Biomedical Engineering Reference
In-Depth Information
with metastatic breast cancer [63]. Some new approaches that can be adapted for
CTC detection are described in the following section.
5.8
Aptamers as an Alternative to Antibodies
Although many of the above-described platforms and methods require the use of
antibodies for specifi c targeting, several challenges and limitations exist with
regards to the use of antibodies. First, the production of antibodies involves raising
them in animals (as opposed to chemical synthesis), and a newly produced batch
may behave quite differently from an older batch (e.g., they may not have the same
level of specifi city or binding characteristics). This is a concern particularly for
in
vitro
diagnostics, where the reproducibility of quantitative measurements is heavily
dependent on the consistent quality of reagents. While it is important to conjugate
antibodies to nanoparticles in a reproducible manner to consistently generate a
certain level of specifi city, the controlled conjugation of antibodies to nanoparticle
surfaces is not always possible. Most importantly, specifi c antibodies against many
of the protein analytes or cell-surface molecules of interest are simply unavailable.
Consequently, one very active area of research is the development of aptamers as
a promising antibody alternative for conjugation to nanoparticles when targeting
cancer cells.
Aptamers may be peptide, DNA, or RNA segments that fold into complex three-
dimensional structures with distinct molecular binding motifs. The aptamers in
all of the following studies were selected using a process known as cell-based
SELEX (systematic evolution of ligands by exponential enrichment), which identi-
fi es sequences with strong binding affi nities for live cancer cells that express a
variety of unique surface markers. The process starts out with a DNA library and
generally involves multiple binding and washing/elution steps (upwards of 25
rounds of selection) with increasing stringency of washes (e.g., longer wash times,
greater wash volumes, increasing numbers of wash cycles), where the unbound
DNA is washed away and the bound DNA is eluted. The eluate containing aptam-
ers with an affi nity for the target cells can be counterselected against control cells,
so as to increase the specifi city of the aptamers.
It has been shown that acute leukemia cells could be detected successfully
by using high- affi nity DNA aptamer-conjugated magnetic nanoparticles [64]. Here,
the aptamer was an 88-base oligonucleotide with binding specifi city for acute
leukemia cells, and the aptamer was attached to iron oxide nanoparticles (65 nm)
and fl uorescent nanoparticles to develop a platform for the collection and imaging,
respectively, of intact leukemia cells from whole blood samples. Smith
et al.
used this platform to demonstrate that multiple cancer cell types can be extracted
from a complex mixture (i.e., fetal bovine serum spiked with CCRF-CEM, Ramos,
and Toledo cancer cells) by a multiple cell extraction procedure using three
different sets of aptamers with specifi city for acute leukemia cells (CCRF-CEM),
Burkitt's lymphoma cells (Ramos), and non-Hodgkin B-cell lymphoma cells
