tape to seal the
well and the cover, and would preferably be easy to manipulate,
either manually or automatically. Ultimately, the process of
experimental set up of the device would be greatly facilitated and
accelerated, while simultaneously eliminating possible risks of
contamination of the eventual crystals. Finally, such device should
be usable for various crystallization processes such as
hanging-drop or sitting-drop processes.
SUMMARY OF THE INVENTION
[0012] In accordance with the present invention, there is provided
a tray comprising a top surface, a bottom, an upright
circumferential wall extending from the top surface to the bottom,
and a plurality of wells extending downwardly from, and being open
at, the top surface, for receiving a precipitating solution; and a
cap provided for each well, each cap comprising locking members for
locking the cap onto the well in a sealed manner. Such tray is
particularly advantageous for growing molecular and macromolecular
crystals.
IN THE DRAWINGS
[0013] FIG. 1 illustrates perspective view of a first cap and well
assembly in accordance with the present invention;
[0014] FIG. 2 illustrates a section view of a preferred embodiment
of a cap;
[0015] FIG. 3 illustrates a perspective view of a tool adapted to
install and remove a cap from the well;
[0016] FIG. 4 illustrates a section view of the tool of FIG. 3;
and
[0017] FIGS. 5, 6 and 7 illustrate other embodiments of the cap and
well assembly according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0018] It is an object of the present invention to provide a
crystal forming device using vapor diffusion method. The device
comprises a well and a transparent cap designed to close the well
and form a sealed volume, the well being sealed without the need to
add a sealing material like grease, oil, adhesive tape and the like
between the well and the cap. The cap is made of transparent
material to allow examination and monitoring of crystal growth, as
well as manipulation of the crystal under a microscope. The present
device therefore represents an important advance in methods for
growing crystals of macromolecules, especially in the field of
hanging-drop and sitting-drop.
[0019] Because of its simplicity, the operations of filling the
well with the precipitating solution, placing a drop of the
macromolecule solution onto the cap and sealing the well by putting
the cap in position over the well can be accomplished by any
competent technician, and not only skilled personnel.
[0020] In a preferred embodiment of the invention, a plurality of
wells are molded together, for example in a tray comprising 4 rows
of 6 wells each, with corresponding transparent caps are provided
thereon. The resulting tray and caps may also be optionally treated
with a hydrophobic agent such as a siliconing agent.
[0021] Because of the transparency of the cap and of the bottom
surface of the well, crystallization can be followed with minimal
handling, and without disturbing the vapor equilibrium within each
well. Further, visualization of the results under the microscope
are simple because the cap is made of a transparent or translucent
(clear) material.
[0022] Preferably, the material of the tray and the cap are the
same, and comprise materials that can be molded easily at a
reasonable cost. The material should be stable for extended periods
of time towards the various chemical products present in the well
and onto the cap. The material should also preferably not absorb
water, and be good optical quality to facilitate work and
observation under a microscope. Example suitable materials include
various thermoplastics such as polystyrene, polypropylene,
polycarbonate, polyacrylate, polymethacrylate,
acrylonitrile-styrene copolymers, nitrile-acrylonitrile-styrene
copolymers, polyphenyleneoxide, phenoxy resins, etc., the most
preferred material being polystyrene.
[0023] It is another object of the present invention to provide a
crystal-forming device that allows the manipulation of the growth
crystals under the microscope without any transfer from the cap,
where solutions can be added directly without any transfer of the
crystals, in a greaseless environment.
[0024] Another major advantage of the device of the present
invention is that once a series of experiments is completed, the
tray is readily reusable, simply by taking another series of caps
containing a drop of a solution containing a macromolecule to be
crystallized, and reinstalling the caps over the wells. Further, a
given cap may be removed from its original well and locked onto
another one containing a different precipitating solution.
[0025] The invention is also concerned with a method for forming
crystals of a macromolecule, the method comprising the steps of
dispensing a precipitating solution in a well; forming a droplet in
a cap comprising locking members to lock the cap over the well; and
locking and sealing the well. In a preferred embodiment, a ring
made of an elastomeric material like polypropylene, an
ethylene-propylene copolymer, Teflon.TM. etc., is preferably
provided between the cap and the well. In a further preferred
embodiment, the well can be filled in advance and tightly sealed,
so that they a tray is provided to a technician in a "ready-to-use"
manner.
[0026] Because of the ergonomics of the present invention, the cap
is engaged easily so that there is no need for special manual
dexterity comparatively to the use of conventional thin, fragile,
microscope coverglasses. The presence of a cavity in the surface of
the cap facing the bottom surface of the well allows the addition
of liquid directly over the drop, after placing the cap upside down
on a table, without the need to transfer the crystals to another
well, thus limiting the manipulations that might ruin the fragile
crystals.
[0027] The use of the cap and well assembly of the present
invention can be automated in a straightforward manner by providing
the extremity of an automated arm with a simple grip element having
an end provided with a structure adapted to releasably grip the
cap. There is no need for the application of grease or the
manipulation of fragile coverglass pieces. The grip element may
also be manipulated manually by a technician, as described
hereinbelow.
[0028] The cap and well assembly of the present invention also
finds applications in the field of cell cultures, molecular or
cellular biology etc.
[0029] In a most preferred embodiment of the invention, the well is
filled beforehand and sealed with the cap. The technician therefore
receives a "ready-to-use" assembly, thus eliminating the
time-consuming operation of filling each well with the appropriate
precipitating solution. The buyer may therefore order as many
assemblies as desired with the same or different precipitating
solutions. For shipping purposes, the cap may be replaced on the
assembly with a film to prevent contact of the precipitating
solution with the cap. Such contact would necessitate the cleaning
of the cap prior to its use. One may also use a cap for shipping
purposes, and a different cap to carry out the experiments. It is
important that the well be sealed to avoid evaporation and spilling
of the precipitating solution, either during shipment of the
pre-filled wells, or during the experiments.
[0030] Referring to the drawings which illustrate preferred
embodiments of the invention, FIG. 1 illustrates a cap and well
assembly 10 which comprises a tray or base plate 12 provided with a
plurality of wells 14 and corresponding caps 16. Assembly 10 may
also include a cover 18 used for shipping or storage purposes. The
preferred form of cover 18 comprises with an insertion (not shown)
at each corner that allows retention of the cover over the caps
without touching them. Cover 18 further allows the storage of
several trays of experiments one on top of the other. Tray 12
comprises a rim 20 extending about the four side walls thereof, and
is provided with finger grip surfaces 22 such as those described in
U.S. Pat. No. 4,038,149, on two opposed side walls for easier
handling of the tray by the technician. Finger grip surfaces 22 are
provided to avoid mishaps, and greatly facilitate handling of
covered and uncovered trays. Cover 18 comprises a section 24
adapted to engage around finger grip surfaces 22 for proper fitting
over assembly 10.
[0031] FIG. 2 illustrates a section view of cap 16. As it can be
seen, cap 16 comprises a cylindrical slot 26 into which is inserted
an O-ring element 28 made of a resilient material. Such material,
although optional, is provided to ensure an appropriate seal when
cap 16 is fitted over upper rim 30 of well 14. The inner surface 32
of slot 26 has a portion or ridge 34 extending passed the planar
surface 36 thereby forming a cavity 38. Surface 36 may be concave
or convex, but the planar configuration illustrated on FIG. 2 is
much preferred. As stated above, the material of cap 16 is such
that it is sufficiently transparent or translucent so that cap 16
can be placed directly under a microscope for observation and/or
manipulation of the crystals.
[0032] Each cap 16 comprises a pair of locking element 40
diametrically opposed to each other and comprising a ridge portion
41. Cap 16 also comprises a further rim 46 provided with a series
of spacer 45 underneath. Once the precipitating solution is poured
into well 14, the technician puts cap 16 upside down on a flat
surface and places a drop of the macromolecule-containing solution
onto surface 36. Cap 16 is then flipped over cautiously, and each
locking element 40 is inserted into a corresponding opening 42
provided onto the upper surface 44 of tray 12 until the abutment of
upper rim 30 of well 14 with the O-ring element 28 inside slot 26
is achieved. Cap 16 is then rotated so that locking elements 40
slide each into a slot 43 having a width smaller than that of
opening 42 and extending on a portion of the periphery of well 14
until the upper surface of portion 41 is entirely under upper
surface 44, thereby efficiently sealing well and maintaining cap 16
in place. In a most preferred embodiment, a section 47 of portion
41 is tapered to facilitate sliding under upper surface 44. To
ensure even better locking and maintenance of the cap in position,
a small bump (not shown) is provided onto section 49 that is
adapted to fit into a corresponding recess (not shown) present
under surface 44 after complete insertion of portion 41 under
surface 44.
[0033] To put cap 16 in place onto well 14, or for removal
therefrom, a tool 48 may be used, as illustrated in FIGS. 3 and 4.
Tool 48 comprises a body 50 divided in a portion 52 shaped in a
manner such as to facilitate holding by the technician or an
automated arm; a cylindrical portion 54 with an external surface 57
having a circumference slightly bigger than that of rim 46, and an
internal surface 59 having a circumference slightly smaller than
that of rim 46. Tool 48 further comprises two diametrically opposed
cap gripping elements 60 each provided with a gripping finger 62.
The gripping element 60 can be provided onto internal surface 59,
directly on rim 66, or onto external surface 57. In operation, tool
48 is placed over cap 16 so that each finger 62 is inserted into a
slot 64 cut into rim 46 until at least a portion 65 of each element
60 is abutted onto rim 46. Tool 48 is then rotated until gripping
fingers 62 are completely engaged under rim 46, and the rotation is
maintained until the locking members 40 are aligned with slots 42.
Cap 16 is then simply pulled up. To reintroduce the cap in
position, the procedure is carried out in an opposite manner. The
external surface 53 of portion 52 should be planar, so that it can
be laid on a table or under a microscope in a stable manner, and
allow the technician to observe and/or work on the crystals. To be
able to work under a microscope directly, surface 53 must comprises
an opening preferably corresponding to the internal diameter of
cylindrical portion 54 (see FIG. 4).
[0034] FIG. 5 illustrates another embodiment of the present
invention. The cap and well assembly 100 which comprises a tray or
base plate (not shown) provided with a plurality of wells 112 and
corresponding cap 114, which comprises a cylindrical slot 116 into
which is inserted an O-ring element 118 made of a resilient
material. As for the previous embodiment illustrated, the O-ring,
although optional, is provided to ensure an appropriate seal.
[0035] Each cap 114 comprises a pair of locking element 120
diametrically opposed to each other and comprising a ridge portion
122. Cap 114 is locked into position on the tray by inserting each
locking element 120 into a corresponding opening 124 provided onto
the upper surface 126 of the tray until the lower surface 128 of
cap 114 lies flat onto the tray upper surface 126. Cap 114 is then
rotated so that locking elements 120 slide into a slot 130 having a
width smaller than that of opening 124 and extending on a portion
of the periphery of well 112 until the upper surface of ridge
portion 122 is entirely under upper surface 126, thereby
efficiently sealing well and maintaining cap 114 in place.
[0036] FIG. 6 illustrates another simple variation of the present
invention, wherein the upper surface 152 of a well 150 comprises a
slot 154 along its circumference and adapted to receive an O-ring
element 156 coupled to a cap 158. The section of slot 154 is such
that is slightly smaller than that of element 156, so that upon
insertion into the slot, a tight seal is formed by the locking of
cap 158 to well 150 without the need of any adhesive or grease.
[0037] FIG. 7 illustrates yet another embodiment of the invention,
wherein the cap 170 is screwed on the well 172.
[0038] The present cap and well assembly is particularly suitable
for both hanging-drop or sitting-drop crystallization methods. With
respect to the sitting-drop method, although not specifically
illustrated in the drawings, anyone skilled in the art will readily
appreciate that any conventional drop support can be inserted or
molded into the well. Examples of such sitting-drop support include
the Micro-Bridges.TM. or the glass sitting drop rods manufactured
and sold by Hampton Research (Laguna Hills, Calif.).
[0039] Each well is carefully filled with a selected equilibrating
solution. Subsequently, a selected protein drop is deposited on the
cap. The shape and the texture of the lower surface can be varied
to obtain optimum results for a particular protein solution being
crystallized, for example, when lower surface tension solutions,
including protein solutions containing detergents, are used. The
addition of the equilibrating solution and the protein drops to the
device may be carried out either manually or through commercial
automated pipetting apparatus, and the sealing of the cap over the
solution may also be carried out manually or in an automated
manner.
[0040] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present description as come
within known or customary practice within the art to which the
invention pertains, and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
* * * * *