A microfluidic device comprising: an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, the fluidic circuit comprising a flow region and sequestration pens (124, 126, 128, 130, 224, 226 or 228), wherein at least one surface of the flow region is modified with a plurality of first covalently bound surface modifications, each of the first covalently bound surface modifications comprising: a first linking group, and a first moiety, wherein the first moiety is a first surface contact moiety covalently linked to the first linking group; wherein at least one surface of each of the sequestration pens (124, 126, 128, 130, 224, 226 or 228) is modified with a plurality of second covalently bound surface modifications, each of the second covalently bound surface modifications comprising: second linking group, and a second moiety, wherein the second moiety is a second surface contact moiety or a reactive moiety covalently linked to the second linking group, the reactive moiety configured to react with a surface modifying reagent; and wherein the first moiety is different from the second moiety. an enclosure comprising a base, a cover, and microfluidic circuit material defining a fluidic circuit therein, the fluidic circuit comprising a flow region and sequestration pens (124, 126, 128, 130, 224, 226 or 228), wherein at least one surface of the flow region is modified with a plurality of first covalently bound surface modifications, each of the first covalently bound surface modifications comprising: a first linking group, and a first moiety, wherein the first moiety is a first surface contact moiety covalently linked to the first linking group; wherein at least one surface of each of the sequestration pens (124, 126, 128, 130, 224, 226 or 228) is modified with a plurality of second covalently bound surface modifications, each of the second covalently bound surface modifications comprising: second linking group, and a second moiety, wherein the second moiety is a second surface contact moiety or a reactive moiety covalently linked to the second linking group, the reactive moiety configured to react with a surface modifying reagent; and wherein the first moiety is different from the second moiety. a first linking group, and a first moiety, wherein the first moiety is a first surface contact moiety covalently linked to the first linking group; wherein at least one surface of each of the sequestration pens (124, 126, 128, 130, 224, 226 or 228) is modified with a plurality of second covalently bound surface modifications, each of the second covalently bound surface modifications comprising: second linking group, and a second moiety, wherein the second moiety is a second surface contact moiety or a reactive moiety covalently linked to the second linking group, the reactive moiety configured to react with a surface modifying reagent; and wherein the first moiety is different from the second moiety. second linking group, and a second moiety, wherein the second moiety is a second surface contact moiety or a reactive moiety covalently linked to the second linking group, the reactive moiety configured to react with a surface modifying reagent; and wherein the first moiety is different from the second moiety.