Biomedical Engineering Reference
In-Depth Information
(10) Run the original NM and the NM-biomolecule in 1-1.5% agarose gel at
100V for 30-40 min to verify conjugation efficiency.
(11) Store the purified NM-biomolecule conjugates at 4 °C until use.
4.4.1.2   Two-Step Conjugation
Some NM conjugations with biomolecules require the two-step process to
maintain colloidal stability in aqueous solution. The following step-by-step
protocol is recommended for the two-step conjugation protocol. 85 This step is
recommended for iron oxide magnetic NMs or other NMs with carboxyl groups
on the surface.
Conjugating a protein or DNA or other molecules to NMs must be optimized
for each type of biomolecule. An NM to biomolecule molar ratio of 1:1 to 1:50
is recommended depending upon the need of the studies. Low molar ratios are
recommended for expensive biomolecules.
4.4.1.2.1   Chemicals
(1) 1-1.25 nmol NMs with carboxyl groups (QD-COOH) on the surface in
100-125 µL DI water.
(2) 2 mg of EDC in 0.5 mL buffer A.
(3) 1 mg NHS (sulfo- N -hydroxysuccinimide).
(4) Buffer A: 1.5 mL of 0.01 M H 3 BO 3 , pH 5.5.
(5) Buffer B: 2 mL of 0.01 M H 3 BO 3 , pH 8.5.
(6) Buffer C: 0.1 mL of 1 M glycine or 1 M lysine.
(7) Buffer D: 3 mL 0.01 M of 0.01 M H 3 BO 3 , pH 7.2.
(8) Biomolecules (protein, DNA, etc.) in 0.01 M H 3 BO 3 (or PBS), pH 7.0-7.4.
If the biomolecule is in a different buffer and/or contains glycerol, these
must be removed through dialysis, ultracentrifugation, or spin filtration to
replace the buffer with H 3 BO 3 .
4.4.1.2.2   Procedure
(1) Pipet 1-1.25 nmol of the NM-COOH into a low protein-binding centri-
fuge tube.
(2) Mix the 2 mg EDC and 1 mg NHS and add 1 mL buffer D.
(3) Add 300 µL of buffer A to the NMs and vortex to mix well.
(4) Add 80 µL of fresh EDC/NHS solution and vortex to mix well.
(5) Incubate with shaking for 5-20 min.
(6) Add 500 µL of buffer B and mix well.
(7) Add the appropriate amount of biomolecule [that may be a protein such as
an antibody or bovine serum albumin (BSA)] from stock solution (a 10-mg/
mL stock solution is advised but dilute solutions are usable except that the
reagents will also be diluted so the reaction may take longer) and mix well.
(8) React at RT for 1-2 h with constant gentle shaking.
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