Biomedical Engineering Reference
In-Depth Information
the preparation of this type of nanobiosensors. To illustrate the quantification of
immobilized capture probes on the surface of IOMNPs, an immunoassay for the
well-studied mouse immunoglobulin G (IgG) will be used.
4.6.1.1 Chemicals
(1)
1-1.25 nmol NMs with carboxyl groups (IOMNP-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.
(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)
Goat anti-mouse IgG (GAM) in 0.01 M H
3
BO
3
(or PBS), pH 7.0-7.4. If
the GAM 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
.
(9)
Donkey anti-goat IgG-horseradish peroxidase (DAG-HRP), 20 µg/mL in
0.01 M H
3
BO
3
(or PBS), pH 7.0.
(10)
Tetramethylbenzidene (TMB).
(11)
Blocking buffer.
4.6.1.2 Procedure for Quantifying the Number of Immobilized
Antibodies (Ab) on the Surface of NMs
A. Preparation of the antibody-modified NM
(1)
Pipet 1 mg of the IOMNP-COOH into a low protein-binding centrifuge
tube. If the IOMNP-COOH is dilute, concentrate (by magnetization or
ultracentrifugation) down to 5 mg/mL.
(2)
In a separate tube, mix the 2 mg EDC and 1 mg NHS and add 1 mL buffer D.
(3)
Add 300 µL of buffer A to the IOMNP-COOH 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 3.4 nmol of the goat anti-mouse IgG to create a 1 NM:100 Ab
ratio and mix well. (Lower or higher NM to Ab ratio may also be used
depending upon the stability of the resulting antibody-modified NM.
This has to be optimized on a case-to-case basis depending upon the
nature and the size of the NM.)
(8)
React at RT for 1-2 h with constant gentle shaking.
(9)
Add 10 µL of buffer C and react for another 15 min.
(10)
Purify by dialysis, ultracentrifugation, or spin filtration to remove excess
reagents.
