Covalently modified surfaces, kits, and methods of preparation and use

1. A microfluidic device comprising:

an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein,

wherein the fluidic circuit comprises a flow region and sequestration pens,

wherein at least one surface of the flow region is modified with a plurality of first covalently bound surface modifications, each comprising

a first linking group, and

a first moiety, wherein the first moiety is a first surface contact moiety;

wherein at least one surface of the sequestration pens is modified with a plurality of second covalently bound surface modifications, each comprising

a second linking group, and

a second moiety, wherein the second moiety is a second surface contact moiety or a second reactive moiety, and

wherein the first linking group and the second linking group are different from each other and/or the first moiety is different from the second moiety.

2. The microfluidic device of claim 1 , wherein the first moiety and the second moiety are each covalently bound to the respective at least one surface via a linking group LG independently selected from —W—Si(OZ)2O— and —OP(O)2O—, wherein W is O, S, or N, and wherein Z is a bond to a silicon atom in an adjacent linking group LG or is a bond to the respective at least one surface.

3. The microfluidic device of claim 1 , wherein the first surface contact moiety comprises one or more of an alkyl, fluoroalkyl, monosaccharide, polysaccharide, alcohol, polyalcohol, alkylene ether, polyelectrolytes, amino, carboxylic acid, phosphonic acid, sulfonate anion, carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety, or cleavable moiety; and/or

wherein the second surface contact moiety comprises one or more of an alkyl, fluoroalkyl, monosaccharide, polysaccharide, alcohol, polyalcohol, alkylene ether, polyelectrolytes, amino, carboxylic acid, phosphonic acid, sulfonate anion, carboxybetaines, sulfobetaine, sulfamic acid, amino acid moiety, or cleavable moiety.

4. The microfluidic device of claim 1 , wherein the first surface contact moiety comprises a polyethylene glycol moiety, a dextran moiety, a proteinaceous moiety, a poly carboxylic acid, a polylysine moiety, or any combination thereof; and/or

wherein the second surface contact moiety comprises a polyethylene glycol moiety, a dextran moiety, a proteinaceous moiety, a poly carboxylic acid, a polylysine moiety, or any combination thereof.

5. The microfluidic device of claim 1 ,

wherein the second reactive moiety is an alkyne moiety, an azide moiety, a carboxylic acid moiety, an amine moiety, an olefinic moiety, a tetrazinyl moiety, a trans-cyclooctenyl moiety, a thiol moiety, a maleimide moiety, a biotin moiety, a streptavidin moiety, a halide moiety, a cyano moiety, isocyanate moiety, an epoxide moiety, a hydroxyamine moiety, or a sulfonyl fluoride moiety.

6. The microfluidic device of claim 1 , wherein each first covalently bound surface modification comprises a linker, wherein the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms; and/or

wherein each second covalently bound surface modification comprises a linker, wherein the linker comprises 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

7. The microfluidic device of claim 6 , wherein the linker of each first covalently bound surface modification further comprises one or two coupling group CG moieties; and/or

wherein the linker of each second covalently bound surface modification further comprises one or two coupling group CG moieties.

8. The microfluidic device of claim 1 , wherein the first covalently bound surface modifications have a structure selected from Formula XXXI, Formula VIII, and Formula IX:

wherein:

LG is —W—Si(OZ) 2 O— or —OP(O) 2 O—;

W is O, S, or N;

Z is a bond to an adjacent silicon atom or is a bond to the surface;

n is an integer of 3 to 21;

L sm is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms and further comprises 0, 1, 2, or 3 coupling groups CG; and

is the surface.

9. The microfluidic device of claim 8 , wherein LG is —W—Si(OZ) 2 O—, and wherein W is O.

10. The microfluidic device of claim 8 , wherein n is 7 to 21.

11. The microfluidic device of claim 8 , wherein the second covalently bound surface modifications have a structure selected from Formula XXX′, Formula V′, Formula VII′, Formula XXXI′, Formula VIII′, and Formula IX′:

wherein:

LG′ is —W′—Si(OZ′) 2 O— or —OP(O) 2 O—;

L′ fm is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms and further comprises 0 or 1 coupling groups CG;

R′ x is a reactive moiety;

W′ is O, S, or N;

Z′ is a bond to an adjacent silicon atom or is a bond to the surface;

n′ is an integer of 3 to 21;

L′ sm is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms and further comprises 0, 1, 2, or 3 coupling groups CG; and

is the surface.

12. The microfluidic device of claim 11 , wherein LG′ is —W′—Si(OZ′) 2 O—, and wherein W′ is O.

13. The microfluidic device of claim 11 , wherein n′ is 7 to 21.

14. The microfluidic device of claim 11 , wherein the reactive moiety R′ x is alkyne, azide, amine, carboxylic acid, biotin, or streptavidin.

15. The microfluidic device of claim 1 , wherein the first moiety is different from the second moiety.

16. The microfluidic device of claim 12 , wherein the first covalently bound surface modifications have a structure selected from Formula XXXI, Formula VIII, and Formula IX, wherein the second covalently bound surface modifications have a structure selected from Formula I′, Formula VIII′ and Formula IX′, and wherein the first covalently bound surface modifications are different from the second covalently bound surface modifications.

17. The microfluidic device of claim 16 , wherein a surface modifying ligand of the first covalently bound surface modifications comprises a structure of Formula X, and wherein a surface modifying ligand of the second covalently bound surface modifications comprises a structure of Formula XI:

wherein:

CG is a coupling group; and L is a linker comprising a bond or 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms.

18. The microfluidic device of claim 1 , wherein the sequestration pens comprise an isolation region and a connection region, wherein the connection region comprises a proximal opening to the flow region and fluidically connects the isolation region to the flow region.

19. The microfluidic device of claim 1 , wherein the second covalently bound surface modifications comprise a surface contact moiety configured to anchor adherent cells.

20. The microfluidic device of claim 1 , wherein the first covalently bound surface modifications comprise a surface contact moiety configured to inhibit migration of motile cells out of the sequestration pens.

21. The microfluidic device of claim 18 , wherein the fluidic circuit further comprises a plurality of sequestration pens each having at least one inner surface modified with the first and/or second covalently bound surface modifications.

22. The microfluidic device of claim 1 , wherein the first covalently bound surface modifications and/or the second covalently bound surface modifications form a monolayer.

23. The microfluidic device of claim 1 , wherein an inner surface of the base and/or an inner surface of the cover of the enclosure comprises glass, silicon, silicon oxide, hafnium oxide, indium tantalum oxide, or aluminum oxide.

24. The microfluidic device of claim 1 , wherein an inner surface of the microfluidic circuit material comprises polydimethylsiloxane (PDMS) or photopatternable silicone (PPS).

25. The microfluidic device of claim 1 , wherein substantially all of inner surfaces of the enclosure are covalently modified.

26. The microfluidic device of claim 1 , wherein the first covalently bound surface modifications have a structure of one of the following formulae:

wherein:

Z is a bond to an adjacent silicon atom or is a bond to the surface, and

is the surface.

27. A method of forming different covalently modified surfaces in a regioselective manner within a microfluidic device, wherein the microfluidic device comprises an enclosure having a base, a cover, and a microfluidic circuit material defining a microfluidic circuit therein, wherein the microfluidic circuit comprises a flow region and a sequestration pen, wherein the sequestration pen comprises an isolation region and a connection region, the connection region comprising a proximal opening to the flow region and fluidically connecting the isolation region to the flow region, and further wherein at least one surface of the flow region and at least one surface of the sequestration pen each comprises a plurality of covalently bound reactive moieties, the method comprising:

flowing a first modifying reagent through the flow region under conditions such that the first modifying reagent does not enter the isolation region of the sequestration pen; and

reacting the first modifying reagent with the covalently bound reactive moieties on at least one surface of the flow region, thereby forming a first modified surface within the flow region, wherein the first modified surface does not extend into the isolation region of the sequestration pen.

28. The method of claim 27 , wherein the first modifying reagent has a structure of one of the following formulae:

V—(CH 2 ) n -surface modifying ligand  Formula I;

V-L sc -surface modifying ligand  Formula XXXII;

wherein:

V is —P(O)(OH) 2 or —Si(T) 2 W;

T is independently OH, OC 1-6 alkyl, or halo;

R is C 1-6 alkyl;

n is an integer of 3 to 21; and

L sc is a linker comprising 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, and further comprises 0, 1, 2, or 3 coupling groups CG.

29. The microfluidic device of claim 1 , wherein the second covalently bound surface modifications have has a structure of one of the following formulae:

wherein:

W′ is O, S, or N;

Z′ is a bond to an adjacent silicon atom or is a bond to the surface;

n′ is an integer of 3 to 21;

Z is a bond to an adjacent silicon atom or is a bond to the surface; and

is the surface.