U.S. patent application number 12/269464 was filed with the patent office on 2009-03-12 for laser desorption substrate.
This patent application is currently assigned to 3M Innovative Properties Company. Invention is credited to Bathsheba E. Chong Conklin, Brinda B. Lakshmi.
Application Number | 20090069177 12/269464 |
Document ID | / |
Family ID | 33490372 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090069177 |
Kind Code |
A1 |
Lakshmi; Brinda B. ; et
al. |
March 12, 2009 |
LASER DESORPTION SUBSTRATE
Abstract
Articles and methods for the high-energy desorption/ionization
of various compositions are disclosed. Methods of the invention
utilize porous substrates, optionally in combination with one or
more surface coatings and fillers, to provide enhanced desorption
of analytes. Such enhanced desorption is particularly useful in
fields of analysis such as mass spectrometry. This enhanced
desorption has various utilities. For example, use of the porous
substrate may allow desorption to be performed without the use of
chemical matrices.
Inventors: |
Lakshmi; Brinda B.;
(Woodbury, MN) ; Chong Conklin; Bathsheba E.;
(Saint Paul, MN) |
Correspondence
Address: |
3M INNOVATIVE PROPERTIES COMPANY
PO BOX 33427
ST. PAUL
MN
55133-3427
US
|
Assignee: |
3M Innovative Properties
Company
|
Family ID: |
33490372 |
Appl. No.: |
12/269464 |
Filed: |
November 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10457651 |
Jun 9, 2003 |
7462494 |
|
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12269464 |
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Current U.S.
Class: |
502/402 |
Current CPC
Class: |
Y10T 436/24 20150115;
Y10T 428/249953 20150401; H01J 49/0418 20130101 |
Class at
Publication: |
502/402 |
International
Class: |
B01J 20/26 20060101
B01J020/26 |
Claims
1-26. (canceled)
27. A porous polymeric article comprising: a polymeric substrate
having a first surface; a plurality of pores on the first surface
of the polymeric substrate; and a coating over at least a portion
of the plurality of pores; wherein the porous polymeric article is
configured for receiving analytes and subsequent desorption of the
analytes.
28. The porous polymeric article of claim 27, wherein the coating
is substantially nonvolatile.
29. The porous polymeric article of claim 27, wherein the coating
comprises diamond-like glass.
30. The porous polymeric substrate or claim 27, wherein the
polymeric substrate further comprises graphite particles.
31. The porous polymeric substrate of claim 27, wherein the
polymeric substrate further comprises carbon particles.
32. The porous polymeric substrate of claim 27, wherein the
polymeric substrate is formed by thermally induced phase
separation.
33. The porous polymeric substrate of claim 27, wherein the
polymeric substrate comprises high density polyethylene.
34. The porous polymeric substrate of claim 27, wherein the
polymeric substrate is configured and arranged for holding a sample
during mass spectrometry analysis.
35. A porous polymeric article comprising: a polymeric substrate
containing a filler, the substrate having a first surface; and a
plurality of pores on the first surface of the polymeric substrate;
wherein the polymeric article is configured for receiving of
analytes and subsequent desorption of the analytes.
36. The porous polymeric substrate of claim 35, wherein the filler
comprises metal particles, metal oxides, carbon particles, or a
combination thereof.
37. The porous polymeric substrate of claim 35, wherein the
polymeric substrate further comprises carbon particles.
38. The porous polymeric substrate of claim 35, wherein the
polymeric substrate is formed by thermally induced phase
separation.
39. The porous polymeric substrate of claim 35, wherein the
polymeric substrate comprises high-density polyethylene.
40. The porous polymeric substrate of claim 35, wherein the
polymeric substrate is configured and arranged for holding a sample
during mass spectrometry analysis.
41. A porous polymeric article comprising: a polymeric substrate
comprising a thermally induced phase separated film containing a
particulate filler; and a plurality of pores in the polymeric
substrate; wherein the polymeric article is configured for
receiving of analytes and subsequent desorption of the
analytes.
42. The porous polymeric article of claim 41, wherein the polymeric
substrate comprises polyethylene.
43. The porous polymeric article of claim 41, wherein the polymeric
substrate comprises high-density polyethylene.
44. The porous polymeric article of claim 41, wherein the
particulate filler comprises metal particles, metal oxides, carbon
particles, or a combination thereof.
45. The porous polymeric article of claim 41, wherein the porous
polymeric article further comprises a coating of diamond-like
glass.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to a substrate for use in
the retention and subsequent desorption of molecules. More
specifically, the invention is directed to a substrate for use in
receiving and releasing samples to be used in analytical processes,
such as mass spectrometry.
BACKGROUND
[0002] Matrix-assisted laser desorption and ionization (MALDI) mass
spectrometry has developed into an important tool for the analysis
of numerous compositions, especially complex biological materials.
MALDI uses a chemical matrix to suspend and retain one or more
analytes prior to subjecting the matrix and analytes to laser
desorption and ionization. Prior to the development of current
organic matrices used in MALDI, it was difficult to ionize intact
analyte molecules without molecular fragmentation.
[0003] Numerous matrices have been developed over the years to
fulfill the poorly understood requirements for successful laser
absorption and analyte ionization without fragmentation of the
analyte. The use of these matrices has become important because
they have permitted the analysis of macromolecules that would
otherwise not be readily observable using laser desorption and
ionization methods.
[0004] MALDI has been successfully used to identify peptides,
proteins, synthetic polymers, oligonucleotides, carbohydrates, and
other large molecules. Unfortunately, traditional MALDI has
drawbacks for the analysis of many small molecules because signals
from the chemical matrix interfere with signals from analyte
molecules. Chemical matrices have many other undesirable
consequences besides signal interference. For example, matrices can
complicate sample preparation, and the additional processing steps
and materials risk the introduction of contaminants into the
sample. Both the matrix and analyte must typically be dissolvable
in the same solvent, further complicating sample preparation. The
matrix can also make it more difficult to interface separation
techniques, and inhomogeneous sample spots can lead to a sweet-spot
phenomenon wherein higher amounts of analyte and matrix crystals
aggregate along the perimeter of the sample drop, leading to
reduced reproducibility of spectra.
[0005] The co-crystallization process of sample and matrix is also
often harsh, risking the denaturation or aggregation of proteins.
Additionally, it is not always clear which matrix is appropriate
for a given sample. For example, matrices that are effective for
peptides and proteins often do not work for oligonucleotides or
polymers. Furthermore, different matrices may be required in the
positive-ion detection mode and the negative-ion detection mode.
Thus, an exhaustive trial and error search can be required to find
the optimal matrix.
[0006] Another difficulty with MALDI is that the currently used
desorption substrates are typically metal plates. These metal
plates are expensive and they typically must be cleaned after use
so that they can be reused. Cleaning the metal plates is time
consuming and presents the possibility of carryover contamination,
and also does not allow for using the substrate as a storage device
for archiving the analyte samples for additional analysis.
[0007] Therefore, a need exists for a method and apparatus for
reducing or eliminating the need for matrices. In 1999, a
matrix-free method was described by Wei et al. in U.S. Pat. No.
6,288,390. Wei discloses the use of silicon wafers that have been
electrochemically etched with an HF/ethanol solution under
illumination and constant current. The sample, in solvent, is
applied directly to the silicon without the addition of any matrix.
This method, labeled desorption/ionization on silicon (DIOS),
allowed for the ionization of molecules within the mass range of
100 to 6000 Da without the interference caused by a matrix. Some
spectra obtained using DIOS, however, have been difficult to
reproduce, and the shelf life of the DIOS chips is often short.
Also, DIOS chips are relatively expensive due to the high cost of
the materials and processes used in their manufacture.
[0008] Therefore, a need remains for an apparatus and method that
provides enhanced laser desorption in comparison to conventionally
used techniques. There is also a need for an analyte desorption
substrate that is sufficiently inexpensive so that it can be used
and then discarded or archived.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to articles and methods
for the high-energy desorption/ionization of various compositions.
A first implementation of the invention includes a porous polymeric
article containing a polymeric substrate having a first
surface;
[0010] a plurality of pores on the first surface of the polymeric
substrate; and a coating over at least a portion of the plurality
of pores; wherein the porous polymeric article is configured for
receiving of analytes and subsequent desorption of the
analytes.
[0011] Methods of the invention utilize porous substrates,
optionally in combination with one or more surface coatings and
fillers, to provide enhanced desorption of analytes. Such enhanced
desorption is particularly useful in fields of analysis such as
mass spectroscopy. This enhanced desorption has various utilities.
For example, use of the porous substrate may allow desorption to be
performed without the use of chemical matrices. In some matrixless
implementations, particularly when a small molecule (such as those
with a molecular weight of less than 1000) is being analyzed, the
methods of the invention may achieve superior performance over that
of conventional matrix based methods (for example, higher signal to
noise ratios and/or better resolution).
[0012] Alternatively, the porous substrate may allow desorption to
be performed in the presence of matrix, but with superior
performance compared to standard matrix based methods using
conventional desorption substrates. For example, using the porous
substrate, an applied analyte/matrix droplet may dry in a more
uniform manner than without a porous substrate. Also, in some
implementations lower levels of matrix may be used, thereby
reducing signal noise from the matrix. Such behavior is
advantageous in allowing the use of automated sample deposition,
location, and analysis. Also, use of the porous substrate may
result in fewer ionic adducts (such as potassium and sodium) being
formed, resulting in a simpler and easier to interpret
spectrum.
[0013] Specific implementations of the invention are directed to an
article having a porous surface. The article contains a polymeric
substrate with a plurality of pores, and in certain implementations
a nonvolatile coating over at least a portion of the plurality of
pores. The present invention also provides for a desorption
substrate that is made from relatively inexpensive raw materials
and can be economically produced such that it may be used and
disposed of, or alternatively used as a storage device for
archiving analyte samples.
[0014] The methods and articles of the invention have many
applications, including use in proteomics, which is the study of
protein location, interaction, structure and function and seeks to
identify and characterize the proteins present in both healthy and
diseased biological samples. Other applications include DNA
analysis, small molecule analysis, automated high throughput mass
spectrometry, and combinations with separation techniques such as
electrophoresis, immobilized affinity chromatography, or liquid
chromatography.
[0015] Additional features and advantages of the invention will be
apparent from the following detailed description of the invention
and the claims. The above summary of principles of the disclosure
is not intended to describe each illustrated embodiment or every
implementation of the present disclosure. The detailed description
that follows more particularly exemplifies certain embodiments
utilizing the principles disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The invention will be more fully explained with reference to
the following drawings.
[0017] FIG. 1a is a mass spectrum of prazosin with matrix on
conventional MALDI-target.
[0018] FIG. 1b is a mass spectrum of prazosin without matrix on
graphite containing micro-porous high density polyethylene film
plus a diamond like glass coating.
[0019] FIG. 2a is a mass spectrum of prazosin without matrix on
graphite containing micro-porous high density polyethylene film
without a diamond like glass coating.
[0020] FIG. 2b is a mass spectrum of prazosin without matrix on
graphite containing micro-porous high density polyethylene film
plus a diamond like glass coating.
[0021] FIG. 3a is a mass spectrum of neurotensin without matrix on
graphite containing micro-porous high density polyethylene film
without a diamond like glass coating.
[0022] FIG. 3b is a mass spectrum of neurotensin without matrix on
graphite containing micro-porous high density polyethylene film
plus a diamond like glass coating.
[0023] FIG. 4a is a mass spectrum of prazosin without matrix on
micro-porous high density polyethylene film containing tungsten
particles and without a diamond like glass coating.
[0024] FIG. 4b is a mass spectrum of prazosin without matrix on
micro-porous high density polyethylene film containing tungsten
particles and a diamond like glass coating.
[0025] FIG. 4c is a mass spectrum of neurotensin without matrix on
micro-porous high density polyethylene film containing tungsten
particles and without a diamond like glass coating.
[0026] FIG. 4d is a mass spectrum of neurotensin without matrix on
micro-porous high density polyethylene film containing tungsten
particles and a diamond like glass coating.
[0027] FIG. 5a is a mass spectrum of a combination of chemical
samples on graphite micro-porous high density polyethylene film
plus a diamond like glass coating (positive ionization mode).
[0028] FIG. 5b is a mass spectrum of a combination of chemical
samples on graphite micro-porous high density polyethylene film
plus a diamond like glass coating (negative ionization mode).
[0029] FIG. 6 is a mass spectrum of a mixture of compounds on
graphite micro-porous high density polyethylene film plus diamond
like glass coating.
[0030] While principles of the invention are amenable to various
modifications and alternative forms, specifics thereof have been
shown by way of example in the drawings and will be described in
detail. It should be understood, however, that the intention is not
to limit the invention to the particular embodiments described. On
the contrary, the intention is to cover all modifications,
equivalents, and alternatives falling within the spirit and scope
of the disclosure and claims.
DETAILED DESCRIPTION
A. General Configuration
[0031] The present invention is directed to methods and articles
for the analysis of various compositions, in particular those
utilizing high-energy desorption/ionization of a sample. For
example, laser desorption and ionization of samples for mass
spectroscopy are suitable applications of the invention. The
apparatus serves to achieve, promote or enhance useful desorption
and ionization without fragmentation. In addition to providing
analyses without the complications of signal due to the matrix, in
some implementations, such as when a small molecule is being
analyzed, the methods of the invention may achieve superior
performance (as manifested by, for example, higher signal to noise
values) compared to traditional methods and devices.
[0032] Various aspects of the invention, including surface
structure and topology, coating compositions, substrate materials
and other aspects of the invention will now be described in greater
detail.
B. Porous Article
[0033] Substrates made in accordance with the invention typically
have a porous surface and include one or more surface coatings
and/or particulate fillers. In certain embodiments of the invention
the substrate comprises a high density polyethylene (HDPE) which
has a carbon based filler, such as graphite or carbon black. The
thermoplastic polymeric structure may be substantially homogeneous
throughout there may be a porosity gradient in the structure, but
is typically finely porous or porous. The particulate filler,
whether graphite, carbon black, metal, or another material, may be
substantially uniformly distributed throughout the article or the
particulate filler may have a gradient density throughout the
article.
[0034] The porous particulate-filled substrate may be provided as,
for example, films, sheets, or webs. When the substrate is in the
form of a film, the film may be uniaxially or biaxially oriented.
The substrates of the invention often have a network of
interconnected passageways to provide communicating pores, with
high effective pore size range, low fluid flow resistance, broad
pore size control and with up to 50 or more volume percent filler
loading.
[0035] The substrate is typically formed by thermally induced phase
separation, also known as TIPS, such as that taught in U.S. Pat.
No. 4,539,256 entitled "Microporous sheet material, method of
making and articles made therewith", incorporated herein by
reference in its entirety. This thermodynamic, non-equilibrium
phase separation may be either liquid-liquid phase separation or
liquid-solid phase separation.
[0036] As used herein, the term "thermoplastic polymer" refers to
conventional polymers, both crystalline and non-crystalline, which
are melt processable under ordinary melt processing conditions.
[0037] As used herein, the term "crystalline", as used with regard
to the thermoplastic polymer, includes polymers which are at least
partially crystalline.
[0038] As used herein, the term "amorphous", as used with regard to
the thermoplastic polymer, includes polymers without substantial
crystalline ordering such as, for example, polymethylmethacrylate,
polysulfone, and atactic polystyrene.
[0039] As used herein, the term "melting temperature" refers to the
temperature at which the thermoplastic polymer, in a blend of
thermoplastic polymer and compatible diluent, will melt.
[0040] As used herein, the term "crystallization temperature"
refers to the temperature at which the thermoplastic polymer, in a
melt blend of thermoplastic polymer and compatible diluent, will
crystallize.
[0041] As used herein, the term "equilibrium melting point", as
used with regard to the thermoplastic polymer, refers to the
commonly accepted melting point temperature of the thermoplastic
polymer as found in published literature.
[0042] As used herein, "particle" refers to submicron or low
micron-sized particles, also termed "particulate filler" herein,
such particles having a major axis no larger than five microns.
[0043] As used herein, "discretely dispersed" or "colloidal
suspension" means that the particles are arrayed substantially as
individual particles throughout a liquid or solid phase.
[0044] Thermoplastic polymers useful in the present invention
include olefinic, condensation and oxidation polymers. One
particularly suitable polymer is high density polyethylene (HDPE).
Representative olefinic polymers include high and low density
polyethylene, polypropylene, polyvinyl-containing polymers,
butadiene-containing polymers, acrylate containing polymers such as
polymethyl methacrylate, and fluorine containing polymers such as
polyvinylidene fluoride. Condensation polymers include polyesters
such as polyethylene terephthalate and polybutylene terephthalate,
polyamides, polycarbonates and polysulfones. Polyphenylene oxide is
representative of the oxidation polymers which can be used. Blends
of thermoplastic polymers may also be used.
[0045] The compatible diluent is a material which is capable of
forming a solution with the thermoplastic polymer when heated above
the melt temperature of the polymer and which phase separates from
the polymer on cooling. The compatibility of the liquid with the
polymer can be determined by heating the polymer and the liquid to
form a clear homogeneous solution. If a solution of the polymer and
the liquid cannot be formed at any liquid concentration, then the
liquid is generally inappropriate for use with that polymer. In
practice, the liquid used may include compounds, which are solid at
room temperature but liquid at the melt temperature of the polymer.
Generally, for non-polar polymers, non-polar organic liquids with
similar room temperature solubility parameters are generally useful
at the solution temperatures. Similarly, polar organic liquids are
generally useful with polar polymers. When blends of polymers are
used, useful liquids are those that are compatible diluents for
each of the polymers used. When the polymer is a block copolymer
such as styrene-butadiene, the liquid selected must be compatible
with each type of polymer block. Blends of two or more liquids can
be used as the compatible diluent as long as the selected polymer
is soluble in the liquid blend at the polymer melt temperature and
the solution formed phase separates on cooling.
[0046] Various types of organic compounds have been found useful as
the compatible diluent, including aliphatic and aromatic acids,
aliphatic, aromatic and cyclic alcohols, aldehydes, primary and
secondary amines, aromatic and ethoxylated amines, diamines,
amides, esters and diesters, ethers, ketones and various
hydrocarbons and heterocyclics. When the polymer selected is
polypropylene, aliphatic hydrocarbons such as mineral oil, esters
such as dibutyl phthalate and ethers such as dibenzyl ether are
useful as the compatible diluent.
[0047] When high density polyethylene is the polymer, an aliphatic
hydrocarbon such as mineral oil or and aliphatic ketone such as
methyl nonyl ketone or an ester such as dioctyl phthalate are
useful as the compatible diluent. Compatible diluents for use with
low density polyethylene include aliphatic acids such as decanoic
acid and oleic acid or primary alcohols such as decyl alcohol. When
the polymer is polyvinylidene fluoride, esters such as dibutyl
phthalate are useful as the compatible diluent. When the polymer
selected is nylon 11, esters such as propylene carbonate, ethylene
carbonate, or tetramethylene sulfone are useful as the compatible
diluent. When the polymer selected is polymethylmethacrylate,
useful compatible diluents include, 1,4-butanediol and lauric acid.
A compatible diluent for use with the polymer polyphenylene oxide
is, for example, tallowamine.
[0048] In certain embodiments, the particulate filler is arrayed in
the structure. For example, when the structure is spherulitic,
particles are in both the spherulites and in the fibrils between
them. Although the particles are firmly held in the polymeric
structure, they are substantially exposed after removal of the
compatible diluent. In a structure, the distribution of particles
is uniform wherever the polymer phase occurs. The particles
substantially exist as individual, and not agglomerated, particles
throughout the porous structure. Agglomerates of 3 to 4 particles
may occur, but their frequency is typically no more than in the
compatible diluent dispersion prior to melt blending with the
polymer. The average particle spacing depends upon the volume
loading of the particle in the polymer.
[0049] The compatible diluent is removed from the material to yield
a particle-filled, substantially liquid-free, porous thermoplastic
polymeric material. The compatible diluent may be removed by, for
example, solvent extraction, volatilization, or any other
convenient method, and the particle phase remains entrapped to a
level of at least about 90 percent, more preferably 95 percent,
most preferably 99 percent, in the porous polymer structure.
[0050] The particle-filled porous structures of this invention can
be oriented, i.e., stretched beyond their elastic limit to
introduce permanent set or elongation and to ensure that the
micropores are permanently developed or formed. Orientation can be
carried out either before or after removal of the compatible
diluent. This orientation of the structures aids in controlling
pore size and enhances the porosity and the mechanical properties
of the material without changing the particle uniformity and degree
of agglomeration in the polymer phase. Orientation causes the
porous structure to expand such that the porosity increases.
[0051] Particle-filled porous films of the invention may be
uniaxially or biaxially oriented in accordance with the teachings
of Shipman in U.S. Pat. No. 4,539,256 incorporated by reference in
its entirety. The particle-filled porous material of the invention
may also be further modified, either before or after removal of the
compatible diluent, by depositing various materials on the surface
thereof using known coating or deposition techniques. For example,
the particle-filled porous material may be coated with metal by
vapor deposition or sputtering techniques or by materials such as
adhesives, aqueous or solvent-based compositions, and dyes. Coating
can be accomplished by such conventional coating techniques as, for
example, roller coating, spray coating, dip coating, and the
like.
C. Filler Material
[0052] The porous substrate of the invention normally include at
least some filler particles, frequently a carbonaceous materials
such as, for example, carbon black or graphite; and metals, such as
gold, silver, and tungsten. The particles useful in the present
invention are generally capable of forming a colloidal dispersion
with the compatible diluent. The particle size is often less than 5
microns, more commonly less than 3 microns in size, and frequently
less than about 1 micron in size. Useful particles besides
carbonaceous materials include metals such as, for example, lead,
platinum, tungsten, gold, bismuth, copper, and silver, metal oxides
such as, for example, lead oxide, iron oxide, chrome oxide,
titania, silica and alumina, and blends thereof. In general,
materials that are good energy dispersers are beneficial,
particular those that absorb light at the same wavelength as the
energy used to laser desorb the analyte. For example, if the laser
has a wavelength of 337 nm, it is typically desirable to have the
particles at least partially absorb light at this wavelength.
[0053] The amount of filler particles in the thermoplastic polymer
depends upon the amount of filler in the compatible diluent prior
to melt blending and upon the relative amount of thermoplastic
polymer and compatible diluent in the blend. The amount of
particles colloidally dispersed in the compatible diluent depends
upon how well the particles are wet by the diluent, the surface
area of the particles, and the proper choice of a dispersing aid or
surfactant. Generally, for non-porous particles, a dispersion
containing 40-50 volume percent particles can be achieved. The
amount of filler in the polymer can he much greater than the amount
of filler in the compatible diluent when the melt blend has a
higher concentration of liquid than polymer.
[0054] The actual polymer concentration selected from within the
predetermined concentration range for the diluent-polymer system
being used is limited by functional considerations. The polymer
concentration and molecular weight should be sufficient to provide
the porous structure which is formed on cooling with adequate
strength for handling in further processing steps. The polymer
concentration should be such that the viscosity of the
diluent-polymer melt solution is suitable for the equipment used to
shape the article.
[0055] Generally, the polymer concentration in the compatible
diluent is about 10 to 80 weight percent, which corresponds to a
compatible diluent concentration of 20 to 90 weight percent. When
high compatible diluent concentrations, i.e. 80 to 90 percent, are
used in conjunction with high volume percent of filler in the
compatible diluent, a very high, e.g., about 95 weight percent,
concentration of the particulate filler in the thermoplastic
polymer, relative to the diluent, can be achieved. For example, if
the blend is 90:10 diluent/polymer by volume and the liquid is 40
percent particulate filler by volume, then the resulting filled
porous article is, surprisingly, 80 percent particulate filler by
volume after the diluent is removed. That the particle-filled
porous thermoplastic polymeric articles of the invention can
contain such large amounts of particulate filler is unexpected
because it is believed that particle-filled thermoplastic articles
made by standard extrusion processes achieve only about 20 percent
filler by volume.
D. Surface Treatment
[0056] The porous films of the present invention may be
advantageously used in combination with one or more coatings
applied on top of the porous film to provide enhanced desorption.
Coatings may also serve other purposes; for example, coatings may
provide a protective or abrasion-resistant barrier.
[0057] Useful coatings include organic materials such as graphite,
carbon black, the families of materials referred to as Diamond-Like
Carbon (DLC), as described in U.S. Pat. No. 6,265,068, and
Diamond-Like Glass (DLG), as described in PCT publication WO
0166820 entitled Diamond-Like Glass Thin Films, and incorporated
herein by reference, silanes and silane derivatives, and parylene.
Other useful coatings according to the present invention include
inorganic materials such as metals; for example aluminum, gold,
silver, nickel, titanium, palladium, and platinum; metal oxides,
for example titanium dioxide, silicon oxide and zirconium oxide,
and alloys of metals or metal oxides, such as inconel or indium tin
oxide.
[0058] Such surface coatings are generally nonvolatile under
conditions used for laser desorption. That is, the coating either
exhibits negligible volatility, or the entities that are
volatilized are so low in molecular weight (for example, carbon
clusters which may be emitted from graphite, or aluminum ions which
may be emitted from aluminum) that they do not interfere with the
analyte being measured. In this regard, the coatings are
distinguished from conventional matrices. While matrix materials
known in the art for MALDI applications are typically thought of as
"nonvolatile" in that they have a slow evaporation or sublimation
rate under ambient conditions they are volatilized to a significant
extent in the actual laser desorption process, and the volatilized
species have molecular weight such that they may interfere with or
obscure the analyte signal.
[0059] Coatings may be applied to the porous film via various
methods, including vapor coating, sputter coating, plasma coating,
vacuum sublimation, chemical vapor deposition, cathodic arc
deposition, and so on. These methods are particularly suited for
coating of metals and metal oxides. Coatings such as graphite are
most easily applied by obtaining the graphite as a dispersion and
applying it to the substrate by any of the well-known methods for
liquid coating (knife coating, spray coating, dip coating, spin
coating, etc.).
[0060] It can be advantageous to provide the coating in a
discontinuous manner as opposed to a continuous coating over the
entire porous surface. For example, the coating can be provided at
discrete locations, such as spots. In the case of multilayer
coatings, one coating may be discrete while the other may be
continuous, according to the needs of the particular instance.
Discontinuous coatings may serve several functions. For example,
they may serve to demarcate the particular area in which the
analyte sample is to be deposited, and then to allow the area to be
located once the film with sample is placed in the mass
spectrometer. A coating may also be used which provides a
discontinuity in the surface energy of the porous film to
advantageously contain a deposited analyte sample within a desired
area, and to prevent wicking or spreading of the sample over an
undesirably wide area.
[0061] Such coatings may be applied in a discrete manner via any
number of methods. If the coating is applied via vapor coating, a
mask, such as a perforated screen or film, may be used to limit the
coating to the areas defined by the mask. In the case in which it
is desired to have multilayer, registered discrete coatings (for
example spots containing superimposed multilayer coatings), the
mask can be attached to the film (for example via an adhesive)
during coating of the different layers such that the layers are
superimposed in registration. The mask is then removed after the
final coating process. In an alternative embodiment, the perforated
mask itself can remain on the film, in which case it will serve to
provide wells that serve to contain the analyte droplet that is
placed in the wells. It is also possible to provide a perforated
layer for this purpose independently of any role il defining the
coating. In the case of coatings such as graphite, well-known
liquid coating methods such as gravure coating can be used to
deposit the graphite in a discontinuous manner.
E. Device Assembly and Features
[0062] The present invention comprises a porous substrate, and
optional coatings useful for enhanced desorption, particularly in
mass spectroscopy. In typical use the film is attached to a
standard metal plate for insertion into a mass spectrometry
instrument. As such, a number of useful embodiments of the
invention exist. It is advantageous to provide the porous substrate
with a layer of adhesive applied to the back (non-porous) side, to
facilitate attachment to the metal plate. The adhesive can be a
laminating adhesive or double-faced tape. The laminating adhesive
can be attached to the underside of the porous film, with a release
liner remaining in place on the bottom of the adhesive. The user
can then simply remove the release liner and attach the film
directly to the plate by means of the adhesive. Alternatively, a
separate piece of laminating adhesive can be supplied to the user,
who can then apply the adhesive to the metal plate, remove the
liner, and attach the porous film to the top of the adhesive.
[0063] The adhesive should be carefully selected such that it does
not harbor or generate any impurities, which might contaminate the
porous substrate. In addition, it may be desirable in some cases
for the adhesive to be electrically conductive. Such conductive
adhesives are readily available, for example conductive adhesive
9713 available from 3M of Maplewood, Minn. The adhesive may be
selected such that it is permanently attached to the underside of
the porous film; alternatively, it may be removable.
[0064] Typically, the porous film, optionally with attached
adhesive underneath, will be packaged for delivery to the customer.
This packaging may consist of any means that protects the film and
does not act to impart contaminating impurities to the film. For
example, the film could be packaged in a plastic bag or plastic
case. As an additional protective measure, a protective liner may
be placed atop the upper (porous) surface of the film.
F. Sample Preparation and Methods of Using the Substrates
[0065] The present invention is particularly well suited to mass
spectrometry analysis. Analyte spots deposited on a substrate are
hit with short laser pulses to desorb and ionize the sample. Ions
are formed and then accelerated by one or more electric fields
before arriving at a detector. The time it takes to reach the
detector, or the location on the detector at which the particles
strike, can be used to determine the mass of the particles.
Time-of-flight analysis (TOF) is one mass spectrometry method that
can be used. For molecules under 10,000 Da, a reflectron mode is
used to condense the kinetic energy distribution of the ions
reaching the detector. This method was developed to increase the
resolution of mass spectroscopy and is used primarily for molecules
under 10,000 Da. This higher resolution often results in a drop in
sensitivity and a limited mass range.
G. EXAMPLES
Examples 1-4
[0066] A micro-porous high density polyethylene film (MPF) was
produced using the thermally-induced phase separation technique
described in U.S. Pat. No. 4,539,256. The film was produced using
Finathene.RTM. 1285 high density polyethylene (AtoFina
Petrochemicals Co. Houston, Tex.) with an initial mineral oil
content of 74%. The mineral oil was extracted using a suitable
solvent. The porosity of the resulting film was approximately 80%
with an average pore size of approximately 0.26 microns.
[0067] To increase the hydrophilicity of the film, the following
procedure was used. A 5 cm.times.5 cm piece of the film was clamped
between two aluminum plates with the uppermost plate having 64
through-holes, 1 mm in diameter, spaced similar to a conventional
MALDI metal plate. The clamped sample was then coated with a
hydrophilic Diamond-Like Glass (DLG) coating using a Plasma-Therm
vapor coater according to the methods described in PCT publication
WO0166820 by exposing the sample to a DLG plasma on one side under
the following conditions: 10 seconds of oxygen plasma, 30 seconds
of oxygen and tetramethylene silane mixture, followed by 20 seconds
of an oxygen plasma. The resulting film had 64 circular DLG
spots.
[0068] Circles were then gently drawn on the membrane using a sharp
tip razor blade to indicate where the analyte solutions would be
deposited. A solution (10 .mu.g/.mu.L in 1:1 H.sub.2O:MeOH) of the
drug molecule prazosin (419.9 Mw) was spotted (0.5 .mu.L) onto the
circular DLG regions on the film and air dried. A solution (0.1
.mu.g/.mu.L in 1:1 H.sub.2O:MeOH) of the peptide neurotensin
(1672.9 Mw) was spotted (0.5 .mu.L) onto the circular DLG regions
on the film and air dried. Prazosin belongs to a class of medicines
called anti-hypertensives. It is used to treat high blood pressure
(hypertension). Neurotensin, on the other hand, is an endogenous
trideca-peptide neurotransmitter, which influences distinct central
and peripheral physiological functions in mammals. Reflectron mode
was used for all tests. To show that the porous substrates of the
invention can be used with and without the use of a traditional
matrix, Examples 1-4 were tested using the above drug and peptide
analytes with the addition of 0.5 .mu.L matrix solution
(.alpha.-cyano 4-hydroxycinnamic acid--CHCA, 1:1
acetonitrile:water, 0.1% TFA). As comparative examples, the same
drug and peptide molecules were analyzed using a traditional steel
MALDI plate with CHCA matrix.
[0069] For each analysis, 2 spots/replicates were run and visually
compared to each other for similarity in Resolution (R) and Signal
to Noise (S/N) ratio. Resolution is the ability of a mass
spectrometer to distinguish between ions of different
mass-to-charge ratios. Greater resolution corresponds directly to
the increased ability to differentiate ions of similar molecular
weights. Resolution is usually defined as R=m/Dm in which Dm is the
mass difference between two adjacent peaks that are just resolved
and m is the mass of the first peak. In MALDI-TOF (time of flight)
measurements, Dm is the width of the peak at half maxima (FWHM) of
the peak corresponding to m. S/N is the ratio of the amplitude of
the desired signal to the amplitude of noise signals at a given
point in time. One factor that affects S/N is the concentration of
the analyte. S/N usually increases with increasing analyte
concentration. If the 2 spots did not compare well with each other,
the analysis was rerun using a freshly prepared film. Table 1 below
shows the use of DLG-coated porous films with and without matrix as
LDI substrates compared to a traditional steel LDI plate. The LDI
mass spectra of prazosin with matrix on a conventional MALDI target
(C1) and prazosin without matrix on graphite loaded MPF with a DLG
coating (E7) are shown in FIGS. 1a and 1b.
TABLE-US-00001 TABLE 1 (Comparatives) (Examples) (Examples)
Conventional MALDI MPF with MPF without Mol. plate with matrix
matrix + DLG matrix + DLG Molecule Wt. R S/N R S/N R S/N Prazosin
419.9 C1: 4070 9330 1: 3060 3900 3: 4540 1340 Neurotensin 1672.9
C2: 10870 1800 2: 3430 1700 4: Not observed* *No ion peak was
detected for the analyte
Examples 5-8
[0070] Examples 5-8 demonstrate the use of particle-loaded porous
films as LDI substrates. The films were prepared as in Examples 1-4
above except GM9255 high density polyethylene was used (Hoescht
Celanese). Approximately 22% by weight of graphite (TimCal America
Inc., Westlake, Ohio) was compounded into the film using a 30%
dispersion of the graphite in mineral oil. The membrane was clamped
between two metal frames and placed in a methyl ethyl ketone bath
for 15 minutes to remove the mineral oil. DLG spots were applied
using the same method as in Examples 1-4 above. Analytes were
spotted onto the substrates as in Examples 1-4. Testing was done
with and without DLG coatings. A matrix was not used. The test
results using the graphite-loaded films are shown in Table 2 below.
The LDI mass spectra for prazosin without DLG (E5) and with DLG
(E7) are shown in FIGS. 2a and 2b. The LDI mass spectra for
neurotensin without DLG (E6) and with DLG (E8) are shown in FIGS.
3a and 3b.
Examples 9-12
[0071] Examples 9-12 demonstrate the use of particle-loaded porous
films as an LDI substrate. The films were prepared as in Examples
5-8 above except FINA 1285 high density polyethylene and
non-conductive carbon (Columbian Chemicals, Marietta, Ga.) was
compounded into the films. The membrane was clamped between two
metal frames and placed in a methyl ethyl ketone bath for 15
minutes to remove the mineral oil. DLG spots were applied using the
same method as in Examples 1-4 above. Analytes were spotted onto
the substrates as in Examples 1-4. Testing was done with and
without DLG coatings. A matrix was not used. The test results using
the carbon-loaded films are shown in Table 2 below.
TABLE-US-00002 TABLE 2 (Examples) (Examples) graphite MPF
(Examples) (Examples) carbon MPF w/o matrix, w/o graphite MPF w/o
carbon MPF w/o w/o matrix + DLG matrix + DLG matrix, w/o DLG DLG
Molecule R S/N R S/N R S/N R S/N Prazosin 5: 3520 3810 7: 3240
18000 9: 2100 950 11: 1200 150 Neurotensin 6: 3500 100 8: 5000 200
10: 5000 50 12: 6100 100
Examples 13-16
[0072] Examples 13-16 demonstrate the use of metal particle-loaded
porous films as an LDI substrate. The films were prepared as in
Examples 5-8 above except 5-8% metal powder was compounded into the
films. PbO (lead oxide, Hammond Lead Products, Hammond, Ind.) and W
(tungsten, Union Carbide Corp, Danbury, Conn.) were used as the
metal powders. The membrane was clamped between two metal frames
and placed in a methyl ethyl ketone bath for 15 minutes to remove
the mineral oil. DLG spots were applied using the same method as in
Examples 1-4 above. Prazosin and neurotensin were used as the test
analytes and were spotted onto the substrates as in Examples 1-4.
Testing was done with and without a DLC coating. A matrix was not
used for any of the tests. The test results using the metal
particle-loaded films without matrix are shown in Table 3 below and
the LDI mass spectra for prazosin and neurotensin without DLG (E13)
and with DLG (E14) are shown in FIGS. 4a, 4b, 4c and 4d. For
reference, the R and S/N ratio for prazosin are 4070 and 9330 on
metal plate with matrix, whereas for neurotensin they are 10870 and
1800 respectively.
TABLE-US-00003 TABLE 3 Prazosin Neurotensin Sample Substrate R S/N
R S/N 13 MPF + W 3154 98 7439 18 14 MPF + W + DLG 2678 136 2985 24
15 MPF + PbO 6181 23100 Fragments* 16 MPF + PbO + DLG 3456 357
Fragments* *Lower molecular weight fragments of the ion peaks were
detected
Examples 17-24
[0073] The graphite-loaded MPF described in Examples 5-8 above was
used with DLG spots to analyze a series of 8 synthesized drug
molecules having molecular weights in close proximity to each
other. The series of eight molecules were dissolved individually in
methanol in concentration ranges from 0.1 to 0.3 .mu.g/.mu.L.
Samples were labeled A through H. 1.0 .mu.L of each solution was
spotted onto the MPF and air-dried. A Voyager-DE.TM. STR
BioSpectrometry Workstation, in reflectron mode, with positive and
negative ionization, was used for the testing. The LDI mass spectra
of compound F in both positive and negative ionization modes are
shown in FIGS. 5a and 5b respectively, showing that this technique
can be used to verify the molecular weight of low molecular weight
molecules in the positive and negative ionization modes in the
absence of matrix. The sensitivity and reproducibility of this
technique were further confirmed by analyzing a mixture of four of
the compounds at different concentrations. FIG. 6 shows one of the
three replicates spectrum showing that the ion signals (positive
ionization mode) of A (1.0 .mu.L), D (1.0 .mu.L), F (0.5 .mu.L),
and H (1.5 .mu.L) were detected and consistent in three replicate
runs despite the variation in concentration. The resolution and
signal to noise ratio of the testing of the eight compounds are
shown below in Table 4.
TABLE-US-00004 TABLE 4 Positive Negative Molecular ionization
ionization Example Molecule Weight R S/N R S/N 17 A 336 5800 140
6650 250 18 B 336 4550 120 5860 350 19 C 337 6620 140 4100 160 20 D
337 4560 240 3680 640 21 E 337 Not observed* Not observed* 22 F 341
5740 1750 4410 610 23 G 341 Not observed* 7810 160 24 H 342 6670 40
3960 350 *No ion peak was detected for the analyte
* * * * *