FIELD OF INVENTION;
The present invention relates to a magnetic nanoparticle separating
device developed for separation of specific cell populations from a
mixture of cells in liquid by labelling magnetic nanoparticles conjugated
to antibody specific to the desired cell population.
BACKGROUND OF THE INVENTION;
For many areas of biomedical sciences, specific pure cell populations are
needed for further studies. For example: knowing the percentage of CD4
populations in the whole blood sample for clinical diagnosis in a specific
infection to a pathogen and for various downstream application of cell
based assay etc. Likewise looking for a specific cell populations in a
tissue, cells etc. As the starting working material is the mixture of
different cell types, thus we need a suitable scheme to separate specific
purified cell populations from the mixture of cells. After obtaining the
desired cells, for knowing the purity of the separated cells tools like
fluorescence activated cell sorting (FACS) is used but it is expensive and
technically demanding. Whereas, the simple magnet based separation
provides an alternative inexpensive way to get purified desired specific
cell populations for downstream applications.
Several forms of high gradient magnetic cell separation systems are
described for cell separation in a liquid. Example- U.S. Patent
No.2010/0006509 Al describes a cell separation system generally in the
form of a column based pipette, syringe which means to draw and expel
liquid in the column chamber containing magnetic labeled spheres
[Annexure 1]. In this type of apparatus set up magnetic beads are
suspended into the liquid medium which later meant to retain by the
magnetic spheres. The desired cells then eluted in application to the
magnetic field, which may require a supporting stand for the external
magnetic device and/or an additional column in the separation

procedure. Furthermore, cells thus separated using columns subjected
to mechanical stress, which may have an altered phenotype, possibility
of cell activation or even cell death, which ruins the downstream
applications.
An alternative system is disclosed earlier in US Patent No.
2003/0099954 Al. This system utilized column based system for cell
separation. Most of the system available in market utilizes single tube
separation method as the method explained in US 0099954 Al while in
this described system plurality of the reaction tubes are available;
however it accommodates only one type of the supplied reaction
columns. Accordingly, there is in need for a column free separation
system which could selectively separate specific cell populations from a
complex mixture of cells which can be used without a support stand and
plurality of the assay using standard micro centrifuge (1.5, 2 ml) and 5
ml FACS reaction tubes simultaneously.
In this regard, a magnetic nanoparticle separator device is invented to
have a column free cell separating system utilizing magnetic
nanoparticles developed by us (US20130302508A1, EP2432460A1,
EP2432460A4, AU 2009346580 B2) for separation of specific pure cell
population from a mixture of cell type starting material. This ILS
magnetic nanoparticle cell separator device system utilizes antibody
conjugated super paramagnetic nanoparticles for cell separation.
However, those conventional magnetic separators can often hold reaction
tubes of same size and also does not provide substantial support while
handling the apparatus. Hence, there is always a need to develop a
further improved magnetic separator device useful in bio-medical and
biotechnological field.
The present invention meets the above mentioned need.

OBJECTS OF THE INVENTION;
It is therefore, the principal object of the present invention to provide a
magnetic separator device for separation of specific cell populations from
a mixture of cells in liquid by labelling magnetic nanoparticles
conjugated to antibody specific to the desired cell population.
Another object of the present invention is to provide a magnetic separator
device which provides specific position to hold different sized tubes of
various volumes, so that it facilitates to carry out four reactions
simultaneously.
Yet another object of the present invention to provide a magnetic
separator device which is not in direct contact with the content of the
reaction tube and hence can be used repetitively.
Further object of the present invention is to provide a magnetic separator
device which does not need any supporting stand and involves column
free separation.
Another object of the present invention to provide a magnetic separator
device which can be used for positive selection, negative selection and
depletion of cells as required for specific experiments.
SUMMARY OF THE INVENTION:
An improved magnetic separator device for separation specific cell
populations from mixture of cell comprises: a strong neodymium magnet
(1) at the core, and embodied by a chemical inert translucent plastic
material (5), four positions for holding four reaction tubes of different
sizes; a flat bottom surface (6) for standing said device standing without
supporting magnet has a coating of Ni surface and angular grooves (3),
which are positioned for real-time visualization of the magnetic
nanoparticles from the solution by adherence of the nanoparticles

towards the magnetic field area forming a pellet and the nanoparticle free
supernatant.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
FIG. 1 is the schematic 3-D image of the magnetic nanoparticle separator
device.
FIG. la is the schematic 3-D image of the magnetic nanoparticle
separator device with the reaction tubes.
FIG. 2 is a schematic of the lateral view of the sections for the grooves.
FIG. 3 is the top view of the outer groove for holding the reaction tubes.
FIG. 4 is the lateral view of the section showing the placement for the
magnet.
FIG. 5 is an ergonomic designed curved shape of the device for handgrip
to hold the magnet while separation of the magnetic nanoparticles.
FIG. 6 is the sectional view of the device to encompass the magnet at the
core of the device.
FIG. 7 is a 3-D view of the design for the magnetic nanoparticles
separator device.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
The subject matter disclosed herein relates to a magnetic nanoparticle
separator device useful for specific cell separation from a mixture of cells
in liquid by labelling magnetic nanoparticles conjugated to antibody
specific to the desired cell population. The cell populations thus
separated could be used for downstream applications in various
biological assays like immune cell separation, detection of circulation
tumor marker in the blood etc. Further, in order to accommodate
number of reactions at a time in the experiment, the magnetic device is
designed to carry forward four reactions simultaneously.

In one embodiment of the present subject matter, the device has
placement for four reaction tubes for multiplexing the assay. When the
reaction tubes are placed in the magnetic separator device, the magnetic
particles collected near the magnetic side of the tube. The magnetic
particles aggregate and held by the wall of the reaction tube towards the
magnetic field in the device [Figure 8 (3)]. Further, other variations of the
scheme could be employed to make to hold different tube size. The device
could also be useful for specific purification of infectious agents like
malaria in the blood circulation. A designed magnetic nanoparticle
separator device is invented for the application in many areas of
biomedical field.
According to an implementation of the present subject matter, a
magnetic separator is provided which can hold four tubes simultaneously
for separation. The magnetic separator has been designed in such a way
so that it can hold different sized tubes such as 1.5 ml and 2 ml micro
centrifuge tubes, 5 ml flow cytometric tube and other variations of the
scheme that could be employed to accommodate 15 ml and 50 ml of
falcon tubes and other similar kind of tubes of various volumes. The
reaction tubes are used in the experiment is a closed system, i.e micro
centrifuge tube; flow cytometric tube etc, where the reaction tubes can be
disposed after the experiment. The magnetic separator device is not in
direct contact with the content of the reaction tube used in the
experiment. The magnetic separator device is a system meant for re-use.
It should be noted that the description and figures merely illustrate the
principles of the present subject matter. It should be appreciated by
those skilled in the art that conception and specific embodiment
disclosed may be readily utilized as a basis for modifying or designing
other structures for carrying out the same purposes of the present
subject matter. It should also be appreciated by those skilled in the art

that by devising various arrangements that, although not explicitly
described or shown herein, embody the principles of the present subject
matter and are included within its spirit and scope. Furthermore, all
examples recited herein are principally intended expressly to be for
pedagogical purposes to aid the reader in understanding the principles of
the present subject matter and the concepts contributed by the
inventor(s) to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and conditions.
The novel features which are believed to be characteristic of the present
subject matter, both as to its organization and method of operation,
together with further objects and advantages will be better understood
from the following description when considered in connection with the
accompanying figures.
These and other advantages of the present subject matter would be
described in greater detail with reference to the following figures. It
should be noted that the description merely illustrates the principles of
the present subject matter. It will thus be appreciated that those skilled
in the art will be able to devise various arrangements that, although not
explicitly described herein, embody the principles of the present subject
matter and are included within its scope.
Fig 8, illustrates such different size tubes in the magnetic separator. The
tubes in said separator are in parallel position. The magnetic separator
device provides a rectangular shaped neodymium block magnet of N48
grade with coating of nickel on the surface and also embodied by the
chemically inert translucent plastic material such as Polymers of -
polypropylene, polyethylene, polystyrene, polyvinyl chloride, acrylic,
glossy, corrugated plastic etc. Further, the invention also provides a
scheme for other variations based on the reaction tube requirements.

The magnet has North and South polarity along the side for separation,
allowing uniform separation of the cells in all four tubes.
Further, this invention has an advantage in terms that there is no need
of a supporting stand for the device and the column free separation
system further allows to have an inexpensive, compact cell separation
system.
The device separates the desired cells separation from liquid from
antibody conjugated magnetic nanoparticles. The positions for holding
the tubes are placed in order to provide uniform strength for separation
of the cells from the magnetic nanoparticles during the process.
Fig 4 illustrates the particular placement of the magnet at the core of the
device.
Fig 6 also illustrates the view where the magnet is encompassed at the
core of the device.
Further, the said device is designed ergonomically or curved shaped for
better hand grip while decanting the liquid.
Furthermore, from Fig 2 and 3, it can be illustrated that grooves are
provided in the lateral side of the device, which helps to visualize the real
time separation of the magnetic nanoparticles in the solution in the
device.
The grooves are angular in shape and are positioned for real-time
visualization of the magnetic nanoparticles from the solution by
adherence of the nanoparticles towards the magnetic field area forming a
pellet and the nanoparticle free supernatant.
Fig 5 illustrates clearly this curved shape of the device for handgrip to
hold the magnet while separation of the magnetic nanoparticles.

Fig 1 illustrates the complete design of the proposed magnetic separator
device in accordance with an embodiment of the present subject matter.
In the said embodiment, a strong neodymium magnet is provided the
magnetic nanoparticle separator embodiment has the said magnet in
device which has overall strength of the magnetic field: the surface
magnetism is approximately 0.4 Tesla (4000 Gauss); and weighs
approximately 120 grams (approx. 4.25 oz.) and grade: N48 (NiCuNi).
Further, the provision for the four tube positions surrounding the
magnet at the (N/S) polarity is such that it increases the efficacy of cells
separation in each tube position (clearly shown in Figure 8). The
positions of the tubes are made to place at the magnetic side of the
polarity in N/S direction, so that all four tubes will have uniform
separation.
The cell separation using the novel magnetic nanoparticle separator
allows scheme for positive selection, negative selection and depletion of
cells as required for specific experiments.
Further, the current invention provides schemes for other variations of
the design based on the specific requirement of the reaction tube used in
the experiment.

A table has provided to prove the enhanced efficacy of the separator
according to the embodiment of the invention from conventional arts.


The non-limiting advantages of the present invention are as follows:
i) The specific curved ergonomic design of the magnetic separator is
intended for optimized performance.
ii) The designed device supports both self-standing on plain surface and
also it provides a curved ergonomic shape for handling to decant to
separate the magnetic nanoparticles from the cells in a liquid in the
device.
iii) The designed device intends to separate the specific cell populations
from using the ligand conjugated-GMO-MNPs (7) from one to four
experimental reaction tubes simultaneously.
The designed device is made to hold the experimental tubes and also
have the groove to visualize the tubes in real-time separation of the
specific cell population from the ligand conjugated GMO-MNPs (7) in
liquid medium
Although embodiments for the present subject matter have been
described in language specific to structural features, it is to be
understood that the present subject matter is not necessarily limited to
the specific features described. Rather, the specific features and methods
are disclosed as embodiments for the present subject matter. Numerous
modifications and adaptations of the system or device of the present
invention will be apparent to those skilled in the art, and thus it is
intended by the appended claims to cover all such modifications and
adaptations which fall within the scope of the present subject matter.

WE CLAIM;
1. An improved magnetic separator device for separation specific cell
populations from mixture of cell comprises:
a strong neodymium magnet (1) at the core, and embodied by a
chemical inert translucent plastic material (5), four positions for holding
four reaction tubes of different sizes;
angular grooves (3), which are positioned for real-time visualization
of the magnetic nanoparticles from the solution by adherence of the
nanoparticles towards the magnetic field area forming a pellet and the
nanoparticle free supernatant; and
a flat bottom surface (6) for standing said device standing without
supporting magnet has a coating of Ni surface.
2. The improved magnetic separator device as claimed in claim 1,
wherein the said magnet has North and South polarity along the side for
separation, allowing uniform separation of the cells in all four tubes.
3. The improved magnetic separator device as claimed in claim 1,
wherein said translucent plastic material comprises Polymers-
polypropylene, polyethylene, polystyrene, polyvinyl chloride, acrylic,
glossy, corrugated plastic etc.
4. The improved magnetic separator device as claimed in claim 1,
wherein said tubes are in parallel position.
5. The improved magnetic separator device as claimed in claim 1,
wherein the said magnet in device which has overall strength of the
magnetic field: the surface magnetism is approximately 0.4 Tesla (4000
Gauss); and weighs approximately 120 grams (approx. 4.25 oz.) and
grade: N48 (NiCuNi).

6. The improved magnetic separator device as claimed in claim 1,
wherein said tubes are made to place at the magnetic side of the polarity
in N/S direction, so that all four tubes shall have uniform separation.
7. The improved magnetic separator device as claimed in claim 1
provides an ergonomically curved shape area to handgrip the device and
also for handling to decant the magnetic nanoparticles from the cells in a
liquid.