U.S. patent application number 16/253141 was filed with the patent office on 2019-12-26 for polymer substrates formed from carboxy functional acrylamide.
The applicant listed for this patent is LIFE TECHNOLOGIES AS, LIFE TECHNOLOGIES CORPORATION. Invention is credited to Elisabeth BREIVOLD, Geir FONNUM, M. Talha GOKMEN, Wolfgang HINZ, Lene HUSABOE, Synne LARSEN, Lily LU, Alfred LUI, Pontus LUNDBERG, Steven M. MENCHEN, Astrid Evenroed MOLTEBERG, Prasanna Krishnan THWAR.
Application Number | 20190390018 16/253141 |
Document ID | / |
Family ID | 56464309 |
Filed Date | 2019-12-26 |
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United States Patent
Application |
20190390018 |
Kind Code |
A1 |
FONNUM; Geir ; et
al. |
December 26, 2019 |
POLYMER SUBSTRATES FORMED FROM CARBOXY FUNCTIONAL ACRYLAMIDE
Abstract
A polymer substrate, such as a polymer particle, is formed from
a carboxyl functional monomer. In an example, the carboxyl
functional monomer has a protection group in place of the OH of the
carboxyl group. Once the monomer is polymerized, such a protection
group can be removed, providing a polymer network with carboxyl
functional sites. Such sites can be used to attach other
functionality to the polymer substrate.
Inventors: |
FONNUM; Geir; (Oslo, NO)
; MENCHEN; Steven M.; (Fremont, CA) ; GOKMEN; M.
Talha; (Fjellhamar, NO) ; LUNDBERG; Pontus;
(Strommen, NO) ; THWAR; Prasanna Krishnan; (San
Jose, CA) ; LUI; Alfred; (Sunnyvale, CA) ; LU;
Lily; (Foster City, CA) ; HINZ; Wolfgang;
(Killingworth, CT) ; HUSABOE; Lene; (Strommen,
NO) ; BREIVOLD; Elisabeth; (Jessheim, NO) ;
MOLTEBERG; Astrid Evenroed; (Fetsund, NO) ; LARSEN;
Synne; (Oslo, NO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES AS
LIFE TECHNOLOGIES CORPORATION |
Carlsbad
Carlsbad |
CA
CA |
US
US |
|
|
Family ID: |
56464309 |
Appl. No.: |
16/253141 |
Filed: |
January 21, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15871863 |
Jan 15, 2018 |
10189956 |
|
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16253141 |
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15200533 |
Jul 1, 2016 |
9868826 |
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15871863 |
|
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62188389 |
Jul 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08J 3/075 20130101;
C08F 230/08 20130101; C08F 8/12 20130101; C08F 230/08 20130101;
C08J 2333/26 20130101; C08F 222/38 20130101; C08F 220/70 20130101;
C08F 220/58 20130101; C08F 230/08 20130101; C08F 220/58 20130101;
C08F 220/58 20130101; C08F 222/38 20130101 |
International
Class: |
C08J 3/075 20060101
C08J003/075; C08F 220/70 20060101 C08F220/70; C08F 8/12 20060101
C08F008/12; C08F 220/58 20060101 C08F220/58 |
Claims
1. A compound of the formula: ##STR00005## wherein R.sub.1 is an
alkyl group having between 3 and 10 carbons or is a polyether group
having between 1 and 10 ether units, wherein R.sub.2 is a linear or
branched alkyl group having between 3 and 8 carbons or a silyl
group, and wherein R.sub.3 is hydrogen or an alkyl group having
between 1 and 6 carbons.
2. The compound of claim 1, wherein R.sub.1 is an alkyl group
having between 3 and 6 carbons.
3. The compound of claim 2, wherein R.sub.1 is an alkyl group
having between 3 and 5 carbons.
4. The compound of claim 1, wherein R.sub.1 is a polyether group
having 2 to 6 units.
5. The compound of claim 1, wherein the units of R.sub.1 include
ethylene oxide or propylene oxide units.
6. The compound of claim 1, wherein R.sub.2 is a branched alkyl
group.
7. The compound of claim 6, wherein R.sub.2 is a branched alkyl
group having between 3 and 5 carbons.
8. The compound of claim 7, wherein R.sub.2 is a branched alkyl
group having 4 carbons.
9. The compound of claim 1, wherein R.sub.3 is hydrogen.
10. The compound of claim 1, wherein R.sub.3 is a methyl or ethyl
group.
11. A population of particles having a coefficient of variance of
not greater than 5% and comprising a polymer derived from
polymerization of a compound of the formula: ##STR00006## wherein
R.sub.1 is an alkyl group having between 3 and 10 carbons or is a
polyether group having between 1 and 10 ether units, wherein
R.sub.2 is a linear or branched alkyl group having between 3 and 8
carbons or a silyl group, and wherein R.sub.3 is hydrogen or an
alkyl group having between 1 and 6 carbons; and wherein after
removal of R.sub.2, the particle absorbs at least 300 wt % and not
greater than 10.sup.6 wt % water based on the weight of the polymer
when exposed to water.
12. The population of particles of claim 11, wherein the polymer is
further derived from the polymerization of protected acrylamide or
protected hydroxyalkyl acrylamide with the compound.
13. The population of particles of claim 11, wherein the polymer is
further derived from the polymerization of a crosslinker with the
compound.
14. The population of particles of claim 13, wherein the
crosslinker comprises a diacrylamide.
15. The population of particles of claim 14, wherein the
diacrylamide includes N,N'-(ethane-1,2-diyl)bis(2-hydroxyl
ethyl)acryl amide, N,N'-(2-hydroxypropane-1,3-diyl)diacrylamide, a
protected derivative thereof, or a combination thereof.
16. The population of particles of claim 11, wherein R.sub.1 is an
alkyl group having between 3 and 6 carbons.
17. The population of particles of claim 16, wherein R.sub.1 is an
alkyl group having between 3 and 5 carbons.
18. The population of particles of claim 11, wherein R.sub.1 is a
polyether group having 2 to 6 units.
19. The population of particles of claim 11, wherein the units of
R.sub.1 include ethylene oxide or propylene oxide units.
20. The population of particles of claim 11, wherein R.sub.2 is a
branched alkyl group.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. application Ser.
No. 15/871,863 filed Jan. 15, 2018, which is a divisional of U.S.
application Ser. No. 15/200,533 filed Jul. 1, 2016 (now U.S. Pat.
No. 9,868,826), which application claims the benefit under 35
U.S.C. of .sctn. 119(e) of U.S. Provisional Application No.
62/188,389 filed Jul. 2, 2015. The entire contents of the
aforementioned applications are incorporated by referenced
herein.
FIELD OF THE DISCLOSURE
[0002] This disclosure, in general, relates to carboxyl functional
acrylamide, polymer substrates formed from such carboxyl functional
acrylamide, and methods for forming such polymer substrates.
BACKGROUND
[0003] Polymeric particles are increasingly being used as
components in separation techniques and to assist with detecting
analytes in both chemical and biological systems. For example,
polymeric particles have been used in chromatographic techniques to
separate target molecules from a solution. In another example,
polymeric particles having a magnetic coating are utilized in
magnetic separation techniques. More recently, polymeric particles
have been used to enhance ELISA-type techniques and can be used to
capture polynucleotides.
[0004] Many such techniques and uses of particles rely on
functionalizing the polymer. However, functionalizing the polymer
presents challenges relating to control of the number of sites
having the desired functionality and access through the polymer
network to the sites to be functionalized.
[0005] As such, an improved polymeric particle and method for
manufacturing such a polymeric particle would be desirable.
SUMMARY
[0006] In an exemplary embodiment, a polymer substrate, such as a
polymer particle, is formed from a carboxyl functional monomer. In
an example, the carboxyl functional monomer has a protection group
in place of the OH of the carboxyl group. Once the monomer is
polymerized, such a protection group can be removed, providing a
polymer network with carboxyl functional sites. Such sites can be
used to attach functionality to the polymer substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The present disclosure may be better understood, and its
numerous features and advantages made apparent to those skilled in
the art by referencing the accompanying drawings.
[0008] FIG. 1 includes an illustration of an exemplary process flow
for manufacturing an exemplary polymeric particle.
[0009] FIG. 2 includes an illustration of an exemplary sequencing
method utilizing polymeric particles.
[0010] The use of the same reference symbols in different drawings
indicates similar or identical items.
DETAILED DESCRIPTION
[0011] In an exemplary embodiment, a polymer substrate, such as a
polymer particle, is formed from a carboxyl functional monomer. In
an example, the carboxyl functional monomer has a protection group
in place of the OH of the carboxyl group. The protection group can
protect the OH group during the polymerization reaction or can
render the monomer more miscible with hydrophobic phases. Once the
monomer is polymerized, the protection group can be removed,
providing a polymer network with carboxyl functional sites. Such
sites can be used to attach functionality to the polymer substrate,
such as oligomer primers.
[0012] In a particular example, a monomer solution can be
distributed to a dispersed hydrophobic phase within a hydrophilic
or aqueous continuous phase. In an example, the dispersed
hydrophobic phase can be formed from a hydrophobic polymer bead.
The monomer solution can include a protected carboxyl functional
monomer, such as a protected carboxyl functional acrylamide.
Optionally, the monomer solution can further include other
monomers, crosslinkers, porogens, catalysts, or any combination
thereof.
[0013] In an example, the monomer can include a protected carboxyl
functional acrylamide monomer. In particular, the protected
carboxyl functional acrylamide includes a protection group
protecting the hydrophilic OH of the carboxyl functionality. The
protection group can protect the OH group, preventing reaction
during polymerization or rendering the monomer more miscible with
hydrophobic phases. In particular, the protection group is
cleavable from the monomer or from a polymer network formed from
the monomer. For example, the protection group can be acid
cleavable, in particular, at a pH that does not cause hydrolysis of
the polymer network.
[0014] For example, the protection group can include a silyl group.
In another example, the protection group can include a linear or
branched alkyl group having at least three carbons. For example,
the alkyl group can include 3 to 8 carbons, such as 3 to 6 carbons
or 3 to 5 carbons. In particular, the protection group can be a
branched alkyl group, such as a branched alkyl group having between
3 and 5 carbons, such as 4 carbons.
[0015] For example, the monomer can have the formula (I):
##STR00001##
wherein R.sub.1 is an alkyl group having between 3 and 10 carbons,
is a polyether group having between 1 and 10 ether units, or is
another non-ionic polar group, wherein R.sub.2 is a linear or
branched alkyl group having between 3 and 8 carbons or is a silyl
group, and wherein R.sub.3 is hydrogen or an alkyl group having
between 1 and 6 carbons. In a particular example, R.sub.1 is an
alkyl group having between 3 and 10 carbons or is a polyether group
having between 1 and 10 ether units. For example, R.sub.1 can be an
alkyl group having 3 to 6 carbons, such as 3 to 5 carbons. In
another example, R.sub.1 can be a polyether group including units,
such as including ethylene oxide or propylene oxide units, in a
range of 2 to 6 units, such as 2 to 4 units. In a further example,
R.sub.1 can be a non-ionic polar group, for example, including an
amide. In an example, R.sub.2 is a branched alkyl group, for
example, having 3 to 5 carbons, such as 4 carbons. In particular,
R.sub.2 can be an isopropyl, isobutyl, sec-butyl, or tert-butyl
group, or any combination thereof. The silyl group can be a
trialkyl silyl group, an organo disilyl group, or an organo
trisilyl group. For example, the trialkyl silyl group can be a
trimethyl silyl or a triethyl silyl group. In a further example,
R.sub.3 is hydrogen. In another example, R.sub.3 is a methyl or
ethyl group.
[0016] In an example, the monomer can have the formula (II):
##STR00002##
wherein R.sub.1 is an alkyl group having between 3 and 10 carbons
or is a polyether group having between 1 and 10 ether units, and
wherein R.sub.2 is a linear or branched alkyl group having between
3 and 8 carbons or is a silyl group. For example, R.sub.1 can be an
alkyl group having 3 to 6 carbons, such as 3 to 5 carbons. In
another example, R.sub.1 can be a polyether group including units,
such as including ethylene oxide or propylene oxide units, in a
range of 2 to 6 units, such as 2 to 4 units. In an example, R.sub.2
is a branched alkyl group, for example, having 3 to 5 carbons, such
as 4 carbons. In particular, R.sub.2 can be an isopropyl, isobutyl,
sec-butyl, or tert-butyl group, or any combination thereof. The
silyl group can be a trialkyl silyl group, an organo disilyl group,
or an organo trisilyl group. For example, the trialkyl silyl group
can be a trimethyl silyl or a triethyl silyl group.
[0017] In a particular example, the protected carboxyl functional
monomer can be acrylamidobutanoate protected with a tert-butyl
protection group and having the formula (III):
##STR00003##
[0018] In an example, the protected carboxyl functional monomers of
formulas (I), (II), or (III) can be formed by reacting a protected
amino alkanoic acid hydrochloride, such as an amino alkanoic alkyl
ester hydrocholoride, with acryloyl chloride. For example,
stoichiometric quantities of an amino alkanoic alkyl ester
hydrocholoride, such as aminobutyric acid t-butyl ester
hydrochloride, in a dichloromethane solvent can be mixed with a
potassium carbonate solution in water at a temperature in a range
of -10.degree. C. to 10.degree. C., such as -5.degree. C. to
5.degree. C. An acryloyl chloride solution can be added and the
mixture stirred under the same thermal conditions. The mixture can
be extracted with a solvent, such as dichloromethane. The solvent
can be removed under reduced pressure or vacuum.
[0019] In an example, the monomer described above can be
polymerized to form a polymer substrate. For example, the polymer
substrate can be a polymer coating or film. In another example, the
polymer substrate can be a polymer particle or bead. For example, a
polymer particle can be formed using emulsion polymerization or can
be formed in a dispersed hydrophobic phase within a hydrophilic
continuous phase.
[0020] For example, as illustrated in FIG. 1, a method 100 for
forming a polymer particle includes providing a seed particle 102,
which is promoted to form a dispersed phase 104. A protected
monomer or monomers are added to the suspension and preferably
reside in the dispersed phase 104 formed from a promoted seed
particle. The monomer or monomers and optionally a crosslinker are
polymerized to form a polymeric particle 108. The polymeric
particle 108 can be stripped of the seed polymer from the seed
particle to form the polymeric particle 110. The protection groups
on the polymeric particle 110 are removed to form a hydrophilic
particle 112. The hydrophilic particle 112 can be activated to form
a conjugated particle 114.
[0021] The seed particle 102 can include a seed polymer. In an
example, the seed polymer is hydrophobic. In particular, the seed
polymer can include a styrenic polymer, an acrylic polymer, an
acrylamide, another hydrophobic vinyl polymer, or any combination
thereof. In an example, the seed particle 102 is monodisperse, for
example, having a coefficient of variance of not greater than 20%.
Coefficient of variance (CV) is defined as 100 times the standard
deviation divided by the average, where "average" is mean particle
diameter and standard deviation is standard deviation in particle
size. Alternatively, the "average" can be either the z-average or
mode particle diameter. In accordance with usual practice, CV is
calculated on the main mode, i.e. the main peak, thereby excluding
minor peaks relating to aggregates. Thus some particles below or
above mode size may be discounted in the calculation which may, for
example, be based on about 90% of total particle number of
detectable particles. Such a determination of CV is performable on
a CPS disc centrifuge. In particular, a population of seed
particles 102 can have a coefficient of variance of not greater
than 10%, such as not greater than 5.0%, not greater than 3.5%, not
greater than 3%, not greater than 2.5%, not greater than 2%, or
even not greater than 1.0%. Further, the seed particle 102 can have
an initial particle size of not greater than 0.6 .mu.m. For
example, the initial particle size can be not greater than 0.45
.mu.m, such as not greater than 0.35 .mu.m, or even not greater
than 0.15 .mu.m, but at least 0.001 .mu.m. Alternatively, larger
seed particles having an initial particle size of at least 3 .mu.m,
such as at least 5 .mu.m, at least 10 .mu.m, at least 20 .mu.m, or
at least 50 .mu.m, can be used to form larger polymeric particles.
In an example, the initial particle size may be not greater than
100 .mu.m.
[0022] The seed particle 102 can be promoted within an aqueous
suspension to form a promoted dispersed phase 104. In particular,
promoting the seed particles includes mixing a solvent and a
promoter with the seed particle within the aqueous suspension to
form the dispersed phase. Promoted seed particles more readily
absorb hydrophobic components. The solvent can be water-miscible.
For example, the solvent can include an aldehyde or ketone, such as
formaldehyde, acetone, methyl ethyl ketone, diisopropyl ketone,
dimethyl formamide, or combinations thereof; an ether solvent, such
as tetrahydrofuran, dimethyl ether, or combinations thereof; an
ester solvent; a heterocyclic solvent, such as pyridine, dioxane,
tetrahydrofurfuryl alcohol, N-methyl-2-pyrrolidone, or combinations
thereof; or combinations thereof. In an example, the solvent can
include a ketone, such as acetone. In another example, the solvent
can include an ether solvent, such as tetrahydrofuran. In an
additional example, the solvent can include a heterocyclic solvent,
such as pyridine.
[0023] The promoter or promoting agent can be hydrophobic and have
a low water solubility, such as a water solubility of not greater
than 0.01 g/l at 25.degree. C. For example, the promoter can
include dioctanoyl peroxide, dioctyladipate, n-butyl phthalate,
dodecanol, polystyrene with molecular weight below 20 kD, or a
combination thereof. In an example, the dioctanoyl peroxide can
also perform as an initiator for a polymerization reaction. The
promoter can also be a low molecular weight polystyrene, for
example, made in a separate polymerization step using a low
monomer/initiator ratio or the addition of chain transfer reagents
during the seed polymerization. The promoter is typically
emulsified in a high pressure homogenizer.
[0024] The aqueous suspension can also include a surfactant. The
surfactant can be an ionic surfactant, an amphoteric surfactant, or
a non-ionic surfactant. The ionic surfactant can be an anionic
surfactant. In another example, the ionic surfactant can be a
cationic surfactant. An exemplary anionic surfactant includes a
sulfate surfactant, a sulfonate surfactant, a phosphate surfactant,
a carboxylate surfactant, or any combination thereof. An exemplary
sulfate surfactant includes alkyl sulfates, such as ammonium lauryl
sulfate, sodium lauryl sulfate (sodium dodecyl sulfate, (SDS)), or
a combination thereof; an alkyl ether sulfate, such as sodium
laureth sulfate, sodium myreth sulfate, or any combination thereof;
or any combination thereof. An exemplary sulfonate surfactant
includes an alkyl sulfonate, such as sodium dodecyl sulfonate;
docusates such as dioctyl sodium sulfosuccinate; alkyl benzyl
sulfonate; or any combination thereof. An exemplary phosphate
surfactant includes alkyl aryl ether phosphate, alkyl ether
phosphate, or any combination thereof. An exemplary carboxylic acid
surfactant includes alkyl carboxylates, such as fatty acid salts or
sodium stearate; sodium lauroyl sarcosinate; a bile acid salt, such
as sodium deoxycholate; or any combination thereof.
[0025] An exemplary cationic surfactant includes primary, secondary
or tertiary amines, quaternary ammonium surfactants, or any
combination thereof. An exemplary quaternary ammonium surfactant
includes alkyltrimethylammonium salts such as cetyl
trimethylammonium bromide (CTAB) or cetyl trimethylammonium
chloride (CTAC); cetylpyridinium chloride (CPC); polyethoxylated
tallow amine (POEA); benzalkonium chloride (BAC); benzethonium
chloride (BZT); 5-bromo-5-nitro-1,3-dioxane;
dimethyldioctadecylammonium chloride; dioctadecyldimethylammonium
bromide (DODAB); or any combination thereof.
[0026] An exemplary amphoteric surfactant includes a primary,
secondary, or tertiary amine or a quaternary ammonium cation with a
sulfonate, carboxylate, or phosphate anion. An exemplary sulfonate
amphoteric surfactant includes
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); a
sultaine such as cocamidopropyl hydroxysultaine; or any combination
thereof. An exemplary carboxylic acid amphoteric surfactant
includes amino acids, imino acids, betaines such as cocamidopropyl
betaine, or any combination thereof. An exemplary phosphate
amphoteric surfactant includes lecithin. In a further example, the
surfactant can be a non-ionic surfactant such as a polyethylene
glycol-based surfactant.
[0027] Returning to FIG. 1, a monomer or monomers added to
suspension preferably naturally reside in the dispersed phase 104
formed from the promoted seed particle. A crosslinker, such as a
hydrophobic crosslinker can also be added to the aqueous suspension
and preferentially can reside in the dispersed phase. In an
example, the crosslinker has a water solubility of not greater than
10 g/l. Further, a porogen can be added to the aqueous suspension
and preferentially can reside within the dispersed phase. In a
further example, the dispersed phase can include acrydite
oligonucleotides, such as an ion-exchanged acrydite
oligonucleotide. As illustrated in FIG. 1, the monomer and
optionally, the crosslinker are polymerized to form a polymeric
particle 108.
[0028] The monomer can include a protected carboxyl functional
acrylamide, as described above. In addition to the protected
carboxyl functional monomer, one or more comonomers can be included
to preferentially reside in the dispersed phase 104 and polymerize
with the protected carboxyl functional monomer. The comonomer can
be a radically polymerizable comonomer such as a vinyl-based
comonomer. In particular, the comonomer can include a hydrophilic
monomer coupled to a hydrophobic protection group. In an example,
the hydrophilic comonomer can include acrylamide, vinyl acetate,
hydroxyalkylmethacrylate, or any combination thereof. In a
particular example, the hydrophilic comonomer is an acrylamide,
such as an acrylamide including hydroxyl groups, amino groups,
carboxyl groups, or a combination thereof. In an example, the
hydrophilic comonomer is an aminoalkyl acrylamide, an acrylamide
functionalized with an amine terminated polypropylene glycol (VI,
illustrated below), an acrylopiperazine (VII, illustrated below),
or a combination thereof. In another example, the acrylamide
comonomer can be a hydroxyalkyl acrylamide, such as hydroxyethyl
acrylamide. In particular, the hydroxyalkyl acrylamide can include
N-tris(hydroxymethyl)methyl)acrylamide (IV, illustrated below),
N-(hydroxymethyl)acrylamide (V, illustrated below), or a
combination thereof.
##STR00004##
[0029] In a particular example, the hydrophilic comonomer includes
hydroxyl groups or includes amines. A hydrophobic protection group
shields the hydrophilicity of the comonomer, for example, by
bonding to a hydroxyl group or an amine group. Such protection
groups are referred to herein as hydroxyl or hydroxy protection
groups when bonding to a hydroxyl group. In particular, the
hydrophobic protection group is removable, such as through
cleaving, for example, acid cleaving. The hydrophobic group can be
selected to cleave under acidic conditions that do not result in
the hydrolysis of the underlying polymer or portions thereof. For
example, for pH values lower than 6, when an acrylamide polymer is
present, the hydrophobic protection group cleaves at a pH higher
than a pH at which the amide portion of the acrylamide hydrolyzes.
For pH values higher than 9, the hydrophobic protection group
cleaves at a pH lower than a pH at which the amide portion of the
acrylamide hydrolyzes.
[0030] An exemplary hydrophobic protection group includes an
organometallic moiety. For example, the organometallic moiety can
form a silyl ether functional group. The silyl ether functional
group can be derived from a halogenated silyl compound, such as a
compound of the general formulation
R.sub.1Si(R.sub.2)(R.sub.3)(R.sub.4), wherein R.sub.1 is a halogen,
such as chlorine and R.sub.2, R.sub.3, and R.sub.4 are
independently selected from hydrogen, alkyl groups such as methyl,
ethyl, propyl, butyl, aryl group, silyl groups, ether derivatives
thereof, or any combination thereof. An exemplary silyl ether
functional group is derived from tert-butyldimethylsilyl chloride,
trimethylsilyl chloride, triethylsilyl chloride, tripropylsilyl
chloride, tributylsilyl chloride, diphenyl methyl silyl chloride,
chloro(dimethyl)phenyl silane, or a combination thereof. In a
particular example, the protected monomer includes
N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide or tBDMS-HEAM,
N-(2-((triethylsilyl)oxy)ethyl)acrylamide or TES-HEAM, or a
combination thereof. In another example, the hydrophobic protection
group can include an organic moiety. An exemplary organic moiety
can include an alkyloxycarbonyl group moiety, such as
t-butyloxycarbonyl, fluorenylmethyloxycarbonyl, or a combination
thereof. In an example, such an organic moiety can be a hydrophobic
protection group bound to an amine functional group, such as an
amine functional group of an amine functionalized acrylamide or
copolymer thereof.
[0031] In a further example, a mixture of the carboxyl functional
monomer and a comonomer, such as a mixture of carboxyl functional
acrylamide monomer and hydroxyalkyl acrylamide comonomer or a
mixture of carboxyl functional acrylamide monomer and amine
functionalized acrylamide comonomer, can be used. In an example,
the carboxyl functional acrylamide monomer can be included in a
ratio relative to hydroxyalkyl acrylamide or amine functionalized
acrylamide comonomer in a range of 2:1 to 1:1000, such as a range
of 1:1 to 1:500, a range of 1:2 to 1:500, a range of 1:5 to 1:500
or even a range of 1:10 to 1:200.
[0032] The protected monomer and comonomer (together "protected
monomers") can be included in an amount relative to the initial
seed polymer, expressed as a ratio of weights (protected
monomers:seed polymer), in a range of 500:1 to 1:2, such as a range
of 200:1 to 1:1, a range of 100:1 to 5:1, a range of 90:1 to 10:1,
or even a range of 80:1 to 30:1. Alternatively, the protected
monomers can be included in an amount in a range of 10:1 to 1:2,
such as a range of 5:1 to 1:2, or even a range of 2:1 to 1:2.
[0033] The dispersed phase can also include a crosslinker. In an
example, the crosslinker is included in a mass ratio of protected
monomer to crosslinker in a range of 15:1 to 1:2, such as a range
of 10:1 to 1:1, a range of 6:1 to 1:1, or even a range of 4:1 to
1:1. The crosslinker can have a low water solubility (e.g., less
than 10 g/l), resulting in a preference for the dispersed phase. In
particular, the crosslinker can be a divinyl crosslinker. For
example, a divinyl crosslinker can include a diacrylamide, such as
N,N'-(ethane-1,2-diyl)bis(2-hydroxyl ethyl)acrylamide,
N,N'-(2-hydroxypropane-1,3-diyl)diacrylamide, or a combination
thereof. In another example, a divinyl crosslinker includes
ethyleneglycol dimethacrylate, divinylbenzene, hexamethylene
bisacrylamide, trimethylolpropane trimethacrylate, a protected
derivative thereof, or a combination thereof. In a further example,
the crosslinker can be protected with a hydrophobic protection
group, such as a hydroxyl protection group. In particular, the
hydrophobic protection group can be an organometallic moiety. For
example, the organometallic moiety can form a silyl ether
functional group. An exemplary silyl ether functional group can be
derived from tert-butyldimethylsilyl chloride, trimethylsilyl
chloride, triethylsilyl chloride, tripropylsilyl chloride,
tributylsilyl chloride, diphenyl methyl silyl chloride,
chloro(dimethyl)phenylsilane, or a combination thereof. An
exemplary protected diacrylamide crosslinker includes
N,N'-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)eth-
yl)acrylamide,
N,N'--(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyediacrylamide,
N,N'-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,
N,N'--(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,
silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide
such as
N,N'(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or a
combination thereof. In another example, the protection group can
include an alkyloxycarbonyl group moiety, such as
t-butyloxycarbonyl, fluorenylmethyloxycarbonyl, or a combination
thereof. In particular, a crosslinker including a hydroxyl group
can be protected with a protection group, such as those described
above in relation to the protected monomer.
[0034] In addition, polymerizing the hydrophilic monomer having a
hydrophobic protection can include polymerizing in the presence of
a porogen. An exemplary porogen includes an aromatic porogen. In
example, the aromatic porogen includes benzene, toluene, xylene,
mesitylene, phenethylacetate, diethyladipate, hexylacetate,
ethylbenzoate, phenylacetate, butylacetate, or a combination
thereof. The porogen typically has a Solubility parameter of 15-20.
In another example, the porogen is an alkanol porogen, such as
dodecanol. The porogen can be included in amounts relative to the
organic phase within the reactive system in a range of 1 wt % to 99
wt %, such as a range of 30 wt % to 90 wt % or even a range of 50
wt % to 85 wt %.
[0035] Optionally, a polymerization initiator can be included. An
exemplary polymerization initiator can initiate polymerization
through free radical generation. An exemplary polymerization
initiator includes an azo initiator, such as oil soluble azo
initiators. Another initiator can include ammonium persulfate. A
further exemplary initiator can include tetramethylethylenediamine.
In an example, the polymerization initiator can be included in an
amount of 0.001 wt % to 3 wt % based on the weight of the dispersed
phase.
[0036] Following polymerization, the polymeric particle 108 can be
stripped of the seed polymer to form the polymeric particle 110
still having the hydrophobic protection groups. For example, the
seed polymer can be extracted using a solvent, such as an aldehyde
or ketone, such as acetone, methyl ethyl ketone, diisopropyl
ketone, butylacetate, cyclohexanone, dimethyl formamide, or a
combination thereof; a phthalate solvent, such as, n-butyl
phthalate; an ether solvent, such as tetrahydrofuran, diisopropyl
ether, methyl tertbutyl ether, dimethyl ether, diethyl ether, or a
combination thereof; an ester solvent, such as ethyl acetate, butyl
acetate, or a combination thereof; a heterocyclic solvent, such as
pyridine, dioxane, tetrahydrofurfuryl alcohol, or a combination
thereof; halogenated solvents, such as dichloro methane, chloroform
or a combination thereof; or a combination thereof. Alternatively,
the seed polymer can be extracted following the conversion of the
polymeric particle to a hydrophilic particle. For example, the seed
polymer can be extracted following deprotecting the polymer of
particle, such as removing the silyl groups on the polymer
resulting from the protected monomer.
[0037] As illustrated in FIG. 1, the polymeric particle 110, once
the seed polymer is extracted, can be converted to a hydrophilic
polymeric particle by removing at least a portion of the
hydrophobic protection groups. For example, the hydrophobic
protection groups can be acid-cleaved from the polymeric particles.
In particular, such removing can remove substantially all of the
hydrophobic protection groups from the polymeric particle, such as
removing at least 80% of the hydrophobic protection groups, or even
at least 90% of the hydrophobic protection groups.
[0038] In an example, the hydrophobic protection groups are
acid-cleaved through the addition of an acid, such as an organic
acid. In particular, the organic acid can have a pKa in a range of
3.0 to 5.5. For example, the organic acid can include acetic acid,
lactic acid, citric acid, or any combination thereof.
Alternatively, inorganic acids can be used. For example, a sulfuric
acid solution can be used.
[0039] Once at least a portion of the hydrophobic protection groups
is removed, a hydrophilic particle 112 is formed. The hydrophilic
particle includes carboxyl functionality. In an example, the
hydrophilic particle 112 can be a hydrogel particle including a
hydrogel polymer. A hydrogel is a polymer that can absorb at least
20% of its weight in water, such as at least 45%, at least 65%, at
least 85%, at least 100%, at least 300%, at least 1000%, at least
1500%, or even at least 2000% of its weight in water, but not
greater than 10.sup.6%.
[0040] Prior to converting to a hydrophilic particle, the particles
can have a positive log(p) value. Following conversion, the
particles can have a negative log(p) value. The converted particles
can preferentially reside in aqueous or hydrophilic phases relative
to hydrophobic phases.
[0041] The hydrophilic polymer 112 can be activated to facilitate
conjugation with a target analyte, such as a polynucleotide. For
example, functional groups on the hydrophilic particle 112 can be
enhanced to permit binding with target analytes or analyte
receptors. In a particular example, functional groups of the
hydrophilic polymer can be modified with reagents capable of
converting the hydrophilic polymer functional groups to reactive
moieties that can undergo nucleophilic or electrophilic
substitution.
[0042] In particular, the hydrophilic polymer 112 has carboxyl
functionality that can be activated to facilitate conjugation, for
example to biomolecules, such as nucleic acids. In an exemplary
embodiment, the hydrophilic polymer includes a polyacrylamide
polymer network having alkanoic acid moieties or ester derivatives
thereof, which can react with succinimidyl compounds, such as a
succinimidyl uronium compound or a succinimidyl phosphonium
compound, to provide succinimidyl alkanoate moieties on the
polyacrylamide network Amine-terminated nucleic acids, such as
amine-terminated oligonucleotides, can react with the succinimidyl
alkanoate moieties to capture the nucleic acid to the polymer
network through an alkylamide moiety.
[0043] For example, a bead substrate can be formed of a
polyacrylamide polymer network that is functionalized with an
alkanoic acid moiety or an ester derivative thereof. In particular,
the polyacrylamide polymer network can be formed from
copolymerization of acrylamide monomers having carboxyl moieties or
ester derivatives thereof and acrylamide monomers having hydroxyl
or amine moieties. The ratio of the carboxyl functional monomer to
the acrylamide monomer including hydroxyl or amine moieties
influences the availability of conjugation sites that are reactive
to succinimidyl compounds, such as succinimidyl uronium or
succinimidyl phosphonium. When conjugated with amine-terminated
biomolecules, such as an amine-terminated nucleic acid (e.g., an
amine-terminated oligonucleotide), the polymeric bead can include a
polyacrylamide polymer network having alkylamide moieties directly
linked to nitrogen of the amide moiety on the acrylamide backbone
of the polyacrylamide network and linked to the biomolecule, such
as the nucleic acid.
[0044] The succinimidyl compound, for example, can be a
succinimidyl uronium compound or a succinimidyl phosphonium
compound. In a particular example, the succinimidyl compound is a
succinimidyl uronium compound. The succinimidyl uronium compound
can be an O-type succinimidyl uronium or an N-type succinimidyl
uronium. In particular, the succinimidyl uronium is an O-type
succinimidyl uronium. In an example, the O-type succinimidyl
uronium is an N-hydroxy succinimidyl uronium. In another example,
the succinimidyl compound is a succinimidyl phosphonium
compound.
[0045] In embodiments formed with a comonomer including hydroxyl
groups, hydroxyl groups on the hydrophilic particle 112 can be
activated by replacing at least a portion of the hydroxyl groups
with a sulfonate group or chlorine. Exemplary sulfonate groups can
be derived from tresyl, mesyl, tosyl, or fosyl chloride, or any
combination thereof. Sulfonate can act to permit nucleophiles to
replace the sulfonate. The sulfonate may further react with
liberated chlorine to provide chlorinated groups that can be used
in a process to conjugate the particles. In another example, amine
groups on the hydrophilic polymer 112 can be activated.
[0046] For example, target analyte or analyte receptors can bind to
the hydrophilic polymer through nucleophilic substitution with the
sulfonate group. In particular example, target analyte receptors
terminated with a nucleophile, such as an amine or a thiol, can
undergo nucleophilic substitution to replace the sulfonate groups
on the surface of the hydrophilic polymer 112. As a result of the
activation, a conjugated particle 114 can be formed.
[0047] In another example, the sulfonated particles can be further
reacted with mono- or multi-functional mono- or multi-nucleophilic
reagents that can form an attachment to the particle while
maintaining nucleophilic activity for oligonucleotides comprising
electrophilic groups, such as maleimide. In addition, the residual
nucleophilic activity can be converted to electrophilic activity by
attachment to reagents comprising multi-electrophilic groups, which
are subsequently to attach to oligonucleotides comprising
nucleophilic groups.
[0048] In another example, a monomer containing the functional
group can be added during the polymerization. The monomer can
include, for example, an acrylamide containing a carboxylic acid,
ester, halogen or other amine reactive group. The ester group may
be hydrolyzed before the reaction with an amine
oligonucleotide.
[0049] Other conjugation techniques include the use of monomers
that comprise hydrophobic protecting groups on amines during
particle synthesis. De-protection of the amine group makes
available a nucleophilic group that can be further modified with
amine reactive bi-functional bis-electrophilic reagents that yield
a mono-functional electrophilic group subsequent to attachment to
the polymer particle. Such an electrophilic group can be reacted
with oligonucleotides having a nucleophilic group, such as an amine
or thiol, causing attachment of the oligonucleotide by reaction
with the vacant electrophile.
[0050] In another example incorporating amino functional comonomers
in the particle 112, nucleophilic amino groups can be modified with
di-functional bis-electrophilic moieties, such as a di-isocyanate
or bis-NHS ester, resulting in a hydrophilic particle reactive to
nucleophiles. An exemplary bis-NHS ester includes bis-succinimidyl
C2-C12 alkyl esters, such as bis-succinimidyl suberate or
bis-succinimidyl glutarate.
[0051] Other activation chemistries include incorporating multiple
steps to convert a specified functional group to accommodate
specific desired linkages. For example, the sulfonate modified
hydroxyl group can be converted into a nucleophilic group through
several methods. In an example, reaction of the sulfonate with
azide anion yields an azide substituted hydrophilic polymer. The
azide can be used directly to conjugate to an acetylene substituted
biomolecule via "CLICK" chemistry that can be performed with or
without copper catalysis. Optionally, the azide can be converted to
amine by, for example, catalytic reduction with hydrogen or
reduction with an organic phosphine. The resulting amine can then
be converted to an electrophilic group with a variety of reagents,
such as di-isocyanates, bis-NHS esters, cyanuric chloride, or a
combination thereof. In an example, using di-isocyanates yields a
urea linkage between the polymer and a linker that results in a
residual isocyanate group that is capable of reacting with an amino
substituted biomolecule to yield a urea linkage between the linker
and the biomolecule. In another example, using bis-NHS esters
yields an amide linkage between the polymer and the linker and a
residual NHS ester group that is capable of reacting with an amino
substituted biomolecule to yield an amide linkage between the
linker and the biomolecule. In a further example, using cyanuric
chloride yields an amino-triazine linkage between the polymer and
the linker and two residual chloro-triazine groups one of which is
capable of reacting with an amino substituted biomolecule to yield
an amino-triazine linkage between the linker and the biomolecule.
Other nucleophilic groups can be incorporated into the particle via
sulfonate activation. For example, reaction of sulfonated particles
with thiobenzoic acid anion and hydrolysis of the consequent
thiobenzoate incorporates a thiol into the particle which can be
subsequently reacted with a maleimide substituted biomolecule to
yield a thio-succinimide linkage to the biomolecule. Thiol can also
be reacted with a bromo-acetyl group.
[0052] Alternatively, acrydite oligonucleotides can be used during
the polymerization to incorporate oligonucleotides. An exemplary
acrydite oligonucleotide can include an ion-exchanged
oligonucleotides.
[0053] Covalent linkages of biomolecules onto refractory or
polymeric substrates can be created using electrophilic moieties on
the substrate coupled with nucleophilic moieties on the biomolecule
or nucleophilic linkages on the substrate coupled with
electrophilic linkages on the biomolecule. Because of the
hydrophilic nature of most common biomolecules of interest, the
solvent of choice for these couplings is water or water containing
some water soluble organic solvent in order to disperse the
biomolecule onto the substrate. In particular, polynucleotides are
generally coupled to substrates in water systems because of their
poly-anionic nature. Because water competes with the nucleophile
for the electrophile by hydrolyzing the electrophile to an inactive
moiety for conjugation, aqueous systems generally result in low
yields of coupled product, where the yield is based on the
electrophilic portion of the couple. When high yields of
electrophilic portion of the reaction couple are desired, high
concentrations of the nucleophile are required to drive the
reaction and mitigate hydrolysis, resulting in inefficient use of
the nucleophile. In the case of polynucleic acids, the metal
counter ion of the phosphate can be replaced with a lipophilic
counter-ion, in order to help solubilize the biomolecule in polar,
non-reactive, non-aqueous solvents. These solvents can include
amides or ureas such as formamide, N,N-dimethylformamide,
acetamide, N,N-dimethylacetamide, hexamethylphosphoramide,
pyrrolidone, N-methylpyrrolidone, N,N,N',N'-tetramethylurea,
N,N'-dimethyl-N,N'-trimethyleneurea, or a combination thereof;
carbonates such as dimethyl carbonate, propylene carbonate, or a
combination thereof; ethers such as tetrahydrofuran; sulfoxides and
sulfones such as dimethylsulfoxide, dimethylsulfone, or a
combination thereof; hindered alcohols such as tert-butyl alcohol;
or a combination thereof. Lipophilic cations can include
tetraalkylammomiun or tetraarylammonium cations such as
tetramethylamonium, tetraethylamonium, tetrapropylamonium,
tetrabutylamonium, tetrapentylamonium, tetrahexylamonium,
tetraheptylamonium, tetraoctylamonium, and alkyl and aryl mixtures
thereof, tetraarylphosphonium cations such as
tetraphenylphosphonium, tetraalkylarsonium or tetraarylarsonium
such as tetraphenylarsonium, and trialkylsulfonium cations such as
trimethylsulfonium, or a combination thereof. The conversion of
polynucleic acids into organic solvent soluble materials by
exchanging metal cations with lipophilic cations can be performed
by a variety of standard cation exchange techniques.
[0054] In another example, particles can be formed using an
emulsion polymerization technique in which a hydrophobic phase
forms a dispersed phase within a hydrophilic phase. The monomers,
crosslinkers, and other agents and compounds described above that
favor hydrophobic phases tend to reside in the hydrophobic phase in
which polymerization occurs.
[0055] Surfactants, such as those described above can be used in
the hydrophilic phase to support emulsion formation. When a seed
particle is used, the surfactant can be used at a concentration
below the critical micelle concentration. Alternatively, the
surfactant can be used at a concentration greater than the critical
micelle concentration. Emulsion polymerization is typically
performed with a water soluble initiator like potassium or ammonium
persulfate.
[0056] In particular, the above method can produce a plurality of
particles having desirable particle size and coefficient of
variance. The set of particles can include, for example, 100,000
particles, such as 500,000 particles, greater than 1 million
particles, greater than 10 million particles, or even at least
1.times.10.sup.10, but can include not greater than
1.times.10.sup.20 particles. Particles of the plurality of
particles may be hydrophilic polymeric particles, such as hydrogel
particles. In a particular example, the hydrogel particle can be an
acrylamide particle, such as a particle including a crosslinked
carboxyl functional acrylamide polymer or a crosslinked copolymer
of carboxyl functional acrylamide and one or both of hydroxyalkyl
acrylamide or amine functionalized acrylamide.
[0057] The plurality of particles can have a desirable particle
size, such as a particle size not greater than 100 .mu.m, not
greater than 30 .mu.m, or not greater than 3 .mu.m. The average
particle size is the mean particle diameter. For example, the
average particle size may be not greater than 2 .mu.m, such as not
greater than 1.5 .mu.m, not greater than 1.1 .mu.m, not greater
than 0.8 .mu.m, not greater than 0.6 .mu.m, not greater than 0.5
.mu.m, or even not greater than 0.3 .mu.m, but generally at least
0.01 .mu.m, such as at least 0.1 .mu.m. In a particular example,
the average particle size can be in a range of 0.1 .mu.m to 100
.mu.m, such as a range of 0.1 .mu.m to 50 .mu.m or a range of 0.1
.mu.m to 1.1 .mu.m. In some aspects, the above described method
provides technical advantages for production of particles having a
particle size in a range of 5 .mu.m to 100 .mu.m, such as a range
of 20 .mu.m to 100 .mu.m, or a range of 30 .mu.m to 70 .mu.m. In
other aspects, the above described method provides technical
advantages for the production of particles having a particle size
of not greater than 1.1 .mu.m. When the seed is larger, larger
particles can be formed. The size of the particles can be adjusted
based on the size of the seed particle. Using the present method,
the size of the polymeric particle is less dependent on surfactant
selection and concentration than when other methods are used.
[0058] Further, the plurality of particles can be monodisperse and
may have a desirably low coefficient of variance, such as a
coefficient of variance of not greater than 20%. As above, the
coefficient of variance (CV) is defined as 100 times the standard
deviation divided by average, where "average" is the mean particle
diameter and standard deviation is the standard deviation in
particle size. The "average" alternatively can be either the
z-average or mode particle diameter. In accordance with usual
practice, CV is calculated on the main mode, i.e., the main peak,
thereby excluding minor peaks relating to aggregates. Thus, some
particles below or above mode size may be discounted in the
calculation which may, for example, be based on about 90% of total
particle number of detectable particles. Such a determination of CV
is performable on a CPS disc centrifuge or a coulter counter. For
example, the coefficient of variance (CV) of the plurality of
particles may be not greater than 15%, such as not greater than
10%, not greater than 5%, not greater than 4.5%, not greater than
4.0%, not greater than 3.5%, or even not greater than 3.0%. Such CV
can be accomplished without filtering or other size exclusion
techniques.
[0059] In a further example, a hydrophilic polymeric particle in
water can be not greater than 50 wt % polymer, such as not greater
than 30 wt % polymer, not greater than 20 wt % polymer, not greater
than 10 wt % polymer, not greater than 5 wt % polymer, or even not
greater than 2 wt % polymer.
[0060] In an additional example, the polymeric particle can have a
porosity permitting diffusion of proteins and enzymes. In an
example, the polymeric particles can have a porosity to permit
diffusion of proteins having a size of at least 50 kilodaltons,
such as at least 100 kilodaltons, at least 200 kilodaltons, at
least 250 kilodaltons, or even at least 350 kilodaltons. In a
particular example, the diffusion is limited for proteins having a
10.sup.5 kilodalton size.
[0061] In another example, when conjugated, the polymeric particle
can include a density of polynucleotides, termed nucleotide
density, of at least 7.times.10.sup.4 per .mu.m.sup.3. For example,
the nucleotide density can be at least 10.sup.5 per .mu.m.sup.3,
such as at least 10.sup.6 per .mu.m.sup.3, at least
5.times.10.sup.6 per .mu.m.sup.3, at least 8.times.10.sup.6 per
.mu.m.sup.3, at least 1.times.10.sup.7 per .mu.m.sup.3, or even at
least 3.times.10.sup.7 per .mu.m.sup.3. In a further example, the
nucleotide density can be not greater than 10.sup.15 per
.mu.m.sup.3.
[0062] Such polymeric particles can be used in a variety of
separations techniques and analytic techniques. In particular, the
polymeric particles may be useful in binding polynucleotides. Such
binding polynucleotides may be useful in separating polynucleotides
from solution or can be used for analytic techniques, such as
sequencing. In a particular example illustrated in FIG. 2, such
polymeric particles can be used as a support for polynucleotides
during sequencing techniques. For example, such hydrophilic
particles can immobilize a polynucleotide for sequencing using
fluorescent sequencing techniques. In another example, the
hydrophilic particles can immobilize a plurality of copies of a
polynucleotide for sequencing using ion-sensing techniques.
[0063] In general, the polymeric particle can be treated to include
a biomolecule, including nucleosides, nucleotides, nucleic acids
(oligonucleotides and polynucleotides), polypeptides, saccharides,
polysaccharides, lipids, or derivatives or analogs thereof. For
example, a polymeric particle can bind or attach to a biomolecule.
A terminal end or any internal portion of a biomolecule can bind or
attach to a polymeric particle. A polymeric particle can bind or
attach to a biomolecule using linking chemistries. A linking
chemistry includes covalent or non-covalent bonds, including an
ionic bond, hydrogen bond, affinity bond, dipole-dipole bond, van
der Waals bond, and hydrophobic bond. A linking chemistry includes
affinity between binding partners, for example between: an avidin
moiety and a biotin moiety; an antigenic epitope and an antibody or
immunologically reactive fragment thereof; an antibody and a
hapten; a digoxigen moiety and an anti-digoxigen antibody; a
fluorescein moiety and an anti-fluorescein antibody; an operator
and a repressor; a nuclease and a nucleotide; a lectin and a
polysaccharide; a steroid and a steroid-binding protein; an active
compound and an active compound receptor; a hormone and a hormone
receptor; an enzyme and a substrate; an immunoglobulin and protein
A; or an oligonucleotide or polynucleotide and its corresponding
complement.
[0064] In an example, the polymeric particle can be utilized in a
system with a surface. The system comprises one or more polymeric
particles on a surface. A surface can be a solid surface. A surface
can include planar, concave, or convex surfaces, or any combination
thereof. A surface can comprise texture or features, including
etching, cavitation or bumps. A surface can lack any texture or
features. A surface can include the inner walls of a capillary,
channel, groove, well or reservoir. A surface can be a mesh. A
surface can be porous, semi-porous or non-porous. A surface can be
a filter or gel. A surface can include the top of a pin (e.g., pin
arrays). The surface may be made from materials such as glass,
borosilicate glass, silica, quartz, fused quartz, mica,
polyacrylamide, plastic polystyrene, polycarbonate,
polymethacrylate (PMA), polymethyl methacrylate (PMMA),
polydimethylsiloxane (PDMS), silicon, germanium, graphite,
ceramics, silicon, semiconductor, high refractive index
dielectrics, crystals, gels, polymers, or films (e.g., films of
gold, silver, aluminum, or diamond). A surface can include a solid
substrate having a metal film or metal coat. A surface can be
optically transparent, minimally reflective, minimally absorptive,
or exhibit low fluorescence.
[0065] A plurality of polymeric particles can be arranged in a
random or ordered array on a surface, or a combination of random
and ordered arrays. Ordered arrays include rectilinear and
hexagonal patterns. A surface can include a plurality of sites
arranged in a random or ordered array, or a combination of both.
One or more polymeric particles can be located at one site, some
sites or all sites. Some sites can have one polymeric particle and
other sites can have multiple polymeric particles. At least one
site can lack a polymeric particle. In an array, at least two
polymeric particles can contact each other, or have no contact
between polymeric particles.
[0066] As illustrated in FIG. 2, a plurality of polymeric particles
204 can be placed in a solution along with a plurality of
polynucleotides 202. The plurality of particles 204 can be
activated or otherwise prepared to bind with the polynucleotides
202. For example, the particles 204 can include an oligonucleotide
complementary to a portion of a polynucleotide of the plurality of
polynucleotides 202.
[0067] In a particular embodiment, the hydrophilic particles and
polynucleotides are subjected to polymerase chain reaction (PCR)
amplification. For example, dispersed phase droplets 206 or 208 are
formed as part of an emulsion and can include a hydrophilic
particle or a polynucleotide. In an example, the polynucleotides
202 and the hydrophilic particles 204 are provided in low
concentrations and ratios relative to each other such that a single
polynucleotide 202 is likely to reside within the same dispersed
phase droplets as a single hydrophilic particle 204. Other
droplets, such as a droplet 208, can include a single hydrophilic
particle and no polynucleotide. Each droplet 206 or 208 can include
enzymes, nucleotides, salts or other components sufficient to
facilitate duplication of the polynucleotide. Alternatively,
amplification techniques, such as recombinase polymerase
amplification (RPA) with or without emulsion, can be used.
[0068] In a particular embodiment, an enzyme such as a polymerase
is present, bound to, or is in close proximity to the hydrophilic
particle or hydrogel particle of the dispersed phase droplet. In an
example, a polymerase is present in the dispersed phase droplet to
facilitate duplication of the polynucleotide. A variety of nucleic
acid polymerase may be used in the methods described herein. In an
exemplary embodiment, the polymerase can include an enzyme,
fragment or subunit thereof, which can catalyze duplication of the
polynucleotide. In another embodiment, the polymerase can be a
naturally-occurring polymerase, recombinant polymerase, mutant
polymerase, variant polymerase, fusion or otherwise engineered
polymerase, chemically modified polymerase, synthetic molecules, or
analog, derivative or fragment thereof.
[0069] In an embodiment, the polymerase can be any Family A DNA
polymerase (also known as pol I family) or any Family B DNA
polymerase. In embodiments, the DNA polymerase can be a recombinant
form capable of duplicating polynucleotides with superior accuracy
and yield as compared to a non-recombinant DNA polymerase. For
example, the polymerase can include a high-fidelity polymerase or
thermostable polymerase. In embodiments, conditions for duplication
of polynucleotides can include `Hot Start` conditions, for example
Hot Start polymerases, such as Amplitaq Gold.RTM. DNA polymerase
(Applied Biosciences) or KOD Hot Start DNA polymerase (EMD
Biosciences). Typically, a `Hot Start` polymerase includes a
thermostable polymerase and one or more antibodies that inhibit the
DNA polymerase and 3'-5' exonuclease activities at ambient
temperature.
[0070] In embodiments, the polymerase can be an enzyme such as Taq
polymerase (from Thermus aquaticus), Tfi polymerase (from Thermus
filiformis), Bst polymerase (from Bacillus stearothermophilus), Pfu
polymerase (from Pyrococcus furiosus), Tth polymerase (from Thermus
thermophilus), Pow polymerase (from Pyrococcus woesei), Tli
polymerase (from Thermococcus litoralis), Ultima polymerase (from
Thermotoga maritima), KOD polymerase (from Thermococcus
kodakaraensis), Pol I and II polymerases (from Pyrococcus abyssi)
and Pab (from Pyrococcus abyssi).
[0071] In embodiments, the polymerase can be a recombinant form of
Thermococcus kodakaraensis. In embodiments, the polymerase can be a
KOD or KOD-like DNA polymerase such as KOD polymerase (EMD
Biosciences), KOD "Hot Start" polymerase (EMD Biosciences), KOD
Xtreme Hot Start DNA Polymerase (EMD Biosciences), KOD XL DNA
polymerase (EMD Biosciences), Platinum.RTM. Taq DNA Polymerase
(Invitrogen), Platinum.RTM. Taq DNA Polymerase High Fidelity
(Invitrogen), Platinum.RTM. Pfx (Invitrogen), Accuprime.TM. Pfx
(Invitrogen), Accuprime.TM. Taq DNA Polymerase High Fidelity
(Invitrogen) or Amplitaq Gold.RTM. DNA Polymerase (Applied
Biosystems). In embodiments, the polymerase can be a DNA polymerase
containing analogous mutations to those polymerases discussed
herein.
[0072] In embodiments, duplication of the polynucleotide can
include modulating the duplication conditions. Modulating can
optionally include: increasing or decreasing the polymerase
concentration; increasing or decreasing the nucleotide
concentration; increasing or decreasing a cation concentration;
increasing or decreasing a reaction temperature, time or pH, or the
like. The modulating can include increasing or decreasing the rate
of the reaction, increasing or decreasing the yield of product of
the reaction, or the like. In embodiments, duplication can be
performed in the presence of appropriate buffers or nucleotides
(including nucleotide analogs or biotinylated nucleotides).
[0073] In particular, the polynucleotide to be amplified can be
captured by the polymeric particle. Exemplary methods for capturing
nucleic acid can include: hybridizing a polynucleotide to an
oligonucleotide that is attached to a polymeric particle. In
embodiments, methods for capturing nucleic acids comprise: (a)
providing a polymeric particle attached to a single-stranded
oligonucleotide (e.g., a capture oligonucleotide); (b) providing a
single-stranded polynucleotide; and (c) hybridizing the
single-stranded oligonucleotide to the single-stranded
polynucleotides, thereby capturing the single-stranded
polynucleotide to the polymeric particle. In embodiments, each of
the polymeric particles can be attached with a plurality of
single-stranded oligonucleotides (e.g., capture oligonucleotides).
In embodiments, step (c) can be conducted with a plurality of
single-stranded polynucleotides. In embodiments, at least a portion
of the single-stranded oligonucleotide comprises a nucleotide
sequence that is complementary (or partially complementary) to at
least a portion of the single-stranded polynucleotide.
[0074] In an example, the method further includes amplifying the
polynucleotide into a plurality of polynucleotides and attaching at
least a portion of the plurality of polynucleotides to the
hydrophilic particle, thereby generating a hydrophilic particle
including a plurality of attached polynucleotides. Alternatively,
the method can further include amplifying the polynucleotide into a
plurality of complementary polynucleotides by extending the
oligonucleotide, thereby generating a hydrogel particle including a
plurality of attached polynucleotides.
[0075] In embodiments, methods for nucleotide incorporation
comprise: conducting a nucleotide polymerization reaction on a
polynucleotide that is hybridized to an oligonucleotide that is
attached to a polymeric particle. In embodiments, methods for
nucleotide incorporation comprise: (a) providing a polymeric
particle attached to a single-stranded oligonucleotide (e.g., a
primer oligonucleotide); (b) providing a single-stranded template
polynucleotide; (c) hybridizing the single-stranded oligonucleotide
to the single-stranded template polynucleotide; and (d) contacting
the single-stranded template polynucleotide with a polymerase and
at least one nucleotide under conditions suitable for the
polymerase to catalyze polymerization of at least one nucleotide
onto the single-stranded oligonucleotide, thereby conducting
nucleotide incorporation. In embodiments, each of the polymeric
particles can be attached with a plurality of single-stranded
oligonucleotides (e.g., capture oligonucleotides). In embodiments,
steps (b), (c) or (d) can be conducted with a plurality of
single-stranded polynucleotides. In embodiments, at least a portion
of the single-stranded oligonucleotide comprises a nucleotide
sequence that is complementary (or partially complementary) to at
least a portion of the single-stranded polynucleotide. In
embodiments, a system comprises a single-stranded polynucleotide
hybridized to a single-stranded oligonucleotide which is attached
to a polymeric particle, wherein at least one nucleotide is
polymerized onto the end of the single-stranded
oligonucleotide.
[0076] In embodiments, methods for primer extension comprise:
conducting a primer extension reaction on a polynucleotide that is
hybridized to an oligonucleotide that is attached to a polymeric
particle. In embodiments, methods for nucleic acid primer extension
comprise: (a) providing a polymeric particle attached to a
single-stranded oligonucleotide (e.g., a primer oligonucleotide);
(b) providing a single-stranded template polynucleotide; (c)
hybridizing the single-stranded oligonucleotide to the
single-stranded template polynucleotide; and (d) contacting the
single-stranded template polynucleotide with a polymerase and at
least one nucleotide under conditions suitable for the polymerase
to catalyze polymerization of at least one nucleotide onto the
single-stranded oligonucleotide, thereby extending the primer. In
embodiments, each of the polymeric particles can be attached with a
plurality of single-stranded oligonucleotides (e.g., capture
oligonucleotides). In embodiments, step (b), (c) or (d) can be
conducted with a plurality of single-stranded polynucleotides. In
embodiments, at least a portion of the single-stranded
oligonucleotide comprises a nucleotide sequence that is
complementary (or partially complementary) to at least a portion of
the single-stranded polynucleotide. In embodiments, a system
comprises a single-stranded polynucleotide hybridized to a
single-stranded oligonucleotide which is attached to a polymeric
particle, wherein the single-stranded oligonucleotide is extended
with one or more nucleotides.
[0077] In embodiments, methods for nucleic acid amplification
comprise: conducting a primer extension reaction on a
polynucleotide that is hybridized to an oligonucleotide which is
attached to a polymeric particle. In embodiments, methods for
nucleic acid amplification comprise: (a) providing a polymeric
particle attached to a single-stranded oligonucleotide (e.g., a
primer oligonucleotide); (b) providing a single-stranded template
polynucleotide; (c) hybridizing the single-stranded oligonucleotide
to the single-stranded template polynucleotide; (d) contacting the
single-stranded template polynucleotide with a polymerase and at
least one nucleotide under conditions suitable for the polymerase
to catalyze polymerization of at least one nucleotide onto the
single-stranded oligonucleotide so as to generate an extended
single-stranded oligonucleotide. In embodiments, the method further
comprises: (e) removing (e.g., denaturing) the single-stranded
template polynucleotide from the extended single-stranded
oligonucleotide so that the single-stranded oligonucleotide remains
attached to the polymeric particle; (f) hybridizing the remaining
single-stranded oligonucleotide to a second single-stranded
template polynucleotide; and (g) contacting the second
single-stranded template polynucleotide with a second polymerase
and a second at least one nucleotide, under conditions suitable for
the second polymerase to catalyze polymerization of the second at
least one nucleotide onto the single-stranded oligonucleotide so as
to generate a subsequent extended single-stranded oligonucleotide.
In embodiments, steps (e), (f) and (g) can be repeated at least
once. In embodiments, the polymerase and the second polymerase
comprise a thermostable polymerase. In embodiments, the conditions
suitable for nucleotide polymerization include conducting the
nucleotide polymerization steps (e.g., steps (d) or (g)) at an
elevated temperature. In embodiments, the conditions suitable for
nucleotide polymerization include conducting the nucleotide
polymerization step (e.g., steps (d) or (g)) at alternating
temperatures (e.g., an elevated temperature and a relatively lower
temperature). In embodiments, the alternating temperature ranges
from 60-95.degree. C. In embodiments, the temperature cycles can be
about 10 seconds to about 5 minutes, or about 10 minutes, or about
15 minutes, or longer. In embodiments, methods for nucleic acid
amplification can generate one or more polymeric particles each
attached to a plurality of template polynucleotides comprising
sequences that are complementary to the single-stranded template
polynucleotide or to the second single-stranded template
polynucleotide. In embodiments, each of the polymeric particles can
be attached with a plurality of single-stranded oligonucleotides
(e.g., capture oligonucleotides). In embodiments, step (b), (c),
(d), (e), (f) or (g) can be conducted with a plurality of
single-stranded polynucleotides. In embodiments, at least a portion
of the single-stranded oligonucleotide comprises a nucleotide
sequence that is complementary (or partially complementary) to at
least a portion of the single-stranded polynucleotide. In
embodiments, methods for nucleic acid amplification (as described
above) can be conducted in an aqueous phase solution in an oil
phase (e.g., dispersed phase droplet).
[0078] Following PCR, particles are formed, such as particle 210,
which can include the hydrophilic particle 212 and a plurality of
copies 214 of the polynucleotide. While the polynucleotides 214 are
illustrated as being on a surface of the particle 210, the
polynucleotides can extend within the particle 210. Hydrogel and
hydrophilic particles having a low concentration of polymer
relative to water can include polynucleotide segments on the
interior of and throughout the particle 210 or polynucleotides can
reside in pores and other openings. In particular, the particle 210
can permit diffusion of enzymes, nucleotides, primers and reaction
products used to monitor the reaction. A high number of
polynucleotides per particle produces a better signal.
[0079] In embodiments, polymeric particles from an
emulsion-breaking procedure can be collected and washed in
preparation for sequencing. Collection can be conducted by
contacting biotin moieties (e.g., linked to amplified
polynucleotide templates which are attached to the polymeric
particles) with avidin moieties, and separation away from polymeric
particles lacking biotinylated templates. Collected polymeric
particles that carry double-stranded template polynucleotides can
be denatured to yield single-stranded template polynucleotides for
sequencing. Denaturation steps can include treatment with base
(e.g., NaOH), formamide, or pyrrolidone.
[0080] In an exemplary embodiment, the particle 210 can be utilized
in a sequencing device. For example, a sequencing device 216 can
include an array of wells 218. A particle 210 can be placed within
a well 218.
[0081] In an example, a primer can be added to the wells 218 or the
particle 210 can be pre-exposed to the primer prior to placement in
the well 218. In particular, the particle 210 can include bound
primer. The primer and polynucleotide form a nucleic acid duplex
including the polynucleotide (e.g., a template nucleic acid)
hybridized to the primer. The nucleic acid duplex is an at least
partially double-stranded polynucleotide. Enzymes and nucleotides
can be provided to the well 218 to facilitate detectable reactions,
such as nucleotide incorporation.
[0082] Sequencing can be performed by detecting nucleotide
addition. Nucleotide addition can be detected using methods such as
fluorescent emission methods or ion detection methods. For example,
a set of fluorescently labeled nucleotides can be provided to the
system 216 and can migrate to the well 218. Excitation energy can
be also provided to the well 218. When a nucleotide is captured by
a polymerase and added to the end of an extending primer, a label
of the nucleotide can fluoresce, indicating which type of
nucleotide is added.
[0083] In an alternative example, solutions including a single type
of nucleotide can be fed sequentially. In response to nucleotide
addition, the pH within the local environment of the well 218 can
change. Such a change in pH can be detected by ion sensitive field
effect transistors (ISFET). As such, a change in pH can be used to
generate a signal indicating the order of nucleotides complementary
to the polynucleotide of the particle 210.
[0084] In particular, a sequencing system can include a well, or a
plurality of wells, disposed over a sensor pad of an ionic sensor,
such as a field effect transistor (FET). In embodiments, a system
includes one or more polymeric particles loaded into a well which
is disposed over a sensor pad of an ionic sensor (e.g., FET), or
one or more polymeric particles loaded into a plurality of wells
which are disposed over sensor pads of ionic sensors (e.g., FET).
In embodiments, an FET can be a chemFET or an ISFET. A "chemFET" or
chemical field-effect transistor, includes a type of field effect
transistor that acts as a chemical sensor. The chemFET has the
structural analog of a MOSFET transistor, where the charge on the
gate electrode is applied by a chemical process. An "ISFET" or
ion-sensitive field-effect transistor, can be used for measuring
ion concentrations in solution; when the ion concentration (such as
H+) changes, the current through the transistor changes
accordingly.
[0085] In embodiments, the FET may be a FET array. As used herein,
an "array" is a planar arrangement of elements such as sensors or
wells. The array may be one or two dimensional. A one dimensional
array can be an array having one column (or row) of elements in the
first dimension and a plurality of columns (or rows) in the second
dimension. The number of columns (or rows) in the first and second
dimensions may or may not be the same. The FET or array can
comprise 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7
or more FETs.
[0086] In embodiments, one or more microfluidic structures can be
fabricated above the FET sensor array to provide for containment or
confinement of a biological or chemical reaction. For example, in
one implementation, the microfluidic structure(s) can be configured
as one or more wells (or microwells, or reaction chambers, or
reaction wells, as the terms are used interchangeably herein)
disposed above one or more sensors of the array, such that the one
or more sensors over which a given well is disposed detect and
measure analyte presence, level, or concentration in the given
well. In embodiments, there can be a 1:1 correspondence of FET
sensors and reaction wells.
[0087] Returning to FIG. 2, in another example, a well 218 of the
array of wells can be operatively connected to measuring devices.
For example, for fluorescent emission methods, a well 218 can be
operatively coupled to a light detection device. In the case of
ionic detection, the lower surface of the well 218 may be disposed
over a sensor pad of an ionic sensor, such as a field effect
transistor.
[0088] Exemplary systems involving sequencing via detection of
ionic byproducts of nucleotide incorporation are the Ion Torrent
PGM.TM., Proton.TM., or S5.TM. sequencers (Life Technologies),
which are ion-based sequencing systems that sequences nucleic acid
templates by detecting hydrogen ions produced as a byproduct of
nucleotide incorporation. Typically, hydrogen ions are released as
byproducts of nucleotide incorporations occurring during
template-dependent nucleic acid synthesis by a polymerase. The Ion
Torrent PGM.TM., Proton.TM., or S5.TM. sequencers detect the
nucleotide incorporations by detecting the hydrogen ion byproducts
of the nucleotide incorporations. The Ion Torrent PGM.TM.,
Proton.TM., or S5.TM. sequencers can include a plurality of
template polynucleotides to be sequenced, each template disposed
within a respective sequencing reaction well in an array. The wells
of the array can each be coupled to at least one ion sensor that
can detect the release of H+ ions or changes in solution pH
produced as a byproduct of nucleotide incorporation. The ion sensor
comprises a field effect transistor (FET) coupled to an
ion-sensitive detection layer that can sense the presence of H+
ions or changes in solution pH. The ion sensor can provide output
signals indicative of nucleotide incorporation which can be
represented as voltage changes whose magnitude correlates with the
H+ ion concentration in a respective well or reaction chamber.
Different nucleotide types can be flowed serially into the reaction
chamber, and can be incorporated by the polymerase into an
extending primer (or polymerization site) in an order determined by
the sequence of the template. Each nucleotide incorporation can be
accompanied by the release of H+ ions in the reaction well, along
with a concomitant change in the localized pH. The release of H+
ions can be registered by the FET of the sensor, which produces
signals indicating the occurrence of the nucleotide incorporation.
Nucleotides that are not incorporated during a particular
nucleotide flow may not produce signals. The amplitude of the
signals from the FET can also be correlated with the number of
nucleotides of a particular type incorporated into the extending
nucleic acid molecule thereby permitting homopolymer regions to be
resolved. Thus, during a run of the sequencer multiple nucleotide
flows into the reaction chamber along with incorporation monitoring
across a multiplicity of wells or reaction chambers can permit the
instrument to resolve the sequence of many nucleic acid templates
simultaneously.
[0089] Embodiments of the polymeric particles exhibit technical
advantages when used in sequencing techniques, particularly
ion-based sequencing techniques. In particular, embodiments of the
polymeric particles are non-buffering or enhance read lengths or
accuracy.
[0090] In a further example, the polymeric particles can exhibit
greater uniformity and lower CV without filtering than particles
made through other methods. For example, the above methods can
directly from the polymer particles without applying any kind of
selection process such as filtering or using a centrifuge. In
particular, emulsion polymerization can be used to produce
particles suitable for seed particles. Typically seed particles are
non-crosslinked to be able to adsorb the promoter molecule.
[0091] Further, embodiments of the present method provide for size
control based on the size of the seed particle. Additionally,
embodiments of particles made by such methods provide an increase
in conjugation, such as a 60% to 80% increase in conjugation, over
other methods.
EXAMPLES
Example 1 (Synthesis)
[0092] To an ice-cold suspension of 24.87 g (127.1 mmol)
.gamma.-aminobutyric acid t-butyl ester hydrochloride in 155 mL
dichloromethane is added a solution of 44.18 g (317.7 mmol)
potassium carbonate in 125 mL water. The reaction mixture is
stirred on ice bath for 15 min, followed by the addition of 16 mL
(0.19 mol) acryloyl chloride over a 10 min period. After 30 min
stirring on ice bath, the mixture is extracted with 250 mL
dichloromethane. The organic phase is washed with water,
3.times.250 mL, and saturated aqueous sodium chloride solution, 250
mL. Removal of solvent under reduced pressure affords the crude
product in quantitative yield.
Example 2 (Purification)
[0093] Approximately 27 g crude product is purified by way of
dry-column vacuum chromatography (DCVC), using 400 g silica as
adsorbent. Fractions of 300 mL size is eluted, employing a gradient
of methanol in dichloromethane (0-4%). The fractions containing
pure product is pooled, and subsequent removal of solvent under
reduced pressure gives 22.68 g product (84% overall yield) as a
colorless oil which solidified upon storage.
Example 3 (Purification)
[0094] Crude product (0.57 g) is purified on a small DCVC-column.
Fractions of 50 mL size are eluted, employing a gradient of ethyl
acetate in hexane (5-50%). The fraction containing pure product is
pooled, and subsequent removal of solvent under reduced pressure
gives 0.46 g product (81% recovery).
Example 4 (Purification)
[0095] Crude product (39.6 g) is dissolved in 150 mL ethyl acetate.
Heptane (600 mL) is added, and the stirred solution is slowly
cooled down -40.degree. C., whereupon a colorless precipitate is
formed. The precipitate is isolated on a cold glass sinter funnel,
washed with cold pentane (2.times.200 mL), and dried to a constant
mass of 29.7 g (75% recovery) in a vacuum desiccator. 1H NMR
(CDCl3, 400 MHz) 6.26 (d, 1H), 6.07 (dd, 1H), 5.98 (s, 1H), 5.62
(d, 1H), 3.37 (q, 2H), 2.30 (t, 2H), 1.84 (p, 2H), 1.44 (s,
9H).
Example 5
[0096] A silyl protected acrylamide monomer,
(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM),
and an ester group bearing acrylamide, t-butyl
4-acrylamidobutanoate, are polymerized with
N,N'-(ethane-1,2-diyl)bis(N-(2-(tert-butyldimethylsilyloxy)ethyl)acrylami-
de) (tBDMS-EBHEAM) crosslinker in a dispersed phase formed from
polystyrene particles and is deprotected to form a hydrogel
particle.
[0097] An emulsion is prepared by first dissolving 1.3808 g SDS in
230.00 g water and then adding 11.51 g acetone and 23.00 g
bis(2-ethylhexyl) adipate (DOA). The emulsion is mixed by
ultraturax for 3 minutes, and further homogenized for 5 minutes in
a high pressure Gauline APV-100 homogenizer at 400 Bar.
[0098] An amount of 37.15 g of this emulsion is added to 13.91 g of
seed particles (seed diameter 0.539 .mu.m, 15.41 weight % solids)
in a flask. The mixture is shaken at 40.degree. C. for 40 h in a
shaking bath for activation.
[0099] An SDS-borax solution is prepared by dissolving 0.65 g SDS
and 1.36 g borax to 341.11 g water.
[0100] A monomer emulsion is formed from 48.20 g 2-phenethyl
acetate, 0.0402 g 2,2'-azobis-(2-methylbutyronitrile) (AMBN), 8.70
g tBDMS-HEAM, 1.7448 g tBDMS-EBHEAM, 0.0411 g t-butyl
4-acrylamidobutanoate and 328.38 g SDS-borax solution, mixed by a
high speed mixer (Ystral D-79282) for 3 minutes, and further
homogenized for 4 minutes by a high pressure homogenizer at 400
bar.
[0101] In a jacket reactor, 14.18 g of a water dispersion of
activated seed particles is mixed with 337.0 g of the monomer
emulsion. The mixture is stirred and heated at 40.degree. C. for 2
h. The mixture is further stirred and heated at 40.degree. C. for
another hour while argon gas (150-200 ml/min) is bubbled through
the mixture. The amount of O.sub.2 in the emulsion at the end of
purging is measured to be 0 ppb. The argon flow is stopped, heating
and stirring continues for 10 hours at 70.degree. C.
[0102] The beads are filtered and the supernatant is removed after
centrifugation. The resulting beads are mixed with water, and 35.77
g 1M aqueous H.sub.2SO.sub.4 solution is added to the bead
dispersion, the dispersion is shaken at 60.degree. C. in a water
bath for 90 min and cooled to room temperature. The pH of the gel
dispersion is adjusted to 7.7 with NaOH and THF is added. The
organic phase is discarded and the hydrolyzed beads are cleaned by
centrifugation three times in water followed by crossflow
filtration in NMP.
[0103] In another example, hydrolysis can be done for 3 hours at
60.degree. C. in a water bath or for 18 hours at 40.degree. C. in a
water bath
[0104] In another example, seed particles can have an average
diameter of at least 0.050 .mu.m. In another example, seed
particles can have an average diameter of at max 10 .mu.m, more
typically between 90 nm and 330 nm.
[0105] In a first aspect, a compound has the formula (I) above,
wherein R1 is an alkyl group having between 3 and 10 carbons or is
a polyether group having between 1 and 10 ether units, wherein R2
is a linear or branched alkyl group having between 3 and 8 carbons
or a silyl group, and wherein R3 is hydrogen or an alkyl group
having between 1 and 6 carbons.
[0106] In an example of the first aspect, R1 is an alkyl group
having between 3 and 6 carbons. For example, R1 is an alkyl group
having between 3 and 5 carbons.
[0107] In another example of the first aspect and above examples,
R1 is a polyether group having 2 to 6 units.
[0108] In a further example of the first aspect and above examples,
the units of R1 include ethylene oxide or propylene oxide
units.
[0109] In an additional example of the first aspect and above
examples, R2 is a branched alkyl group. For example, R2 is a
branched alkyl group having between 3 and 5 carbons. In an example,
R2 is a branched alkyl group having 4 carbons.
[0110] In another example of the first aspect and above examples,
R3 is hydrogen.
[0111] In a further example of the first aspect and above examples,
R3 is a methyl or ethyl group.
[0112] In a second aspect, a method of synthesizing a monomer
includes reacting an amino alkanoate alkyl ester hydrochloride with
acryloyl chloride at a temperature in a range of -10.degree. C. to
10.degree. C. to form an alkyl ester of acrylamide alkanoic acid,
wherein the alkanoate group of the amino alkanoate alkyl ester
includes 3 to 10 carbons and the alkyl ester of the amino alkanoate
alkyl ester has 3 to 8 carbons; and extracting the alkyl ester of
acrylamide alkanoic acid using a solvent.
[0113] In an example of the second aspect, the alkanoate group has
3 to 6 carbons. For example, the alkanoate group has 3 to 5
carbons.
[0114] In another example of the second aspect and above examples,
the alkyl ester includes a branched ester.
[0115] In a further example of the second aspect and above
examples, the alkyl ester includes 3 to 5 carbons. For example, the
alkyl ester includes 4 carbons.
[0116] In a third aspect, a population of particles having a
coefficient of variance of not greater than 5% and comprising a
polymer derived from polymerization of a compound of the formula
(I) above, wherein R1 is an alkyl group having between 3 and 10
carbons or is a polyether group having between 1 and 10 ether
units, wherein R2 is a linear or branched alkyl group having
between 3 and 8 carbons or a silyl group, and wherein R3 is
hydrogen or an alkyl group having between 1 and 6 carbons; and
wherein after removal of R2, the particle absorbs at least 300 wt %
and not greater than 10.sup.6% water based on the weight of the
polymer when exposed to water.
[0117] In an example of the third aspect, the polymer is further
derived from the polymerization of protected acrylamide or
protected hydroxyalkyl acrylamide with the compound.
[0118] In another example of the third aspect and above examples,
the polymer is further derived from the polymerization of a
crosslinker with the compound. For example, the crosslinker
comprises a diacrylamide. In an example, diacrylamide includes
N,N'-(ethane-1,2-diyl)bis(2-hydroxyl ethyl)acrylamide,
N,N'-(2-hydroxypropane-1,3-diyl)diacrylamide, a protected
derivative thereof, or a combination thereof.
[0119] In a further example of the third aspect and above examples,
R1 is an alkyl group having between 3 and 6 carbons. For example,
R1 is an alkyl group having between 3 and 5 carbons.
[0120] In an additional example of the third aspect and above
examples, R1 is a polyether group having 2 to 6 units.
[0121] In another example of the third aspect and above examples,
the units of R1 include ethylene oxide or propylene oxide
units.
[0122] In a further example of the third aspect and above examples,
R2 is a branched alkyl group. For example, R2 is a branched alkyl
group having between 3 and 5 carbons. In an example, R2 is a
branched alkyl group having 4 carbons.
[0123] In an additional example of the third aspect and above
examples, R3 is hydrogen.
[0124] In another example of the third aspect and above examples,
R3 is a methyl or ethyl group.
[0125] In a further example of the third aspect and above examples,
the particle absorbs at least 1000 wt % water based on the weight
of the polymer when exposed to water.
[0126] In an additional example of the third aspect and above
examples, the particle has a particle size of not greater than 100
micrometers.
[0127] In a fourth aspect, a method of forming a particle includes
in a disperse phase within an aqueous suspension, polymerizing a
plurality of mer units having the formula (I) above, wherein R1 is
an alkyl group having between 3 and 10 carbons or is a polyether
group having between 1 and 10 ether units, wherein R2 is a linear
or branched alkyl group having between 3 and 8 carbons or a silyl
group, and wherein R3 is hydrogen or an alkyl group having between
1 and 6 carbons; thereby forming a polymeric particle including a
plurality of the hydrophobic protection groups; and converting the
polymeric particle to a hydrophilic particle.
[0128] In an example of the fourth aspect, the hydrophilic particle
is a hydrogel particle.
[0129] In another example of the fourth aspect and the above
examples, the disperse phase further includes a hydrophilic
monomer, the hydrophilic monomer including an acrylamide
monomer.
[0130] In an additional example of the fourth aspect and the above
examples, the dispersed phase further includes a diacrylamide
crosslinker having a hydrophobic protection group.
[0131] In a further example of the fourth aspect and the above
examples, converting the polymeric particle includes removing at
least a portion of the plurality of the R.sub.2 groups from the
polymeric particle. For example, removing at least a portion of the
plurality of the R.sub.2 groups includes acid cleaving at least a
portion of the plurality of the R.sub.2 groups from the polymeric
particle.
[0132] In another example of the fourth aspect and the above
examples, the method further includes promoting a seed particle in
the aqueous suspension to form the dispersed phase. For example,
the mass ratio of monomer:seed particles is in a range of 150:1 to
1:1. In another example, the seed particle includes a seed polymer.
In a further example, the method further includes extracting the
seed polymer after converting the polymeric particle. For example,
the seed polymer is hydrophobic. In an example, the seed polymer
includes a styrenic polymer, an acrylic polymer, an acrylamide,
another vinyl polymer, or a combination thereof. For example,
promoting the seed particle includes mixing a solvent and a
promoting agent with the seed particle. In another example, the
promoting agent includes dioctanoyl peroxide or dioctyladipate or
polystyrene with molecular weight below 20 kD.
[0133] In an additional example of the fourth aspect and the above
examples, the dispersed phase further includes acrylamide,
hydroxyalkyl acrylamide, or a combination thereof, the acrylamide
or hydroxyalkyl acrylamide polymerizing with the plurality of mer
units.
[0134] In a further example of the fourth aspect and the above
examples, polymerizing the plurality of mer units further includes
mixing a crosslinker with the plurality of mer units.
[0135] In an additional example of the fourth aspect and the above
examples, mixing the crosslinker includes mixing the crosslinker at
a mass ratio of monomer:crosslinker in a range of 15:1 to 1:2. For
example, the crosslinker is a divinyl crosslinker. In an example,
the divinyl crosslinker includes a diacrylamide. For example, the
diacrylamide includes
N,N'-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)eth-
yl)acrylamide, N,N'-(2-hydroxypropane-1,3-diyl)diacrylamide, a
protected derivative thereof, or a combination thereof. For
example, the diacrylamide includes
N,N'-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acryla-
mide,
N,N'--(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylam-
ide,
N,N'-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,
N,N'--(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,
silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide
such as
N,N'(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or a
combination thereof. In an example, the divinyl crosslinker
includes ethyleneglycoldimethacrylate, divinylbenzene,
hexamethylene bisacrylamide, trimethylolpropane trimethacrylate, or
a combination thereof.
[0136] In another example of the fourth aspect and the above
examples, polymerizing the plurality of mer units includes mixing a
porogen in the disperse phase. For example, the porogen is an
aromatic porogen. In an example, the aromatic porogen includes
toluene, xylene, mesitylene, phenylenethyl acetate or
ethylbenzoate.
[0137] In a further example of the fourth aspect and the above
examples, the method further includes activating the hydrophilic
particle. For example, activating includes applying a succinimidyl
compound to the hydrophilic particle. In an example, the method
further includes binding an oligonucleotide to the activated
hydrogel polymer. In another example, binding includes nucleophilic
substitution and the oligonucleotide is a nucleophile-terminated
oligonucleotide. For example, a nucleophile of the
nucleophile-terminated oligonucleotide is an amine group. In an
additional example, the method further includes hybridizing a
polynucleotide to the oligonucleotide. For example, the method
further includes amplifying the polynucleotide into a plurality of
polynucleotides and attaching at least a portion of the plurality
of polynucleotides to the hydrogel particle, thereby generating a
hydrogel particle including a plurality of attached
polynucleotides. For example, the method further includes
amplifying the polynucleotide into a plurality of complementary
polynucleotides by extending the oligonucleotide, thereby
generating a hydrogel particle including a plurality of attached
polynucleotides.
[0138] In an additional example of the fourth aspect and the above
examples, the hydrogel particle is one of a plurality of similarly
formed hydrogel particles having an average particle size of at
least 0.01 micrometers and not greater than 3 micrometer in
water.
[0139] In another example of the fourth aspect and the above
examples, the hydrogel particle is one of a plurality of similarly
formed hydrogel particles having an average particle size in a
range of 5 micrometers to 100 micrometers in water.
[0140] In a further example of the fourth aspect and the above
examples, the polymeric particle has a positive log(p) value and,
after converting the hydrophilic particle has a negative log(p)
value.
[0141] The above described methods, systems, compounds, and polymer
particles exhibit desirable technical advantages. Previous systems
and methods utilized silane protected amine or hydroxyl
functionalized acrylamides, which hydrolyze at a pH below 7.
Unprotected carboxyl functionalized acrylamides can cause low pH,
hydrolyzing other components and rendering them immiscible with the
dispersed phase, leading to reduced bead formation. Even when
buffered to pH 9, the carboxylic acid is a salt and does not
dissolve in the oil phase and thus, is not polymerized into the
particle. Accordingly, the above compounds, methods, and systems
advantageously lead to improved bead formation.
[0142] Note that not all of the activities described above in the
general description or the examples are required, that a portion of
a specific activity may not be required, and that one or more
further activities may be performed in addition to those described.
Still further, the order in which activities are listed are not
necessarily the order in which they are performed.
[0143] In the foregoing specification, the concepts have been
described with reference to specific embodiments. However, one of
ordinary skill in the art appreciates that various modifications
and changes can be made without departing from the scope of the
invention as set forth in the claims below. Accordingly, the
specification and figures are to be regarded in an illustrative
rather than a restrictive sense, and all such modifications are
intended to be included within the scope of invention.
[0144] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are intended to cover a non-exclusive inclusion. For
example, a process, method, article, or apparatus that comprises a
list of features is not necessarily limited only to those features
but may include other features not expressly listed or inherent to
such process, method, article, or apparatus. Further, unless
expressly stated to the contrary, "or" refers to an inclusive-or
and not to an exclusive-or. For example, a condition A or B is
satisfied by any one of the following: A is true (or present) and B
is false (or not present), A is false (or not present) and B is
true (or present), and both A and B are true (or present).
[0145] Also, the use of "a" or "an" are employed to describe
elements and components described herein. This is done merely for
convenience and to give a general sense of the scope of the
invention. This description should be read to include one or at
least one and the singular also includes the plural unless it is
obvious that it is meant otherwise.
[0146] Benefits, other advantages, and solutions to problems have
been described above with regard to specific embodiments. However,
the benefits, advantages, solutions to problems, and any feature(s)
that may cause any benefit, advantage, or solution to occur or
become more pronounced are not to be construed as a critical,
required, or essential feature of any or all the claims.
[0147] After reading the specification, skilled artisans will
appreciate that certain features are, for clarity, described herein
in the context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features
that are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any
subcombination. Further, references to values stated in ranges
include each and every value within that range.
* * * * *