Use of non-nucleophilic additives for reduction of surface morphological anomalies in probe arrays
The present invention relates to a formulations and methods for coupling a reactant (or probe precursor) to a functionalized surface for purposes of forming an arrayed sensor. This method includes the steps of: providing a surface having a reactive functional group; and introducing onto the surface, at a plurality of discrete locations, two or more compositions of the invention, which include a different reactant (probe precursor) and a non-nucleophilic additive, wherein such introduction is carried out under conditions effective to allow for covalent binding of the reactant to the surface via the reactive functional group. This results in a probe-functionalized array that substantially overcomes the problem of surface morphological anomalies on the array surface. Use of the resulting arrays in various detection systems is also encompassed.
1. A method of coupling a polypeptide reactant to a functionalized surface comprising:
providing a surface comprising a reactive functional group; and
introducing onto the surface, at a plurality of discrete locations, two or more compositions comprising a different polypeptide reactant and a non-nucleophilic additive wherein glycerol is not present in the two or more compositions, said introducing being effective to allow for covalent binding of the polypeptide reactant to the surface via the reactive functional group in the absence of glycerol,
wherein the non-nucleophilic additive comprises a structure according to formula (I) present in an amount of 0.001 to 3 percent v/v:
R 1 —O—[(CH 2 ) m O] n —R 2 (I)
where
n is an integer from 0 to about 250,
m is an integer from 1 to about 3, and
R 1 and R 2 are independently selected from the group of a C1 to C3 alkyl, or R 1 and R 2 together form a C1 to C3 alkyl, in which case the compound of formula (I) has a cyclic structure, or
wherein the non-nucleophilic additive is dimethylsulfoxide (DMSO) present in an amount of 0.01 to 1 percent v/v.
2. The method according to claim 1 , wherein the non-nucleophilic additive comprises a structure according to formula (I).
3. The method according to claim 2 , wherein the non-nucleophilic additive according to formula (I) has a molecular weight that is about 5000 Da or less.
4. The method according to claim 2 , wherein the non-nucleophilic additive comprises a cyclic structure, where R 1 and R 2 together form a C1 to C3 alkyl.
5. The method according to claim 4 , wherein the non-nucleophilic additive is a crown ether.
6. The method according to claim 2 , wherein the non-nucleophilic additive comprise a non-cyclic structure, where R 1 and R 2 are independently selected from the group of a C1 to C3 alkyl.
7. The method according to claim 2 , wherein the non-nucleophilic additive according to formula (I) is present in an amount of about 0.01 to about 1 percent v/v.
8. The method according to claim 1 , wherein the non-nucleophilic additive is dimethylsulfoxide (DMSO).
9. The method according to claim 1 , wherein the non-nucleophilic additive facilitates dispersion of the polypeptide reactant within the composition but does not participate in covalent bond formation.
10. The method according to claim 1 , wherein the reactive functional group is an aldehyde group, an activated ester, an isothiocyanate, an azide, an alkyne, a silyl halide, or combinations thereof.
11. The method according to claim 10 , wherein the polypeptide reactant comprises an amine, a carboxylic acid, a thiol, an aldehyde, or a primary alcohol.
12. The method according to claim 10 , wherein the polypeptide reactant comprises an amine group.
13. The method according to claim 1 , further comprising combining the additive into a solution comprising the polypeptide reactant, thereby forming the composition, immediately preceding said introducing.
14. The method according to claim 1 , further comprising washing the compositions from the plurality of discrete locations.
15. The method according to claim 1 , wherein the non-nucleophilic additive for each of the two or more compositions is the same.
16. The method according to claim 1 , wherein the non-nucleophilic additive for the two or more compositions is different.
17. The method according to claim 1 , wherein each of the discrete locations has a substantially homogeneous distribution of the polypeptide reactant across said discrete location.
18. The method according to claim 1 , wherein each of the discrete locations exhibits, upon imaging, a spot morphology that exhibits less than about 20 percent pixel variation across the discrete location.
19. A method of coupling a polypeptide reactant to a functionalized surface comprising:
providing a surface comprising a reactive functional group; and
introducing onto the surface, at a plurality of discrete locations, two or more aqueous compositions comprising a different polypeptide reactant and a non-nucleophilic additive wherein glycerol is not present in the two or more aqueous compositions, said introducing being effective to allow for covalent binding of the polypeptide reactant to the surface via the reactive functional group in the absence of glycerol, wherein the non-nucleophilic additive does not participate in covalent bond formation, and
wherein the non-nucleophilic additive comprises a structure according to formula (I) present in an amount of 0.001 to 3 percent v/v:
R 1 —O—[(CH 2 ) m O] n —R 2 (I)
where
n is an integer from 0 to about 250,
m is an integer from 1 to about 3, and
R 1 and R 2 are independently selected from the group of a C1 to C3 alkyl, or R 1 and R 2 together form a C1 to C3 alkyl, in which case the compound of formula (I) has a cyclic structure, or
wherein the non-nucleophilic additive is dimethylsulfoxide (DMSO) present in an amount of 0.01 to 1 percent v/v.
20. The method according to claim 19 , wherein each of the discrete locations has a substantially homogeneous distribution of the polypeptide reactant across said discrete location.
21. The method according to claim 19 , wherein each of the discrete locations exhibits, upon imaging, a spot morphology that exhibits less than about 20 percent pixel variation across the discrete location.