SAMPLE PREPARATION MATRIX, SYSTEM, METHOD OF MAKING, METHOD OF USING, AND KIT COMPRISING MATRIX
Disclosed herein are aspects of a matrix, typically comprising a rigid resin, for purifying samples, such as by separating one or more small molecules from one or more large molecules in a sample. Also disclosed is a method of making the matrix, a method of using the matrix, systems comprising the matrix, and kit aspects. Disclosed aspects comprising a porous size exclusion support and an amine-containing moiety can be used in small and large spin column formats and can be equilibrated with a wide range of buffers.
1 . A matrix, comprising:
at least one amine-containing moiety; and
a porous size exclusion support having a molecular weight cut-off ranging from greater than 30,000 Da to 45,000 Da and comprising 2-hydroxyethyl cellulose crosslinked with a heterocyclic compound having a structure according to Formula I,
wherein:
X is a heteroatom selected from nitrogen, oxygen, or sulfur; and
each of R 1 , R 2 , R 3 , and R 4 is independently selected from aliphatic, heteroaliphatic, haloaliphatic, halo, or glycidyl ether;
wherein the at least one amine-containing moiety is associated with the porous size exclusion support.
2 . The matrix of claim 1 , wherein the heterocyclic compound is epichlorohydrin.
3 . The matrix of claim 1 , wherein the porous size exclusion support is formed by reacting an amount of 2-hydroxyethyl cellulose ranging from greater than 0 grams to 200 grams with an amount of epichlorohydrin ranging from greater than 0 milliliters to 400 milliliters.
4 . The matrix of claim 1 , wherein the porous size exclusion support further comprises at least one modification reagent comprising epoxybutane, epoxyhexane, epoxypropane, dimethylsulfate, episulfide, disulfide, azirine, diazirine, diglycidyl ether, or any combination thereof.
5 . The matrix of claim 1 , wherein the at least one amine-containing moiety is covalently bound to the porous size exclusion support.
6 . The matrix of claim 1 , wherein, prior to association with the porous size exclusion support, the at least one amine-containing moiety is a monoamine, a diamine, or a polyamine.
7 . The matrix according to claim 6 , wherein, prior to association with the porous size exclusion support, the at least one amine-containing moiety has structure according to Formula V
wherein:
(i) at least one of R, R 1 , and R 2 is hydrogen;
(ii) at least one of R, R 1 , and R 2 is aliphatic, heteroaliphatic, aryl, or heteroaryl; and
(iii) the remaining R, R 1 , or R 2 is hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl.
8 . The matrix according to claim 7 , wherein at least one of R, R 1 and R 2 is hydrogen, and the remaining R, R 1 , or R 2 groups independently are C 1 -C 10 aliphatic.
9 . The matrix according to claim 8 , wherein at least one of R, R 1 and R 2 is hydrogen, and remaining R, R 1 and R 2 groups are independently methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, or isohexyl.
10 . The matrix according to claim 6 , wherein, prior to association with the porous size exclusion support, the at least one amine-containing moiety is a diamine having a structure according to Formula VI,
wherein:
(i) R is aliphatic, heteroaliphatic, aryl, or heteroaryl,
(ii) at least one of R 1 , R 2 , R 3 and R 4 is hydrogen, and
(iii) the remaining R 1 , R 2 , R 3 and R 4 are independently hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl.
11 . The matrix of claim 10 , wherein the at least one amine-containing moiety is a diamine having a structure according to Formula VII,
H 2 N—R—NH 2 Formula VII
wherein R is C 1 -C 10 aliphatic, heteroaliphatic, aryl, or heteroaryl.
12 . The matrix according to claim 11 , wherein, prior to association with the porous size exclusion support, the at least one amine-containing moiety is 1,5-diaminopentane and wherein greater than 0 mg 1,5-diaminopentane per mL of the porous size exclusion support to 10 mg 1,5-diaminopentane per mL of the porous size exclusion support is used to form the matrix.
13 . The matrix according to claim 6 , wherein, prior to association with the porous size exclusion support, the at least one amine-containing moiety is a monoamine having a structure according to Formula VIII,
wherein:
R 1 is hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl;
R 2 and R 3 are independently aliphatic, heteroaliphatic, aryl, or heteroaryl;
at least one of R 4 and R 5 is hydrogen, and the remaining R 4 group or R 5 group is hydrogen, aliphatic, heteroaliphatic, aryl, or heteroaryl; and
each of m and n independently is an integer selected from 1 to 10.
14 . The matrix according to claim 13 , wherein the amine-containing moiety is O-(2-aminopropyl)-O′-(2-methoxyethyl)polypropylene glycol.
15 . A device for separating one or more small molecules from one or more large molecules in a sample, the system comprising:
a container comprising a matrix according to claim 1 , wherein the container has a volume ranging from 0.075 mL to 50 mL.
16 . The device of claim 15 , wherein container is a columnar container, a tube, a multi-well tube, a multi-well plate, or a multi-well filter plate.
17 . The device of claim 15 , further comprising a receptacle located below the container to collect a first flowthrough.
18 . The device of claim 17 , wherein the heterocyclic compound is epichlorohydrin; and wherein the porous size exclusion support is formed by reacting an amount of 2-hydroxyethyl cellulose ranging from greater than 0 grams to 200 grams with an amount of epichlorohydrin ranging from greater than 0 milliliters to 400 milliliters.
19 . The device of claim 15 , wherein the porous size exclusion support has a MWCO of 40,000 Da; and wherein the one or more small molecules has a molecular weight less than or equal to 40,000 Da and the one or more large molecules has a molecular weight of 40,000 Da or greater.
20 . The device of claim 19 , wherein the one or more small molecules is a biomolecule; a salt; an unreacted label or partially reacted label or nanoparticle, or a derivative thereof; a dye or derivative thereof; a radioactive ligand or an intermediate thereof; a mass tag; a metal; biotin or a derivative thereof; a crosslinker or derivative thereof; a reducing agent or derivative thereof; or any combination thereof.
21 . A kit for separating one or more large molecules from one or more small molecules in a sample, the kit comprising the device of claim 18 , wherein the device is operably configured to operate by gravity flow, a centrifugal force, a positive pressure, a negative pressure, vacuum, or any combination thereof.
22 . The kit of claim 21 , wherein the kit further comprises a buffer.
23 . A method for separating one or more large molecules from one or more small molecules, the method comprising:
adding a sample comprising the one or more large molecules and the one or more small molecules to the matrix of claim 1 ; and
subjecting the matrix to a gravity flow, a centrifugal force, a positive pressure, a negative pressure, a vacuum, or any combination thereof, wherein the one or more large molecules is excluded by the matrix and is collected as a flow-through.