Biocompatibility and Functionalization - Nanomaterials for Medical Applications - page 101

telomerase activity, showing an absorbance at a wavelength equal to 610 nm.
Functionalized QD and other NMs contain either an amine or a carboxyl group
that offers the possibility of cross-linking or covalent bonding molecules con-
taining a thiol group, 188,189 carboxyl group, hydroxyl group, or an N -hydroxysuc-
cinimydyl ester moiety 125,126 by means of standard bioconjugation reactions. 108
Another approach for NM functionalization uses electrostatic interac-
tions between NMs and adapter biomolecules, or between NMs and proteins,
DNA, etc. 190 These functionalization steps can be repeated to add two or more
functional moieties or to change functionality. One example is streptavidin-
coated QDs (QDstrep) used in combination with biotinylated proteins or Abs
(biotinAb) 191 forming QDstrep-biotinAb. This structure, QDstrep-biotinAb,
depending upon the specificity of the Ab, can be used for (a) specific whole cell
or tissue staining, or (b) specific medical sensing.
3.3.2.1 Protocol for Covalent Conjugation of NPs to QDs
(available at Ocean NanoTech, LLC website,
www.oceananotech.com )
This biomolecule conjuagtion protocol can be used for water-soluble NMs that
contain carboxyl groups (-COOH) on the surface.
(1) Place 50 µL of 4 µM QD (2 nmol) aqueous solution in a low protein binding
1.5 mL centrifuge tube.
(2) Add 300 µL of QD reaction buffer (or 10 mM PBS, pH 7.4) to the QDs and
mix well.
(3) Add 50 µL of EDC solution (2 mg EDC/mL reaction buffer).
(4) Add at least 5 nmol of protein to make a 1 nmol QD to 5 nmol protein. This
ratio can be increased or decreased as necessary such as when the proteins
are too expensive.
(5) Mix well and react for 2 h in a vortex mixer or shaker.
(6) Add 10 µL of quenching buffer and allow it to react for 10-15 min at RT.
(7) Dialyze, spin filter, or use ultracentrifugation to purify.
(8) Resuspend in storage buffer or in 10 mM PBS buffer.
(9) Refrigerate until use.
To keep the activity of the proteins and to avoid contamination, only use auto-
claved buffers during the conjugation process. It is best to perform conjugation
in aseptic conditions as much as possible.
3.3.2.2 Specific Staining of Cancer Cells with QDs attached to Ab
against Cell Membrane Surface Proteins
This protocol can be applied to the specific attachment of NMs that are cova-
lently conjugated with Ab on the cell membrane.
(1) Insert a cover slip in each well of a 6-well culture plate.
(2) Place 5000 cells in each well and add 1 mL culture media.