Sample preparation compositions, devices, systems and methods
The present specification relates to compositions, devices, apparatus, methods, kits and systems for sample preparation (e.g., separation, reduction or removal of small molecules from biomolecules in a sample). Exemplary small molecules that can be separated, reduced or removed have a molecular weight range of <2000 Da. and may include, but are not limited to, dyes, biotin, affinity tags, crosslinkers, reducing agents, labels, nanoparticles, radioactive ligands, mass tags, unreacted molecules and combinations, intermediates and derivatives of the foregoing. Exemplary biomolecules present in a sample, include but are not limited to, proteins, glycoproteins, antibodies, peptides, nucleic acids, polysaccharides, carbohydrates and lipids. Methods, compositions, kits, devices, apparatus and systems of the disclosure may advantageously provide superior separation of small molecule contaminants and additionally reduce time and expenses related to separation of small molecules from larger biomolecules in samples. Biomolecules separated as set forth herein are amenable to better downstream processing.
1 . A composition for separating one or more small molecules from a sample, the composition comprising:
a first size exclusion support and a second size exclusion support, each comprising a material selected from polyacrylamide, a cellulose material, a hydroxyethyl cellulose, and/or derivatives thereof;
a first moiety that is immobilized onto the first size exclusion support; and
a second moiety that is immobilized onto the second size exclusion support;
wherein
(i) the second moiety is different from the first moiety,
(ii) the second size exclusion support comprises the same material as the first size exclusion support and is physically distinct from the first size exclusion support, and
(iii) the first moiety, the second moiety, or both the first moiety and the second moiety can associate with the one or more small molecules to separate the one or more small molecules from the sample.
2 . The composition of claim 1 , wherein the first moiety is an amine-containing polymer.
3 . The composition of claim 2 , wherein the amine-containing polymer is poly(ethylene glycol)diamine.
4 . The composition of claim 1 , wherein the second moiety is a polysaccharide.
5 . The composition of claim 4 , wherein the polysaccharide is dextran.
6 . The composition of claim 1 , wherein the one or more small molecules are associated by charge interaction, hydrophilic interactions, hydrophobic interactions, affinity interaction, hydrogen bonding, Van der Waals forces, or covalent bonding.
7 . The composition of claim 1 , wherein the one or more small molecules independently are selected from an unreacted or partially reacted label, or a derivative thereof; a dye or derivative thereof; biotin or a derivative thereof; a crosslinker or derivative thereof; an unreacted oligonucleotide; or any combination thereof.
8 . The composition of claim 1 , wherein the one or more small molecules have a molecular weight range of less than 2000 Da.
9 . The composition of claim 1 , wherein the one or more small molecules comprises a reactive functional group.
10 . The composition of claim 9 , wherein the reactive functional group is an activated ester.
11 . The composition of claim 10 , wherein the activated ester has a structure according to a formula —COΩ, wherein Ω is an oxysuccinimidyl, an oxysulfosuccinimidyl, or a substituted aryloxy group having one or more fluoro substituents or one or more chloro substituents.
12 . The composition of claim 9 , wherein the one or more small molecules is associated with the first moiety, the second moiety, or both.
13 . The composition of claim 1 , wherein the first moiety and the second moiety independently associate with different small molecules of the one or more small molecules.
14 . A method comprising adding, to a container, (i) a first size exclusion support having a first moiety immobilized on the first size exclusion support, and (ii) a second size exclusion support having a second moiety immobilized on the second size exclusion support; wherein
(a) the second moiety is different from the first moiety,
(b) each of the first and second size exclusion supports comprises a material selected from polyacrylamide, a cellulose material, a hydroxyethyl cellulose, and/or derivatives thereof;
(c) the second size exclusion support comprises the same material as the first size exclusion support and is physically distinct from the first size exclusion support, and
(d) the first moiety, the second moiety, or both the first moiety and the second moiety can associate with the one or more small molecules to separate the one or more small molecules from the sample.
15 . The method of claim 14 , wherein the first moiety is immobilized onto the first size exclusion support by:
providing the first size exclusion support having one or more vicinal diols;
oxidizing the one or more vicinal diols to form one or more aldehyde groups; and
reacting the one or more aldehyde groups with an amine group of the first moiety.
16 . The method of claim 14 , wherein the second moiety is immobilized onto the second size exclusion support by:
providing the second size exclusion support having one or more vicinal diols;
oxidizing the one or more vicinal diols to form one or more aldehyde groups;
reacting the one or more aldehyde groups with an amine-containing compound to generate one or more terminal amines;
introducing an oxidized second moiety having one or more aldehyde groups; and
reacting the one or more aldehyde groups with the one or more terminal amines.
17 . The method of claim 14 , wherein each of the first size exclusion support and the second size exclusion support comprises hydroxyethyl cellulose.
18 . The method of claim 14 , wherein the first moiety is poly(ethylene glycol)diamine.
19 . The method of claim 14 , wherein the second moiety is dextran.
20 . The method of claim 14 , wherein a ratio of the first size exclusion support to the second size exclusion ranges from 1:9 to 9:1.