U.S. patent application number 12/942816 was filed with the patent office on 2011-03-10 for method of scanning a sample plate surface mask in an area adjacent to a conductive area using matrix-assisted laser desorption and ionization mass spectrometry.
Invention is credited to Joseph L. DiCesare.
Application Number | 20110056311 12/942816 |
Document ID | / |
Family ID | 33309822 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110056311 |
Kind Code |
A1 |
DiCesare; Joseph L. |
March 10, 2011 |
Method of Scanning a Sample Plate Surface Mask in an Area Adjacent
to a Conductive Area Using Matrix-Assisted Laser Desorption and
Ionization Mass Spectrometry
Abstract
A method of scanning a sample plate surface mask in an area
adjacent to a conductive area using mass spectrometry is disclosed.
The method comprises the steps of providing a sample plate
including a mask applied with a rough surface to the electrically
conductive surface to produce a sample site comprising a central
portion formed from the electrically conductive surface and a
marginal portion of the mask, preparing an analyte comprising
mixing a biomolecule with an organic solvent, an aqueous solution,
and a matrix selected from the group of
.alpha.-cyano-4-hydroxycinnamic acid and
3,5-dimethoxy-4-hydroxycinnamic acid; applying the analyte to the
sample site; forming at least one crystal of the analyte in an area
on the mask adjacent to the conductive area, and scanning the area
on the mask adjacent to the conductive area with a laser beam.
Inventors: |
DiCesare; Joseph L.;
(Redding, CT) |
Family ID: |
33309822 |
Appl. No.: |
12/942816 |
Filed: |
November 9, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10426226 |
Apr 30, 2003 |
7858387 |
|
|
12942816 |
|
|
|
|
Current U.S.
Class: |
73/863.21 |
Current CPC
Class: |
H01J 49/0418 20130101;
G01N 2001/045 20130101; B01L 2300/165 20130101; G01N 2001/4027
20130101; B01L 3/5085 20130101; Y10T 436/25 20150115; G01N 1/40
20130101; H01J 49/164 20130101; B01L 2300/0829 20130101; Y10T
436/24 20150115; B01L 3/5088 20130101 |
Class at
Publication: |
73/863.21 |
International
Class: |
G01N 1/36 20060101
G01N001/36 |
Claims
1. A method of applying a specimen to sample plate surface mask in
an area adjacent to a conductive area comprising the steps of:
providing a sample plate including a mask applied with a rough
surface to an electrically conductive surface to produce a sample
site comprising a central portion formed from the electrically
conductive surface and a marginal portion of the mask; providing a
liquid chromatography system for preparing a specimen comprising
eluting a biomolecule with an organic solvent, an aqueous solution,
and a matrix and applying the specimen; moving the sample plate
cooperatively with the liquid chromatography system; and applying
the specimen to the sample site.
2. The method of claim 1 wherein the matrix is selected from the
group consisting of .alpha.-cyano-4-hydroxycinnamic acid and
3,5-dimethoxy-4-hydroxycinnamic acid.
3. The method of claim 1, wherein the step of applying the specimen
further comprises the step of applying the specimen at a rate of at
least 1 .mu.l per 1 mm.
4. The method of claim 1, wherein the step of applying the specimen
further comprises the step of applying the specimen within at least
0.2 mm of the periphery of the electrically conductive surface on
the mask.
5. The method of claim 1, wherein the step of providing a liquid
chromatography system for preparing a specimen comprising eluting a
biomolecule, further comprises eluting a biomolecule selected from
the group consisting of oligonucleotides, DNA, RNA, peptide,
polypeptide, oligopeptide, protein, glycoprotein, lipoprotein,
carbohydrate, monosaccharide, disaccharide, polysaccharide, and
combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of allowed U.S. patent
application Ser. No. 10/426,226 filed Apr. 30, 2003, the content of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a method of analyzing a sample
plate in mass spectrometry using a sample plate having a rough
hydrophobic surface.
BACKGROUND OF THE INVENTION
[0003] Matrix-assisted laser desorption and ionization mass
spectrometry (MALDI-MS) is an important analytical tool for the
study and identification of biomolecules, particularly proteins,
peptides, and nucleic acids such as DNA and RNA.
[0004] MALDI-MS results in a mass spectrum that graphically
identifies biomolecules according to peaks that correspond to the
biomolecules' concentration and mass. Using a library of known
peaks, the biomolecules can be identified.
[0005] Various methods exist for the preparation of samples for
analysis by MALDI-MS, including the dried droplet method. In the
dried droplet method an aqueous sample containing the subject
biomolecule is mixed with an organic compound, the matrix, which is
usually suspended in an easily evaporative aqueous-organic solvent.
The resulting liquid mixture containing the biomolecule, the
matrix, aqueous solution, and solvents is referred to herein as the
specimen.
[0006] The specimen is applied to the sample plate in a
predetermined target area and allowed to dry. As the solvent begins
to evaporate, and the biomolecule and matrix become more
concentrated, the matrix molecules crystallize from solution while
drying on the sample plate. The resulting crystals entrap the
biomolecules on and/or within the crystals and in due course
deposit on the sample plate.
[0007] Other methods of applying the specimen to the sample plate
are also known. In the electrospray deposition method, the specimen
is applied to the sample plate as a fine mist of microdroplets that
evaporates very quickly forming the specimen crystals.
[0008] To analyze the biomolecules, the sample plate is inserted
into the sampling compartment of a mass spectrometry instrument. A
voltage is applied to the sample plate to permit the flow of
electric current over the sample plate and prevent the possibility
of an electrical charge buildup. To desorb the crystals, an
ultra-violet (UV) laser scans the target area either by manual
direction or in a predetermined automated fashion to irradiate the
crystals. The laser beam radiation is absorbed by the matrix
molecules, resulting in a vaporization of both the matrix molecules
and the biomolecules. Once in the vapor phase, while still in close
proximity to the target area, a charge transfer occurs as the
matrix molecule loses a proton to the biomolecule. The ionized
biomolecules are then drawn into the mass spectrometer where they
are analyzed. Data processing yields a mass spectrum of a series of
characteristic peaks corresponding to the biomolecules and matrix
molecules. The signature of peaks is used to identify the
biomolecules by reference to known peaks.
[0009] Prior art of interest includes U.S. Pat. No. 6,287,872
(herein incorporated by reference) relating to sample support
plates for the mass spectrometric analysis of large molecules, such
as biomolecules, methods for the manufacture of such sample support
plates and methods for loading the sample support plates with
samples of biomolecules from solutions together with matrix
substance for the ionization of the biomolecules using
matrix-assisted laser desorption (MALDI).
[0010] Also of interest is U.S. Pat. No. 5,958,345 (herein
incorporated by reference) relating to a sample support for holding
samples for use with an analysis instrument. The sample support is
for use with analysis instruments, which rely on a beam of
radiation or accelerated particles and a method for making the
same. The holder includes a frame with one or more orifices covered
by a support surface, typically in the form of a thin polymer film.
The film is divided into hydrophobic and hydrophilic portions to
isolate precise positions where samples can be placed to intersect
a probe beam during analysis.
[0011] MALDI-MS performance suffers chiefly from analysis
insensitivity. The sample plates that are used in MALDI-MS are
typically metallic plates due to the need to apply a voltage across
the plate. Known trays have a smooth hydrophilic surface where the
applied specimen drop spreads over a relatively large area before
drying and forming crystals. Consequently, to effectively irradiate
the crystals the UV laser has to scan this enlarged area requiring
extra time.
[0012] Another drawback of metallic plates is that they
unfortunately often provide unsuitable results due to unintentional
contamination from detergents. Since, metallic plates are also
expensive, they are used repeatedly. Washing between each use may
contaminate subsequent analysis.
[0013] It is known, that specimens are non-homogeneously
distributed on and/or within the lattice that located at the
specimen periphery. It is further known that some of these matrix
crystals bear more biomolecules than others. Thus, as the laser
covers a likely search area at the specimen periphery, it scans
"sweet spots" having a comparatively higher specimen concentration
in the matrices. When irradiated and detected, the sweet spots
provide an inaccurate concentration reading of the biomolecule.
[0014] What is desired, therefore, is a sample plate for MALDI-MS
analysis of a specimen wherein crystals are located in a sample
site.
[0015] What is also desired is a durable and cost effective sample
plate which enables archiving of samples.
[0016] What is also desired is a rough surface that is hydrophobic
to enhance the formation of crystals in a sample site. What is
further desired is a higher ratio of surface area to planar area of
the hydrophobic mask.
SUMMARY OF THE INVENTION
[0017] Accordingly it is an object of the invention to provide a
method of analyzing a specimen.
[0018] Another object of the invention is to provide a method of
sample plate that overcomes known problems of analysis
insensitivity.
[0019] A further object of the invention is to provide a sample
plate wherein crystals are located at predetermined positions.
[0020] A still further object of the invention is to provide a
durable and cost effective sample plate which enables archiving of
samples.
[0021] These and other objects of the invention are achieved by a
method that crystallizes analyte in a reduced area.
[0022] Therein, a method of scanning a sample plate surface mask in
an area adjacent to a conductive area using mass spectrometry is
disclosed. The method comprises the steps of providing a sample
plate including a mask applied with a rough surface to the
electrically conductive surface to produce a sample site comprising
a central portion formed from the electrically conductive surface
and a marginal portion of the mask, preparing an analyte comprising
mixing a biomolecule with an organic solvent, an aqueous solution,
and a matrix, applying the analyte to the sample site; forming at
least one crystal of the analyte in an area on the mask adjacent to
the conductive area, and scanning the area on the mask adjacent to
the conductive area with a laser beam.
[0023] In some embodiment the step of scanning the area on the mask
adjacent to the conductive area with a laser beam further comprises
scanning in a pattern.
[0024] In some embodiments the method further comprises the step of
determining a scanning pattern based on an algorithm having a
confidence level.
[0025] In some embodiments the step of preparing a specimen further
comprises providing a matrix selected from the group consisting of
.alpha.-cyano-4-hydroxycinnamic acid and
3,5-dimethoxy-4-hydroxycinnamic acid.
[0026] In some embodiments the step of preparing a specimen further
comprises providing an organic solvent.
[0027] In some embodiments the step of preparing a specimen further
comprises providing an aqueous solution.
[0028] In some embodiments the step of preparing a specimen further
comprises providing analyte wherein the analyte is at least one
biomolecule.
[0029] In some embodiments the step of preparing a specimen further
comprises providing analyte wherein the analyte is selected from
the group consisting of oligonucleotides, DNA, RNA, peptide,
polypeptide, oligopeptide, protein, glycoprotein, lipoprotein,
carbohydrate, monosaccharide, disaccharide, polysaccharide, and
mixtures thereof.
[0030] In some embodiments the method comprises applying a specimen
to sample plate surface mask in an area adjacent to a conductive
area comprising the steps of: providing a sample plate including a
mask applied with a rough surface to an electrically conductive
surface to produce a sample site comprising a central portion
formed from the electrically conductive surface and a marginal
portion of the mask; providing a liquid chromatography system for
preparing a specimen comprising eluting a biomolecule with an
organic solvent, an aqueous solution, and a matrix and applying the
specimen; moving the sample plate cooperatively with the liquid
chromatography system; and applying the specimen to the sample
site.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1a is an isometric view of a sample plate with a
circular target area in accordance with one embodiment of the
invention.
[0032] FIG. 1b is an isometric view of a sample plate with a
rectangular target area in accordance with one embodiment of the
invention.
[0033] FIG. 2a is an enlarged view taken at area A-A of FIG. 1a of
a section of a circle sample plate in accordance with one
embodiment of the invention.
[0034] FIG. 2b is an enlarged view taken at area A-A of FIG. 1b of
a section of a channel sample plate in accordance with one
embodiment of the invention.
[0035] FIG. 2c is a plan view of a circle sample site that includes
a target area and a mask spot in accordance with one embodiment of
the invention.
[0036] FIG. 3a is a cross-section at section B-B of FIG. 2a of a
section of a circle sample plate in accordance with one embodiment
of the invention.
[0037] FIG. 3b is a cross-section at section B-B of FIG. 2b of a
section of a channel sample plate in accordance with one embodiment
of the invention.
[0038] FIG. 3c is an expanded elevation view of a circle sample
site that includes a target area and a mask spot in accordance with
one embodiment of the invention.
[0039] FIG. 4a is a cross-section at section B-B of FIG. 2a of a
section of a circle sample plate with an electrically conductive
coating applied to the substrate in accordance with one embodiment
of the invention.
[0040] FIG. 4b is a cross-section at section B-B of FIG. 2b of a
section of a channel sample plate with an electrically conductive
coating applied to the substrate in accordance with one embodiment
of the invention.
[0041] FIG. 5a is an enlarged view taken at area A-A of FIG. 1a of
a circle sample plate in accordance with one embodiment of the
invention wherein specimens have been applied on sample sites.
[0042] FIG. 5b is an enlarged view taken at area A-A of FIG. 1b of
a channel sample plate in accordance with one embodiment of the
invention wherein specimens have been applied on sample sites.
[0043] FIG. 6a is a cross-section at section C-C of FIG. 5a of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens have been applied on sample
sites.
[0044] FIG. 6b is a cross-section at section C-C of FIG. 5b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens have been applied on sample
sites.
[0045] FIG. 6c is a cross-section at section D-D of FIG. 5b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens have been applied on sample
sites.
[0046] FIG. 7a is an enlarged view taken at area A-A of FIG. 1a of
a circle sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6a have begun to dry.
[0047] FIG. 7b is an enlarged view taken at area A-A of FIG. 1b of
a channel sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6b have begun to dry.
[0048] FIG. 8a is a cross-section at section E-E of FIG. 7a of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6a have begun to
dry.
[0049] FIG. 8b is a cross-section at section E-E of FIG. 7b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6b have begun to
dry.
[0050] FIG. 8c is a cross-section at section F-F of FIG. 7b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6b have begun to
dry.
[0051] FIG. 9a is an enlarged view taken at area A-A of FIG. 1a of
a circle sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6a have dried.
[0052] FIG. 9b is an enlarged view taken at area A-A of FIG. 1b of
a channel sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6b have dried.
[0053] FIG. 10a is a cross-section at section G-G of FIG. 9a of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6a have dried.
[0054] FIG. 10b is a cross-section at section G-G of FIG. 9b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6b have dried.
[0055] FIG. 10c is a cross-section at section H-H of FIG. 9b of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6b have dried.
[0056] FIGS. 11 and 12 are isometric views of crystals that have
crystallized in a halo effect around a sample site with different
concentrations of matrix formulations in accordance with one
embodiment of the invention.
[0057] FIG. 13 is a cross-sections at section E-E of FIG. 5a of a
section of a sample plate in accordance with one embodiment of the
invention wherein crystals are being scanned by an UV laser.
[0058] FIGS. 14a and 14b are enlarged views of area A-A of FIG. 1a
of a circle sample plate wherein a path, in accordance with one
embodiment of the invention, to irradiate crystals produced using
CHCA or SA is illustrated.
[0059] FIGS. 15a and 15b are enlarged views of one sample site of a
channel sample plate wherein a path, in accordance with one
embodiment of the invention, to irradiate crystals produced using
CHCA or SA is illustrated.
[0060] FIG. 16 is a photograph of a mask having a rough surface in
accordance with one embodiment of the present invention.
[0061] FIG. 17 is a photograph showing crystals that have
crystallized around a sample site in accordance with one embodiment
of the invention.
[0062] FIG. 18 is a photograph showing crystals that have
crystallized around a sample site in accordance with one embodiment
of the invention using a specimen different than that shown in FIG.
17.
[0063] FIG. 19 is a photograph showing DHB crystals in target area
having large surface area.
DETAILED DESCRIPTION OF THE INVENTION
[0064] FIGS. 1a, 2a, 3a, and 4a and FIGS. 1b, 2b, 3b, and 4b are
views of a sample plate with at least one circular target area and
rectangular target area, respectively, in accordance with one or
more embodiments of the invention.
[0065] Therein, FIGS. 1a and 1b are isometric views of sample
plates with a circular target area and rectangular target area,
respectively, in accordance with one or more embodiments of the
invention. Sample plate 10 is characterized by any number of
equally preferred embodiments of target area 24.
[0066] In one embodiment, referred to for simplicity as a circle
sample plate 10, and depicted in FIG. 1a, sample plate 10 has a
plurality of circular target areas 24. In the second embodiment,
referred to for simplicity as a channel sample plate 10, and
depicted in FIG. 1b, sample plate 10 has a plurality of
rectangular, linear, and/or curvilinear target areas 24. Other
embodiments, including combinations of geometries of target areas
24, are also contemplated.
[0067] FIGS. 2a and 2b are enlarged views taken at area A-A of
FIGS. 1a and 1b, respectively, of a section of a sample plate in
accordance with one or more embodiments of the invention. FIGS. 3a
and 4a and FIGS. 3b and 4b are cross-sections at section B-B of
FIGS. 2a and 2b, respectively, of a section of a sample plate in
accordance with one or more embodiments of the invention.
[0068] Sample plate 10 is a sample plate for applying a sample
containing both matrix and biomolecules, referred to for
convenience as specimen 40 (not shown for clarity in FIGS. 1a and
1b), for subsequent analysis in a mass spectrometry instrument
within a sample site 20 (not shown for clarity in FIGS. 1a and 1b).
Specimen 40 may be applied within sample site 20 by using the dried
droplet method by spotting, i.e. in drop form, by streaking, i.e.
in a continuous manner, by spraying, and/or any other form.
Specimen 40 may also be applied within sample site 20 by the
electrospray deposition method.
[0069] Sample plate 10 includes substrate 12 having electrically
conductive surface 12a and mask 14 which is selectively applied to
surface 12a to form a mask that has a rough surface 14a where at
least one target area 24 is located within sample site 20, as will
be explained further herein.
[0070] Sample plate 10 is sized appropriately for usage for
biological laboratory processing using automated and/or manual
processing equipment. Thus, sample plate 10 may be appropriately
sized as microtiter plate size comprising a rectangular plan size
of 116.2 mm by 83 mm and/or any other convenient size. Sample plate
10 may be any suitable thickness for automated and/or manual
processing. In accordance with one embodiment of the invention
sample plate 10 is at minimum 0.5 mm thick with a maximum planar
variance of 50 .mu.m or less. For clarity, herein, sample plate 10
is described in relation to a rectangular plan and generally planar
shape of the plate. However, sample plate 10 may have any plan
shape and/or may have non-planar shapes as are and/or may become
appropriate for usage.
[0071] In accordance with one embodiment of the invention, sample
plate 10 has an indicator, such as a notched corner, that aids in
orienting sample plate 10. Other indicators may instead or in
addition be a central notch; one or more physical, chemical,
optical, and/or electromagnetic markers; and/or any other type of
indicator or indicating and/or orienting means.
[0072] In accordance with one embodiment of the invention, sample
plate 10 has a reference indicator for inventorying or archiving
sample plate 10 before and/or after usage. Such an indicator may be
a bar tag, alpha-numeric reference, chemical and/or luminescent
reference, and/or any indexing and/or archival reference that is
readable by a machine and/or a human, attached to and/or integral
with sample plate 10 on one or more of its surface.
[0073] In accordance with one embodiment of the invention, the
reference indicator is sensitive to one or more wavelengths of the
UV laser used in ionizing the crystals. Therein, the reference
indicator is activated and/or marked by the UV laser leaving a
permanent or semi-permanent reference readable by a machine and/or
a human.
[0074] Substrate 12 is preferably substantially planar and is made
of any solid material and/or combination of material. Substrate 12
has a first surface 12a that is electrically conductive. Surface
12a has an electrical resistance of 100 meg. ohms-per-square or
less.
[0075] As illustrated in FIGS. 3a and 3b, substrate 12 may be made
of electrically conductive materials; as for example using metals,
metal alloys, electro-conductive plastics, and/or combinations
thereof. In accordance with one embodiment of the invention,
surface 12a is made electrically conductive using an electrically
conductive coating 16 that is applied to substrate 12, as depicted
in FIGS. 4a and 4b. Coating 16 may be any type of applied mass that
has an electrical resistance of 100 meg. ohms-per-square or less.
Preferably, coating 16 maintains the substantially planar shape of
substrate 12. Coating 16 may be gold, copper, copper alloy, silver
alloy, silver plating, conductive plastic, or a conductive polymer
coating of any type. Preferably, the polymer coating includes
Baytron P (3,4-polyethylenedioxythiophene-polystyrenesulfonate in
water), CAS # 155090-83-8; polypyrrole, CAS # 30604-81, as a five
percent (5%) water solution, or in a solvent-based solution;
polyaniline as an emeraldine base, CAS # 5612-44-2; polyaniline as
an emeraldine salt, CAS # 25233-30-1; and/or variants of
polythiophenes, polyphenylenes, and/or polyvinylenes.
[0076] Mask 14 is selectively applied to surface 12a to form a mask
that has a rough surface 14a wherein target area 24 is centrally
located within sample site 20. Preferably, mask 14 has a thickness
in the range of 1 to 100 .mu.m and is made of a material that is
relatively more hydrophobic than surface 12a and that maintains a
suitable bond with substrate 12. For example, mask 14 may be made
of polytetrafluoroethylene, commonly known as Teflon.RTM. and
manufactured, sold, and/or licensed by DuPont Fluoroproducts of
Wilmington, Del., or any other suitable material.
[0077] Rough surface 14a is a non-homogenous surface that is
characterized by a coarse and/or an uneven surface quality and that
is lacking uniform surface intensity, regardless whether surface
14a has a regular or repeating pattern or patterns of intensity,
i.e. depth and/or graduations of the surface and/or material
thickness.
[0078] In accordance with one embodiment of the invention, mask 14
may be adulterated, i.e. doped, with one or more marking agents
that upon mass spectrometric analysis is/are detected as one or
more markers as a predetermined analytical result or is detected by
another means such as visual reference by an operator who sees the
color effect of a marking agent. Such marking agents may be used
for instrument calibration; quality assurance of sample
preparation, handling, laboratory procedures, and/or sample
tracking; quality assurance during production of sample plate 10;
and/or handling. Examples of marking agents may be carbon black,
titanium oxide, ferrous oxide, aluminum trioxides, polymeric
materials, coloring materials, and/or others.
[0079] In accordance with one embodiment of the invention, mask 14
is applied to surface 12a with a predetermined rough surface 14a.
For example, mask 14 is applied using a screening application
process resulting in rough surface 14a. Preferably, mask 14 is
applied utilizing Teflon.RTM. with a screen mesh sizes ranging from
30.times.30 .mu.m to 500.times.500 .mu.m such resulting rough
surface being described by the mesh size. Other screen sizes may be
employed equally well. Upon screening, sample plate 10 is allowed
to air dry, and once dry is heated to at least 50 Celsius to bond
mask 14 with substrate 12. Referring to FIG. 16, a microscopic
photograph of mask having rough surface is shown. The mask is made
of black Teflon and is shown having a matted appearance. In this
case, the matted appearance shows repeating substantially square
shaped imperfections to the polymer surface substantially similar
in size and shape as the mesh screen applied to the mask to form
rough surface.
[0080] In accordance with one embodiment of the invention, physical
and/or chemical manipulation of the material of mask 14 is used to
texture and create a rough surface 14a. For example etching,
gouging, scraping, oxidation, photo-oxidation, lithographic
printing, off-set printing, reverse image accessing, and/or any
other means may be used. In manufacturing the invention, rough
surface may be applied to mask while mask is being applied to the
substrate, or after it has been fixed to the surface.
[0081] Sample site 20 includes target area 24 and peripheral margin
22 of mask 14 that surrounds target area 24. Target area 24 is an
area of electrically conductive surface 12a and may have a number
of equally preferred embodiments, including embodiments wherein
target area 24 includes a mask spot or other structure. In
accordance with one embodiment of the invention, target area 24 has
a circular plan area as depicted in FIG. 1a for a circle sample
plate 10. In accordance with one embodiment of the invention,
target area 24 has a rectangular, linear, and/or curvilinear plan
area as depicted in FIG. 1b for a channel sample plate 10. Target
area 24 may also be embodied having other plan areas.
[0082] As will be described further herein, target area 24 serves
to substantially attract specimen 40 while it is in the liquid drop
state. Specimen 40 is attracted to target area 24 because mask 14
is relatively more hydrophobic than target area 24.
[0083] In the circle sample plate 10 depicted in FIG. 1a, sample
site 20 includes target area 24 having a circular plan area of
surface 12a and peripheral margin 22 coincident with the maximum
diameter in plan view with specimen 40 upon spotting on target area
24. Since the size of drops of specimen 40 may vary depending on
investigative need, i.e. using a large drop to increase
investigative sensitivity when biomolecules are in low
concentration, target area 24 may be of different sizes to
accommodate differently sized drops and sample plate 10 may be
selected based upon a diameter of target area 24 that is sized
appropriately for the drop size of specimen 40 that is to be
investigated.
[0084] As is easily understood, the volume of a drop of a liquid
directly correlates to the diameter of any planar section of the
drop. As is further understood, plan and radial dimensions of a
drop of liquid may be predetermined by controlling the drop's
volume and determining the relative hydrophilic and/or hydrophobic
qualities of the surface to which it adheres. Thus, control of drop
size may be achieved using pipetting or any other method to control
the volume of specimen 40.
[0085] It is known that hydrophobic and/or hydrophilic qualities
are relative to the contact angle between a drop and the surface to
which it adheres. An angle of 0.degree. indicates total hydrophilic
wetting of the surface and an angle of 180.degree. indicates total
hydrophobicity of the surface. Teflon.RTM. typically has a contact
angle of 140.degree. to 160.degree. for water.
[0086] In channel sample plate 10 depicted in FIG. 1b, sample site
20 includes target area 24 having a plan area of surface 12a,
characterized by length exceeding width, and a peripheral margin 22
substantially parallel to target area 24 coincident with the
maximum diameter in plan view of specimen 40 or the maximum
diameter in plan view of a plurality of specimen 40. Preferably,
target area 24 has a width of 0.1 to 0.5 mm and sample plate 10 may
be selected based upon a width of target area 24 that is sized
appropriately for the volume of specimen 40 that is to be
investigated.
[0087] In accordance with one embodiment of the invention, sample
site 20 includes target area 24 having a rectangular plan area.
[0088] In accordance with one embodiment of the invention, sample
site 20 includes target area 24 having a curvilinear plan area
comprising a spiral, although other curvilinear plan areas such as
a series of concentric plan areas are also contemplated.
[0089] In accordance with one embodiment of the invention, mask 14
is additionally applied to a central region of target area 24 to
form at least one mask spot 24a. FIG. 2c is a plan view of a sample
site that includes a target area and a mask spot in accordance with
one embodiment of the invention. FIG. 3c is an expanded elevation
view of a sample site that includes a target area and a mask spot
in accordance with one embodiment of the invention. Mask spot 24a
is further explained herein.
[0090] FIGS. 5 through FIGS. 10 depict the crystallization and
crystals produced by the dried droplet method using specimen 40 on
sample plate 10 in accordance with one embodiment of the invention.
FIG. 5a is an enlarged view taken at area A-A of FIG. 1a of a
circle sample plate in accordance with one embodiment of the
invention wherein specimens have been applied on sample sites. FIG.
5b is an enlarged view taken at area A-A of FIG. 1b of a channel
sample plate in accordance with one embodiment of the invention
wherein specimens have been applied on sample sites.
[0091] FIG. 6a is a cross-section at section C-C of FIG. 5a of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens have been applied on sample
sites. FIG. 6b is a cross-section at section C-C of FIG. 5b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens have been applied on sample
sites. FIG. 6b is a cross-section at section D-D of FIG. 5b of a
section of a channel sample plate in accordance with one embodiment
of the invention wherein specimens have been applied on sample
sites.
[0092] FIG. 7a is an enlarged view taken at area A-A of FIG. 1a of
a circle sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6a have begun to dry. FIG. 7b
is an enlarged view taken at area A-A of FIG. 1b of a channel
sample plate in accordance with one embodiment of the invention
wherein specimens of FIG. 6b have begun to dry.
[0093] FIG. 8a is a cross-section at section E-E of FIG. 7a of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6a have begun to dry.
FIG. 8b is a cross-section at section E-E of FIG. 7b of a section
of a channel sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6b have begun to dry. FIG. 8c
is a cross-section at section F-F of FIG. 7b of a section of a
channel sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6b have begun to dry.
[0094] FIG. 9a is an enlarged view taken at area A-A of FIG. 1a of
a circle sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6a have dried. FIG. 9b is an
enlarged view taken at area A-A of FIG. 1b of a channel sample
plate in accordance with one embodiment of the invention wherein
specimens of FIG. 6b have dried.
[0095] FIG. 10a is a cross-section at section G-G of FIG. 9a of a
section of a circle sample plate in accordance with one embodiment
of the invention wherein specimens of FIG. 6a have dried. FIG. 10b
is a cross-section at section G-G of FIG. 9b of a section of a
channel sample plate in accordance with one embodiment of the
invention wherein specimens of FIG. 6b have dried. FIG. 10c is a
cross-section at section H-H of FIG. 9b of a section of a circle
sample plate in accordance with one embodiment of the invention
wherein specimens of FIG. 6b have dried.
[0096] Specimens 40, each consisting of a drop in liquid form, are
applied to sample plate 10 within sample site 20 and contact mask
14. Therein, it is preferred that specimen 40 contact mask 14 at
the sides over a distance of at least 0.1 mm.
[0097] For circle sample plate 10, specimen 40 contacts mask 14 in
plan view at the perimeter of target area 24 by using a drop of
specimen 40 where the drop's maximum diameter exceeds the diameter
of target area 24. Since, it is known that a drop with a volume of
0.5 .mu.l has diameter of approximately 1.0 mm on the hydrophobic
surface of Teflon.RTM., it is preferred that each specimen 40 is
between 0.1 to 4.0 .mu.l in volume.
[0098] For channel sample plate 10, specimen 40 contacts mask 14 in
plan view at peripheral margin 22 while the perimeter of specimen
40 also contacts target area 24. In accordance with one embodiment
of the invention, specimen 40 is applied within sample site 20 on
channel sample plate 10 in a continuous manner, such as by spraying
or streaking specimen 40. Therein, the width or length of the
application of specimen 40 exceeds the width or length of target
area 24, respectively, so that specimen 40 contacts mask 14.
[0099] In accordance with one embodiment of the invention, mask
spot 24a is appropriately sized to form a drop of specimen 40 so
that the drop contacts the side of mask 14 to enhance the
deposition of crystals.
[0100] Specimen 40 includes a biomolecule and a matrix mixed in a
1:1 ratio, by volume. Although other formulations including those
with low solubility may also advantageously be used and the
formulations presented herein are not intended to be limiting, the
matrix may be made according to the following formulations:
[0101] In a first formulation (CHCA formulation),
.alpha.-cyano-4-hydroxycinnamic acid (C.sub.10H.sub.7NO.sub.3) and
an aqueous solution containing a solvent are mixed to produce a
matrix. The solvent preferably is acetonitrile (C.sub.2H.sub.3N)
and is mixed at a ratio of 30% to 50% acetonitrile and 70% to 50%
water, respectively, with 0.1% trifluoroacetic acid
(C.sub.2HF.sub.3O.sub.2), pH 2.3, by volume, to produce a solvent.
CHCA may be present at a concentration of 0.2 mg to 20 mg per 1 ml
of solvent, although a concentration of 1 to 5 mg of CHCA per 1 ml
of solvent is preferred. The first formulation may utilize other
matrices such as other cinnaminic acids or other matrices of low
solubility instead of CHCA.
[0102] In a second formulation (SA formulation),
3,5-dimethoxy-4-hydroxycinnamic acid (C.sub.11H.sub.12O.sub.5),
commonly known as sinapinic acid, and an aqueous solution
containing a solvent are mixed to produce a matrix. The solvent
preferably is acetonitrile (C.sub.2H.sub.3N) and is mixed at a
ratio of 30% to 50% acetonitrile and 70% to 50% water with 0.1%
trifluoroacetic acid (C.sub.2HF.sub.3O.sub.2), pH 2.3, by volume,
to produce a solvent. Sinapinic acid may be present at a
concentration of 0.2 mg to 20 mg per 1 ml of solvent, although a
concentration of 1 to 5 mg of sinapinic acid per 1 ml of solvent is
preferred.
[0103] The CHCA formulation is preferred for analysis of
biomolecules such as peptides and other biomolecules having
molecular weights of less than 10,000 Daltons. The SA formulation
is preferred for biomolecules such as proteins and other
biomolecules having molecular weights of 10,000 Daltons and
more.
[0104] FIGS. 11 and 12 are isometric views of crystals that have
formed in a halo effect in a sample site with different
concentrations of matrix formulations in accordance with one
embodiment of the invention. For simplicity, a sample site 20 of
circle sample plate 10 is depicted. Therein, crystals 42 deposit on
rough surface 14a in margin 22, forming a halo effect around the
perimeter of target area 24. Further crystals 42 form as the sample
solution dries and crystals 42 continue to form on surface 14a on
margin 22 on sample site 20. While crystals 42 are greatest in
number in margin 22, a significantly smaller amount deposit on
target area 24.
[0105] FIG. 11 depicts crystals 42 on sample site 20 of circle
sample plate 10 produced using CHCA matrix solution at a
concentration of 1 mg of CHCA per 1 ml of solvent. Crystals 42
crowd margin 22 near the periphery of target area 24 approximately
forming two concentric crystal rings around the periphery. A third
ring is approximately present in some areas. FIG. 17 is a
photograph of a similar sample showing crystals on sample site of
circle sample plate produced using CHCA matrix solution at a
concentration of 1 mg of CHCA per 1 ml of solvent. Crystals crowd
margin near the periphery of target area forming crystal rings
around the periphery.
[0106] Crystal rings are believed to result from the increase in
matrix concentration during the concomitant decrease in solvent
volume as the solvent evaporates. Crystalline lattices begin to
form and are attracted to rough surface 14a known to induce
crystalline formation. As the specimen drop dries, many matrix
crystalline lattices precipitate from the solution at roughly the
same time. Such precipitation occurs at regular intervals leading
to deposition in ring. As the larger matrix crystals precipitate,
smaller crystals form anew while the specimen drop continues
drying. Eventually these smaller crystals 42 also are unsustainable
in solution and precipitate from solution. In contrast, where a
lower concentration of matrix is used, crystals 42 result in only
one ring. Such crystals 42 are depicted in FIG. 12 where crystal 42
on sample site 20 were produced using CHCA matrix solution at a
concentration of 0.5 mg of CHCA per 1 ml of solvent. FIG. 18 is a
photograph of sample showing crystals on sample site of circle
sample plate produced using 30% ACN matrix solution at a
concentration of 1 mg of per 1 ml of solvent. Crystals crowd margin
near the periphery of target area forming crystal ring around the
periphery.
[0107] To analyze the biomolecule, crystals 42 are irradiated using
a UV laser (not shown for clarity) that scans crystals 42 directly
using the energy of a laser beam. The UV laser generates a laser
beam 48 typically at 337 nm wavelength, which may be any suitable
ultra-violet laser beam such one having an effective beam diameter
of 0.1 to 0.2 mm. As is easily understood, concentrating crystals
42 in margin 22 reduces the area required to be scanned by the
laser in order to irradiate sufficient crystals 42 to obtain
significant irradiation without compromising analysis sensitivity.
Given the beam's relatively small effective diameter, reducing the
requisite scanning area significantly enhances efficiency.
[0108] The reduced area is advantageously illustrated in comparison
to the area that must be irradiated when a traditional formulation
is used to produce specimen 40. To illustrate this example,
2,5-dihydroxybenzoic acid (C.sub.7H.sub.6O.sub.4) known as DHB is
used and is compared to the formulations of the present invention.
In DHB formulations, crystals 42 occur in target area 24. Thus, in
order to irradiate crystals 42 of the DHB formulation, the entire
target area 24 must be scanned. Thus, if target area 24 has a
diameter of 1 mm, the area to be scanned is 0.25.pi. mm.sup.2. In
order to irradiate crystals 42 wherein the matrix is the CHCA or SA
formulation and the sample site 20 has a diameter of 1.2 mm, the
area to be scanned is the 0.11.pi. mm.sup.2. This results in a
required scanning area that is only 44% of the traditional scanning
area. FIG. 19 is a photograph of DHB crystals forming in the
traditional target area, covering a large surface area.
[0109] FIG. 13 is a cross-section at section E-E of FIG. 5a of a
section of a sample plate in accordance with one embodiment of the
invention wherein crystals are being scanned by an UV laser. FIG.
13 illustrates the scanning of crystals 42 that were produced using
the CHCA and SA formulation.
[0110] Laser beam 48 sweeps scanning pattern 50 (not shown for
clarity) wherein it irradiates crystals 42 at a first position,
marked by the letter A in FIG. 13. As pattern 50 continues, at a
second position, marked by the letter B in FIG. 13, laser beam 48
irradiates another crystal 42 and proceeds further, where at a
third position, marked by the letter C in FIG. 13, it irradiates
another crystal 42, and so forth. Therein, it is understood that
scanning pattern 50 may be accomplished by maintaining the laser
stationary and moving plate 10, or by moving the laser and
maintaining plate 10 stationary, and/or a combination of both.
[0111] FIGS. 14a and 14b illustrate scanning pattern 50 in
accordance with one embodiment of the invention. Pattern 50 may be
any variety of patterns, circuit, or other traverse that irradiates
crystal 42 efficiently by minimizing the length of the path while
maximizing the number of crystals 42 that are irradiated.
[0112] FIGS. 14a and 14b are enlarged views of area A-A of FIG. 1
of circle sample plate 10 in accordance with one embodiment of the
invention wherein a scanning path to irradiate crystals 42 produced
using CHCA or sinapinic acid is illustrated. Laser beam 48 (not
shown for clarity) utilizes pattern 50 that is confined by two
predetermined boundaries; inner boundary 52a that is approximate
with the perimeter of target area 24 and an outer boundary 52b that
is within margin 22. Boundaries 52a and 52b may be predetermined
according to experience by an operator, statistical sampling, by an
algorithm, or any other suitable means.
[0113] One embodiment of scanning pattern 50 is illustrated in FIG.
14a. Pattern 50 is a cross-pattern that oscillates between boundary
52a on target area 24 and boundary 52b within sample site 20.
Another embodiment is illustrated in FIG. 14b. Therein, pattern 50
is spiral pattern that starts at boundary 52a and in one or more
circuits ends at boundary 52b. Other patterns or combinations of
patterns may also be used for pattern 50.
[0114] For the cross pattern illustrated in FIG. 14a, an algorithm
may include the number of oscillations, n, required to cover the
area of margin 22 based on a certain confidence level, c, as
expressed by a percentage or a ratio. A confidence level of 1 may
mean certainty that all crystals 42 have been irradiated. Thus,
n=(3.times.r.sub.1.sup.2)/r.sub.2.sup.2.times.c Equation 1
where r.sub.1 is the radius of target area 24 and r.sub.2 is the
effective radius of laser beam 48. Therein, if target area 24 is 1
mm in diameter, laser beam 48 has an effective diameter of 0.1 mm,
and a confidence level of 75% is desired, 56.25 oscillation are
required if boundaries 52a and 52b are at perimeters of margin
22.
[0115] Similarly, pattern 50 may be advantageously employed to more
to reduce the time and travel of UV laser and more efficiently
irradiate crystals 42 on a channel sample plate. FIGS. 15a and 15b
are enlarged views of one sample site of a channel sample plate
wherein a scanning pattern, in accordance with one embodiment of
the invention, to irradiate crystals produced using CHCA or SA is
illustrated.
[0116] Therein, similarly, laser beam 48 utilizes pattern 50 that
is confined by two predetermined boundaries; inner boundary 52a
that is approximate with the perimeter of target area 24 and an
outer boundary 52b that is within or coincident with sample site
20. Boundaries 52a and 52b may be predetermined according to
experience by an operator, statistical sampling, by an algorithm,
and/or any other suitable means. Illustrated in FIG. 15a is a
scanning pattern 50 that alternates between boundaries 52a and 52b,
and illustrated in FIG. 15b is a path that is a spiral pattern 50.
Other patterns may also be used.
[0117] In accordance with one embodiment of the invention, specimen
40 is applied on sample plate 10 using the electrospray deposition
method. Critical to the electrospray deposition method is that
specimen 40 is deposited in a smooth and constant application.
Sample plate 10 moves on a platform while a liquid chromatography
system elutes specimen 40 using a solvent and specimen 40 is
applied by electrospray on sample plate 10 for mass spectrometry
analysis.
[0118] Sample plate 10 is a channel sample plate or a circle sample
plate and cooperatively progresses from one location to another
with a liquid chromatography system. In accordance with one
embodiment of the invention, sample plate is located on a moving
platform that operates at a predetermined speed. Preferably, the
platform is operator controllable and adjustable, and further
includes one or more check mechanisms to ensure a precise
predetermined speed that cooperates with the electrospray
deposition.
[0119] A liquid chromatography system, such as micro-liquid
chromatography system or nano-liquid chromatography system is
provided to elute specimen 40 and apply it by electrospray on
sample plate 10. Typically, the liquid chromatography system
includes a reversed-phase column. The liquid chromatography system
is eluted with a matrix of the CHCA formulation at a concentration
of 1 mg of CHCA per 1 ml of solvent or SA formulation at a
concentration of 1 mg of SA per 1 ml of solvent; although other
matrices may also be used. Therein, the percentage of acetonitrile
varies from 0% to 70% over the course of eluting specimen 40 from
the column during the time period of elution, typically 15 to 60
minutes.
[0120] In accordance with one embodiment of the invention, specimen
40 is applied on channel sample plate 10 by direct application of
specimen 40 in liquid form such as by streaking. Therein, sample
plate 10 moves on a platform while a stationary liquid
chromatography system applies specimen 40. Specimen 40 is applied
to channel sample plate 10 at a continuous rate over a
predetermined length of target area 24, preferably at a rate 1
.mu.l per 1 mm of length of target area 24, while specimen 40 is
applied to circle sample plate 10 at a rate consistent with the
size of target area 24. Preferably, specimen 40 is applied within
sample site 20 that is no more than 0.2 mm from the periphery of
target area 24. Specimen 40 then quickly forms crystals 42 that
deposit on rough surface 14a from where they are irradiated using a
UV laser.
[0121] The present novel invention is also contemplated in
additional embodiments. In accordance with one embodiment of the
invention, sample plate 10 is produced includes sample site 20
wherein mask 14 is selectively applied with rough surface 14a to
surface 12a so that mask 14 is surrounded by surface 12a. Specimen
40 may be applied to sample plate 10 using the dried droplet method
by spotting, streaking, or spraying or by the electro-spray
deposition method. Specimen 40 may also be applied by washing or
submerging sample plate 10 with or in specimen 40. Crystals 42 will
then form on mask 14 in peripheral margin 22 and may be efficiently
irradiated using laser beam 48.
* * * * *