Nanobiosensors - Nanomaterials for Medical Applications - page 136

types of functionalized NMs 6,7,13,21,66,82,83 that are used for the detection of
cells, 12,39,70,74 proteins, 42 DNA, 6,7,13,82,84 and microorganisms. There are two
methods to conjugate biomolecules to NMs: (1) the one-step method and the (2)
two-step method. These methods are described below.
4.4.1.1 One-Step Conjugation
The following step-by-step protocol is recommended for the one-step conjuga-
tion protocol. 6,7,12,41,71 This one-step conjugation is recommended for QDs and
other NMs with carboxyl groups on the surface.
Conjugating a protein or DNA or other molecules to NMs must be opti-
mized 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 as well
as the stability of the NMs. Low molar ratios are recommended for expensive
biomolecules.
4.4.1.1.1 Chemicals
(1) 1-1.25 nmol of NMs with carboxyl groups (NM-COOH) on the surface in
100-125 µL deionized (DI) water.
(2) Freshly prepared solution containing 1 mg of N -ethyl- N '-dimethylaminopropyl-
carbodiimide (EDC) in 0.5 mL buffer A.
(3) Buffer A: 1.5 mL of 0.01 M H 3 BO 3 , pH 5.5.
(4) Buffer B: 0.1 mL of 1 M glycine or 1 M lysine.
(5) Buffer C: 3 mL 0.01 M of 0.01 M H 3 BO 3 , pH 7.2.
(6) 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 .
4.4.1.1.2 Procedure
(1) Pipet 1-1.25 nmol of the NM-COOH into a low protein-binding centri-
fuge tube.
(2) Add 300 µL of buffer A to the NMs and vortex to mix well.
(3) Add the appropriate amount of biomolecule 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.
(4) Add 50 µL of fresh EDC solution and vortex to mix well.
(5) React at room temperature (RT) for 1-2 h with constant gentle shaking.
(6) Add 10 µL of buffer B and react for another 15 min.
(7) Purify by dialysis, ultracentrifugation, or spin filtration to remove excess
reagents.
(8) Wash three times with buffer C.
(9) Reconstitute with buffer C to 4-µM NM or as desired and label as NM-
biomolecule.