IS
FAST, LABEL-FREE DETECTION OF VIRUSES, TOXINS OR EVEN DNA FRAGMENTS
POSSIBLE IN NANOCHANNELS?
Fluids confined in nanometer-sized structures exhibit physical
behaviors not observed in larger structures, such as those of
micrometer dimensions
and above, because the characteristic physical scaling lengths of the
fluid very closely coincide with the dimensions of the
nanostructure itself.
For example, confinement of molecular transport in fluidic channels
with transport-limiting pore sizes of nanoscopic dimensions gives rise
to
unique molecular separation
capabilities. Such
nanofluidic structures
are used widely for separating fluids with disparate
characteristics. Development of bio-nanofluidic technology for
chip-based analysis systems gives possibility of
investigating DNA behavior at the single-molecule
level.
Various molecular separation techniques such as
nanochannel electrophoresis,
microchannel
capillary
electrophoresis and
gel
electrophoresis rely
on difference between velocities of movement of molecules due to
various sizes, charges or combination of those. After sufficient time
has passed, clearly
visible bands of
separated molecules are observed using various possible detection
schemes.
Think about two fishes moving down the stream. If they look identical
and weigh the same, how do we know if any of them have eaten
another small fish for dinner? And that is very important
question, believe me! The only way to answer it is to take
your stopwatch and wait for both fishes to swim down the stream far
enough so that the difference in velocities becomes apparent depending
on time sensitivity of your stopwatch.
In this analogy fish is
antibody,
and the dinner is
antigen in biochemical world.
If there is no
antigenpresent,
single band of
antibody will be moving down the
nanofluidic channel with the velocity
v1.
When
antigen is present, however, part
of the
antibody will form
antibody-antigen complex and will be
moving with a slower velocity
v2, while the rest of
antibody will
be left unbound and will be travelling with the same velocity
v1. As time passes,
this will result in two clear bands separated along the distance of the
separation platform.
If two of the molecules separated are
fluorescently labeled with different dyes they can be images by
fluorescent
microscopy. This is shown in the example below where
model receptor/toxin system has been separated
by Capillary Electrophoresis.
Green-labeled GM1 is
forming a complex with
red-labeled CTB and moves slower than
excess of unbound GM1. Clear
green band of GM1 is followed by
orange band of the complexed
receptor/toxinmixture confirming the presence of toxin in the
system.
This is a main principle of using
separation
assays for
detection purposes of various viruses, toxins, etc. The problem with
all these detection systems that they often must involve labeling
of analytes and binding agents with dyes, and sometimes it may take
long time to see clearly separated bands to be certain that the analyte
is present. Two different flow velocities as shown in example with
fishes are either obvious from visual analysis of images (observing
clear separate bands) or can be determined by manual calculations from
images as a function of time of separation, which is tedious,
time-consuming and quite subjective process dependent on the analyst
doing the calculations.
This is where patent
”Method for multivariate analysis of
confocal temporal image sequences for velocity estimation” comes
handy.
It
allows identifying whether there are two flow velocities present in
images acquired as a function of time of separation from the way
intensities of images themselves change with time.
- First very important benefit of this methodology that
this can be done at the very beginning of experiment when no clearly
visible separation is present with as few as first four
acquired images.
- And the second
benefit is
that no labeling of molecular species is necessary as presence of two
flow velocities can be determined from gray scale intensity of image at
either Green orRed or overall RGB image
converted to grayscale.
We have shown this in “
Detecting
molecular separation in nano-fluidic channels through velocity analysis
of temporal image sequences by multivariate curve resolution”
published in
MICROFLUIDICS
AND NANOFLUIDICS journal.