Preparation of latex beads (SG beads—proto beads)

To prepare latex beads, a mixture of 1.8 g GMA, 1.2 g St, 0.04 g DVB and 110 g
distilled water was put into a 200 ml three-necked round-bottom flask equipped with
a stirrer, a nitrogen gas inlet, and a condenser. Nitrogen gas was bubbled into the
mixture to purge oxygen. The system was kept at 70℃ in a water bath. 10 g distilled
water containing 0.06 g azobisamidinopropane dihydrochloride was added to the
mixture to initiate a soap-free emulsion polymerization. 0.3 g GMA was
supplemented to the reaction mixture 2 h after the initiation of the polymerization
and polymerization was continued for 22 h. the particles were collected by
centrifugation, and washed 3 times with water.
Preparation of matrix for coupling (SGNEGDEN beads)
1 g of SG beads was suspended in 100 ml of 3 M NH 4OH (pH 11), incubated at 70℃
for 24 h, and then washed with water. The products (SGN beads) got were incubated
with 100 mmol EGDE at 30℃ for 24 h at pH 11 (stored at 4℃ in the dark) (SGNEGDE
beads). And then incubated with 3 M NH 4OH (pH 11) at 70℃ for 24 h (SGNEGDEN
beads).
Pretreatment of haem (succination)
In order to be coupled to the beads, haem (0.3 mM) should be treated with equal
amount of N-hydroxysuccinimide in N,N-dimethylformamide at RT for 2 h.
Coupling
Succinated haem (0.3 mM) was incubated with 3 mg SGNEGDEN beads at RT
overnight, and then the haem-immobilized beads were masked with 4% acetic
anhydride in N,N-dimethylformamide containing 10% triethylamine. The immobilized
beads were stored in 50% methanol at 4℃.

GMA: glycidyl methacrylate

St: styrene

DVB: divinylbenzene

EGDE: ethylene glycol diglycidyl ether

N-hydroxysuccinimide

N,N-dimethylformamide