FIELD OF THE INVENTION:
The present invention relates to a microfluidic chip/system directed to Traction Force
Microscopy (TFM) for mapping the cellular traction forces imparted on the adhering
substrate, so as to depict the biophysical state of the cells surviving in the micro-
confinement. Importantly, the method of measurement of bio-cellular adhesion forces or
change in stress in micro-scale precision is adapted to be integrated to a micro-fluidic
platform for evaluating different state of stress. The microfabrication compatible force
measurement device possibly in the form of a chip is capable of monitoring the spatio-
temporal evolution of cellular traction forces for static incubation periods with no media
replenishment as well as dynamic flow conditions that inherently induce cell deformation
and detachment. The present invention would find varied application in pharmaceutical
industries gainfully with regard to in vitro drug screening, where exact biophysical state
of the cell and hence the appropriate time for administering related drug can be
ascertained. The invention can also be applied to commercial scale for micro-scale
adhesion force measurement with required precision, especially within the purview of the
spatio-temporal scales that are experimentally encountered in typical microchannel-
based cell-culture systems.
BACKGROUND ART
The understanding of the dynamics of biological cells in micro-scale conduits, in response
to either chemically changing envirorment or shear stress imparted by the background
flow is of immense importance in designing and optimizing advanced lab-on-a-chip based
biomicrofluidic devices. In biological research, determination of the traction force
imparted by a cell on the adhering surface has been the most important parametric
markers of its biophysical states. Also it has been a well-established conclusion that
change in the strength of cell adhesion is a consequence of either of diseased conditions,
apoptosis, exposure to unfavorable environment, shear stress etc. For all such cellular
events, the activation of intracellular signaling pathways results in either over-expression
or degradation of the adhering molecules, thus affecting the cellular adhesion strength.
Hence, there has been a continuing need in the related art that necessity of a technique
which enables the revelation of local cdhesion forces with microscale precision, especially

within the purview of the spatio-temporal scales that are experimentally encountered in
typical microchannel-based cell-culture systems. One important technique for measuring
the cellular adhesion forces is a fluorescent bead-based Traction Force Microscopy (TFM)
that relies on measuring the resultant displacement of fluorescent beads embedded
inside the substrate or characterizing the force mediated wrinkling of the substrate onto
which the cell applies the traction force.
Two polymers namely Polyacrylamide (PAA) and Polydimethylsiloxane (PDMS) are used
for aforementioned techniques respectively in the existing art. Importantly, due to the
non-linearity and poor reproducibility of the wrinkling patterns, bead based method has
been used more extensively and effectively. In the conventional fluorescent bead based
TFM technique, fluorescent labeled beads are embedded into an easily deformable
substrate and displacement field of the bead-embedded substrate is calculated by
comparing fluorescent-images taken at identical microscopic location, in presence and
absence of cells, adhering to the substrate. Such images are compared by dividing both
of them in small viewports, each spanning over 10 pixel x10 pixel, and locating the
coordinates of the cross-correlation function maximum through a two-dimensional Fast
Fourier Transform (FFT) algorithm. A match can be accepted for a threshold value of 0.98
and subsequently, the displacement field can be evaluated by measuring the changes in
positions of the marker fluorescent beads. Next, from the experimentally obtained
displacement field, the traction field was determined by utilizing the unconstrained
Fourier Transformed Traction Cytometry (FTTC) method.
The polymer used for conventional bead based TFM technique i.e. Polyacrylamide (PAA)
is incompatible with microfabrication technology in the sense that the bonding between a
PAA substrate and PDMS microchannel is very weak. Hence, it has not been possible in
the prevailing art to integrate a TFM system into a microchannel network.
There has been a persistent need to cevelop a TFM technique to measure biophysical
state of cells under dynamic flow condition on lab-on-a-chip based biomicrofluidic devices
e.g. microfluidic conduits, wherein the use of appropriate fluorescent beads and a
substrate materials would provide adequate bond strength making possible integration of
TFM system into a microchannel based network. The present invention seeks to address
the related problem in the existing art using Ultrasoft-Polydimethylsiloxane based
Traction Force Microscopy (UPTFM) technique wherein PDMS base to Cross-linker ratio

has been increased from conventionally used 10:1 to a new 65:1 value, thereby
decreasing the Young's modulus. The invention is further providing means to address
problems relating to the spatial resolution of the TFM by selective use of bead size.
Additionally, the invention provides a basis for surface modification technology of the
soft-PDMS substrate, which imparts cytocompatibility to the TFM substrate. The invention
is thus directed to resolving problems of traction force measurement, especially in spatio-
temporal scales that are experimentally encountered in typical microchannel-based cell-
culture systems, and thus making tie invention capable of being successfully applied in
cell based microfluidic devices used in drug screening or other lab-on-a-chip based
appliances.
OBJECTS OF THE INVENTION
It is thus the basic object of the present invention to provide a microfluidic chip/system
adapted for Traction force Microscopy (TFM) and in particular, a microfabrication
compatible force measurement technique or Traction Force Microscopy that is capable of
measuring precisely the force of adherence of cell to adjacent substrate to quantify the
stability of the cell in biophysical terms.
Another object of the present invention is directed to developing a chip/system for TFM
wherein bonding of a substrate with the microchannel could be strengthened to thereby
facilitate integration of a TFM system into a microchannel network.
A still further object of the present invention directed to developing a chip/system
adapted for spatial resolution of the TFM for application in micro confinement by selective
fluorescent beads.
A still further object of the present invention directed to developing a chip/system for
TFM with possible surface modification technology introduced adapted to impart
cytocompatibility to the TFM subs rate.
A still further object of the present invention directed to developing a chip/system for
TFM wherein the biophysical stata of the cell can be quantified towards designing better
lab-on-a-chip devices with pharmaceutical and biomedical applications and as a result
point of drug administration can be effectively determined using this technology.

A still further object of the present invention directed to developing a chip/system for
TFM wherein exact biophysical state of cell can be monitored before the drug-screening
and by selecting the appropriate cellular state, false-positive results such as when actual
cellular response is not because of the drug administration but because of pre-existing
neighboring environmental conditions, can be significantly eliminated.
A still further object of the present invention directed to developing a system for TFM
wherein exact biophysical state can be quantified by spatio-temporal evolution of cellular
traction forces for static incubation pe-iods with no media replenishment as well as for
dynamic flow conditions that inherently induce cell deformation and detachment.
SUMMARY OF THE INVENTION
Thus according to the basic aspect of the present invention there is provided a
microfluidic chip/system adapted for mapping cellular traction force and/or measuring
cellular state in static or dynamic conditions comprising:
a microchannel provided with a selective UPTFM substrate;
said microchannel having an inlet and an outlet ports for passage of fluid there through;
smaller diameter of marker fluorescent beads in said microchannel adapted for enhanced
spatial resolution of TFM ;and
means adapted such as to capture phase contrast images of the cells in the channel and
also the fluorescent images of the beads.
According to another aspect of the invention there is provided a microfludic chip/system
adapted for mapping cellular traction force and/or measuring cellular state in static or
dynamic conditions comprising:
a microchannel provided with a selective UPTFM substrate;
said microchannel having an inlet and an outlet ports for passage of fluid there through;

smaller diameter of marker fluorescent beads in said microchannel adapted for enhanced
spatial resolution of TFM ;and
a microscope means adapted and operatively connected such as to capture phase
contrast images of the cells in the channel and also the fluorescent images of the beads.
Another aspect of the present invention is directed to a microfluidic system wherein said
UPTFM substrate is obtained of a selective Ultrasoft-PDMS, base to cross-linker ratio
65:1, substrate for TFM.
A further aspect of the present invention is directed to said microfluidic system wherein
said smaller diameter of marker fluorescent beads comprise fluorescent beads of
0.046±0.006 µm.
A still further aspect of the present invention is directed to a microfluidic system wherein
said selective Ultrasoft-PDMS comprises cytocompatibility inducing surface modified
Ultrasoft-PDMS having Aminopropyltrimethoxysilane (APTMS) and Poly-D-Lysine.
According to yet another aspect of the present invention directed to a process for the
manufacture of the microfluidic system comprising:
I) preparing the said ultra-soft polydimethylsiloxane (PDMS) substrate following (a)
mixing base to cross-linker in 65:1 ratio (w/w) , adding the amine modified
fluorescent labeled polystyrene beads to the PDMS mixture, followed by stirring;(b)
placing a small drop of the mixture an one of the edges of a glass slide and casting
a approximately 10 to 100 µm thick film of PDMS-bead mixture on glass coverslip;
(c) leveling the top surface ;(d)subsequently, the PDMS coated coverslips was
incubated at 40 to 60°C preferably about 50°C for 4 to 6 Hrs preferably about 4
hours for desired cross-linking; and (e)finally, treating the same with 0.05%
solution of Y-Aminopropyltrimethoxysilane in water for 15 to 30 minutes preferably
about 15 minutes at 22 to 40 °C preferably about 37°C and with 0.1 mg/ml Poly-D-

Lysine solution for 30 to 60 mimtes preferably about 30 minutes at 20 to 40°C
preferably about 37°C to obtain the substrate ;
II) providing said microchannel which is oxidized and press-bonded against the
substrate forTFM; and
III) providing the required fluidic connections .
A still further aspect of the present invention is directed to a process wherein the said
microchannel dimensions are 50 µm to 3 mm preferably about 2 mm (Width, W) x 50
urn to 1 mm preferably about 70 µm (Height, H) x 0.5 to 5 cm preferably about 1 cm
(Lengh, L).
According to yet another aspect of the present invention is directed to a method for
quantifying the stability of cells in micro confinement using the microfludic system
according to the present invention, comprising the step of involving ultrasoft-
polydimethylsolixane based traction force microscopy to identify the physiological state of
the cell surviving in a micro confinement.
A still further aspect of the present invention is directed to said method for quantifying
the stability of cells in micro confinement according to the present invention comprising
the step of mapping the cellular traction force comprising:
(i) providing the cells inside said microfluidic device;
(ii) incubating in a CO2 incubator ;
(iii) taking out of the incubator and setting under a fluorescence phase-contrast
inverted microscope for analysis;
(iv) replacing the cell culture medium of each microchannel by phosphate buffered
saline and subsequently adding cell culture grade trypsin-EDTA solution ;

(v) capturing the phase contrast images of the cell and the fluorescent images of
bead underneath at selective interval by a cooled monochrome charge
coupled device camera attached to the microscope.
A still further aspect of the present invention is directed to a method for quantifying the
stability of cells in micro confinement wherein the images were further stored and
analyzed by the combination of an mage processing software and Matlab image
processing features with the bead displacement field obtained from the analysis, the
fraction force field being determined usirg equation (5).
A still further aspect of the present invention is directed to a method for measuring the
cellular state in static condition using the microfludic system of the present invention
comprising:
i) injecting the cells into the microfluidic device and incubating for selective
time intervals;
ii) at the end of each time interval, phase contrast images of the cell and the
fluorescent images of bead distribution underneath were acquired at selected
positions of the device and the traction force field evaluated.
A still further aspect of the present invention is directed to a method for measuring the
cellular state in static condition using the microfluidic system of the present invention
wherein the active medium was replaced with a fresh one after every about 12 hours of
incubation whereby about 15 to 100 nl media was available per cell on an average.
According to yet another important aspect of the present invention is directed to a
method for measuring the cellular state in dynamic condition using the microfluidic
device according to the present invention comprising:
(i) injecting the cells inside the microchamels through the inlet port;
(ii) sealing the inlet and outlet ports of the microchannel and the set up was
kept inside a cell culture incubator;

(iii) taking out the set-up after about 4 to 24 Hrs preferably after about four
hours and was connecting :o a pump means preferably a syringe pump filled
with cell compatible solution preferably PBS (pH 7.4) solution;
(iv) subjecting the adherent cells to different flow rates and
(v) capturing the resulting phase contrast and fluorescence images.
A still further aspect of the present invention is directed to said method for measuring
the cellular state in dynamic condition wherein the dynamic deformation of the cells on
an imposed pressure driven flow were grabbed at selected intervals by phase contrast
microscope.
According to a further aspect of the present invention is directed to a chip device adapted
to quantify the biophysical state of a cell comprising:
a microchannel provided with a selective UPTFM substrate;
said microchannel having an inlet and an outlet ports for passage of fluid there through;
smaller diameter of marker fluorescent beads in said microchannel adapted for enhanced
spatial resolution of TFM ;and
means adapted to capture phase contrast images of the cells in the channel and also the
fluorescent images of the beads.
The present invention and its objects and advantages are described in greater details
with reference to the accompanying non I miting illustrative figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Figure 1: is the schematic illustration of the conventional fluorescent bead based TFM
technique.

Figure 2 : is the schematic illustration of an embodiment of experimental set-up of lab-
on-a-chip devices for UPTFM according to the present invention.
Figure 3: is the illustrative images of changes in MTF during static incubation period for
different values of average volume of media available per cell during seeding.
Figure 4: is the Traction Force evolution using UPTFM according to the present invention
during dynamic flow condition.
DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO THE
ACCOMPANYING FIGURES
Reference is first invited to the accompanying Figure 1 that schematically illustrates the
conventional fluorescent bead based TFM technique for determining the traction force
field.
One known conventional technique in the existing art for measuring the cellular adhesion
forces is a fluorescent bead-based Traction Force Microscopy (TFM) that relies on
measuring the resultant displacement of fluorescent beads embedded inside the
substrate or characterizing the force mediated wrinkling of the substrate onto which the
cell applies the traction force. To implerrent such force measurement technique, usually
two polymers namely Polyacrylamide(PAA) and Polydimethylsiloxane (PDMS) have been
used for aforementioned beads or substrate respectively. Importantly, due to the non-
linearity and poor reproducibility of the wrinkling patterns, bead based method has been
used more extensively and effectively in conventional TFM. The fundamental steps of
conventional TFM technique involve embedding at first, the fluorescent labeled beads into
an easily deformable substrate and displacement field of the bead-embedded substrate is
then calculated by comparing fluorescent-images taken at identical microscopic location,
in presence and absence of cells, adhering to the substrate. Usually, fluorescent beads
having 1 urn diameter are conventionally used for embedding in to PDMS substrate
wherein base to Cross-linker ratio used is about 60:1 for the stated measurement
purpose. Images are compared by dividing both them in small view ports, each spanning
over 10 pixel x 10 pixel, and locating the coordinates of the cross-correlation function
maximum through a two-dimensional Fast Fourier Transform (FFT) algorithm. A match

can be accepted for a threshold value of 0.98 and subsequently, the displacement field
can be evaluated by measuring the changes in positions of the marker fluorescent beads.
Next, from the experimentally obtained displacement field, the traction field was
determined by utilizing the unconstrained Fourier Transformed Traction Cytometry
(FTTC) method. In this method, the traction vector field which is effectively defined as
the localized force per unit area on the substrate, is evaluated from the experimentally
obtained displacement field. From Mathematical point of view, it is called inverse problem
of the Boussinesq's solution in semi-infinite medium, which fundamentally deduces a
solution of the displacement field due to the traction force applied onto a point. Assuming
that Green's function for mapping the traction to the displacement field is represented by
the tensor the displacement vector u can be related to the traction vector
by the following convolution:

, in this equation, is the position vector of the point where the displacement is being
evaluated in response to the point force.
Subsequently, in Fourier space, the relation is described by the transformation of
equation (1), given as follows

In this relation, the Fourier transform by wave-vector is presented by putting a tilde
over the relevant vectors. In pertinence :o equation (2), the expression for in real
space is given in terms x and y components of the displacement with zero normal
traction by the matrix

where σ and E are the Poisson's ratio and the Young's modulus of the substrate,
respectively. In the Fourier space, the transformed kernel is however obtained in terms
of x and y component of the wave-vector ( i.e., kx and ky) as


Owing to the diagonality of the transformed kernel the determination of traction field
is simplified as the following inverse relation
(5)
where FT2" is the two-dimensional inverse Fourier transformation operator.
Accompanying Figure 1 illustrates the images of the displacement field based on
deformity of bead embedded substrate end also the image determining the traction force
field, based on the abovestated unconstrained Fourier Transformed Traction Cytometry
(FTTC) method.
Reference is now invited to the accompanying Figure 2, that schematically illustrates an
embodiment of experimental set-up of lab-on-a-chip devices for UPTFM according to the
present invention. The present invention provide means for integrating the TFM
technique in micro-channel network to determine the biophysical state of cells, for
favorable application of the system and method for drug screening or other lab-on-a-chip
based biomicrofluidic devices to ascertair point of administration of drug.
It is important that the very polymer used for bead based TFM technique i.e. PAA being
incompatible with microfabrication technology and the bonding a PAA substrate with
PDMS microchannel being a weak poss bility. The present invention thus attempts to
integrate a TFM system into a microchannel network by selecting the size of fluorescent
beads and also the appropriate base to cross-linker ratio of the PDMS substrate. In the
preferred embodiment as illustrated in :he accompanying Figure 2, present invention
seeks to address this problem using Ultrasoft-Polydimethylsiloxane based (mixed in 65:1
base to cross-linker ratio) Traction Force Microscopy (UPTFM) technique.
Also the spatial resolution of the conventional TFM has been attempted to improve in two
ways for microfluidic compatibility- either for static incubation or under dynamic flow
conditions. Firstly, the PDMS base to Cross-linker ratio has been increased from earlier
used 60:1 to a new value of 65:1, thereby decreasing the Young's modulus. Secondly,
the diameter of fluorescent beads used in the present technique is only 0.046±0.006 µm
in comparison to the previously used 1 µm diameter fluorescent beads. (0.046±0.006
urn) which can be placed very close to each other (~ 8 in 1 µm2 area) allowing enhanced
spatial resolution of TFM.

Additionally, the development includes a novel soft-PDMS surface modification
technology using Aminopropyltrimethoxysilane (APTMS) and Poly-D-Lysine (PDL), which
makes ultrasoft PDMS surface complying with biological cell adhesion and growth.
Importantly, the UPTFM exact biophysical state of cell can be monitored before the drug-
treatment and by selecting the appropiate cellular state, false positive results can be
significantly eliminated.
Thus the salient inventive aspects of the present invention of the method of UPTFM may
be summarized as follows:
-Unique Ultrasoft-PDMS (base tc cross-linker ratio 65:1) substrate for TFM;
-Incorporation of this substrate into a PDMS microchannel system, which
enables end-users to measure biophysical state of the cell, adhering to
microchannel wall;
-Smaller diameter of marker fluorescent beads (0.046±0.006 µm) which can be
placed very close to each other (~ 8 in 1 µm2 area) allowing enhanced spatial
resolution of TFM;
-Cytocompatibility inducing surface modification of the Ultrasoft-PDMS utilizing
Aminopropyltrimethoxysilane (APTMS) and Poly-D-Lysine;
It is clearly apparent from the accompanying Figure 2, that illustrates the experimental
set up to carry out TFM technique on a lab-on-a-chip based device for integrating with
biomicrofluidic application of UPTFM according to the present invention so as to measure
the exact biophysical state of cells in microfluiclic confinement. The set-up shows a
microfabricated channel produced using ohotolithography having desired size/geometry.
Working fluid is flown at desired rate, being connected to a Syringe Pump filled with
phosphate buffered saline (PBS) (pH 7A) solution, within said microchannel over the
fluorescent bead embedded substrate surface in x-y plane, in particular modifying the
ultrasoft PDMS surface complying with biological cell adhesion and growth. UV light rays
through a blue exciting filter are made to illuminate any adhered cell on substrate from
bottom of channel through the objective and normal light is incident through lense on cell
from top such that the deformation/displacement is either viewed through eye piece or
fluorescent-images taken at identical rni:roscopic location, in presence and absence of
cells, adhering to the substrate in order to ascertain the displacement field. The traction
force field is then obtained from the disp acement field by using Fourier Transformation.

The working and the manner of implementation of the embodiment according to the
present invention is described in further details in the later paragraphs.
The biophysical state of the cell can be quantified towards designing better lab-on-a-chip
devices with pharmaceutical and biomedical applications. Point of drug administration can
be effectively determined using this technology. Moreover, a major concern in drug
screening technology due to the frequent occurrence of false-positive results where
actual cellular response is not because of the drug administration but because of pre-
existing neighboring environmental corditions, are duly addressed. Respectively, with
relevance to the aforementioned issues, exact biophysical state of cell can be monitored
before the drug-treatment and by selec:ing the appropriate cellular state, false positive
results can be significantly eliminated.
Thus the application of the UPTFM technique is novel in the application of same in the use
of traction force microscopy to quantify the stability of the cell in biophysical terms.
Biophysical stability of a cell is a major issue for the Cell culture based microfluidic
devices used in drug screening or other Iab-on-a-chip based appliances. Using the UPTFM
technique developed according to the present invention, biophysical state of a cell can be
determined with respect to the cell-substatum adhesion for following two cases:
-During the static incubation period i.e. no flow of nutritional medium, when the growth
and survival of cell is severely limited by toxic metabolites accumulation, exhaustion of
nutritionally important components and changes in pH and osmomolarity;
-During the dynamic flow condition when the cell is stressed by flow imparted shear
forces.
Consequently, the TFM technique has teen devised for mapping the cellular traction
forces imparted on the adhering substrate, so as to depict the physiological state of the
cells surviving in the micro-confinemert. The TFM is integrated with a microfluidic
platform for evaluating different states of stress in adherent mouse skin fibroblast L929
cells. Utilizing this technique, the spatio-temporal evolution of cellular traction forces are
monitored for static incubation periods with no media replenishment, as well as for
dynamic flow conditions that inherently induce cell deformation and detachment. This is
an important parameter to pharmaceutical industries with respect to in vitro drug

screening, where exact biophysical sta ;e of the cell and hence, the appropriate time of
drug administration, can be ascertained using UPTFM technology according to the present
invention.
TESTING PROCEDURE FOR UPTFM IN THE LAB-ON-A CHIP BASED DEVICE:
To carry out the Traction Force Microsopy for measurement of force of adherence of a
cell to the adjacent substrate integrated to a microfluidic device on laboratory scale to
ascertain the point of drug administntion avoiding false-positive results under the
conditions of either static incubation without media replenishment or dynamic flow

Inlet and outlet ports were punched by a blunt end 18-gauge needle. The fabricated
microchannel was oxidized and was gently press-bonded against the substrate for TFM.
Fluidic connections were made by insetting 18-gauge blunt end needles in the punched
holes and by press fitting silicone tub ng to the open end of needles. The fabricated
microchannel dimensions were 2 mm (Width, W) x 70 µm (Height, H) x 1 cm (Lengh, L).
b. Mapping Cellular Traction Force:
For experimental measurement of traction force of cell adherence with substrate, L929
Cells with a concentration of 104 cells/ml were injected inside the microfluidic device.
These cells were incubated in a CO2 incubator at 37°C and at 5% CO2. After 6 hours, the
devices were taken out of the incubator and were set under a fluorescence phase-
contrast inverted microscope for analysis. The cell culture medium of each microchannel
is replaced by phosphate buffered saline (PBS, pH 7.4). Subsequently, cell culture grade
trypsin-EDTA solution was added. Phase contrast images of the cell and the fluorescent
images of bead underneath were captured at 30 seconds interval by a cooled
monochrome charge coupled device camera attached to the microscope. Images were
further stored and analyzed by the combination of an image processing software and
Matlab image processing features. While bead displacement field is obtained from the
analysis, the fraction force field is determined using equation (5). The maximum
adhesion stress for the L929 cell population was measured to fall within the range of
260-300 pN/µm2.
c. Measuring Cellular state in Static and Dynamic Condition:
i) Static Incubation:
L929 Cells with different concentrations varying within 104-105 cells/ml are injected into
the microfluidic device and are incubated for 0.5, 1, 1.5, 2, 4, 6, 9, 12, 18, 24, 30, 36,
42 hours of time intervals. A few selective positions on microfluidic devices are marked at
the fabrication level for alignment. At the end of each time interval, phase contrast
images of the cell and the fluorescent images of bead distribution underneath are
acquired at those selected positions and :he traction force field is evaluated. While the

temporal changes in Maximum Traction Force (MTF) imparted by a cell is studied,
identical patterns of behavior have been found, irrespective of the cell density. MTF is
found to be increasing during the initial stages, thereafter remaining with a saturated
value over a period of time, and finally fall sharply after long incubation time owing to a
reduced cytocompatibility of the medium. Importantly also, the commencement of
declining phase is observed to be a function of the cell density. When the active medium
is replaced with a fresh one after every 12 hours of incubation while 30 nl media is made
available per cell on an average, cells are found to stay in the second phase for much
longer time duration.
The accompanying Figure 3 illustrate the above mentioned changes in MTF during static
incubation period for different values of average volume of media available per cell
during seeding.
ii) Dynamic Flow Condition;
L929 cells are injected inside the microchannels through the inlet port. Thereafter the
inlet and outlet ports of the microchannel are sealed and the set up is kept inside a cell
culture incubator. After about four hours the set up is taken out of the incubator and is
connected to a Syringe Pump filled with PBS (pH 7.4) solution. Adherent cells are
subjected to different flow rates and the resulting phase contrast and fluorescence
images are captured. Dynamic deformation of L929 cells on an imposed pressure driven
flow is grabbed at 65 ms interval by phase contrast microscope. In independent
experiments, traction forces at equilibrium phases of the cells under different background
flow conditions are mapped. Small increments in the traction has been noticed for all
shear rates less than critical value in all parts of a cell. In contrast, for shear rates
greater than , the traction is found to decrease significantly with time in the upstream
end of the cell whereas the same underwent a marginal increment in the neighbourhood
of the opposite end. Consequently, the change in shape and the peeling-off process is
first noticed to begin at the upstream end.

The accompanying Figure 4 illustrates the Traction Force evolution during dynamic flow
condition as detailed above.
It is thus possible by way of the present invention to develop a lab-on-a-chip based
microfluidic UPTFM device to measure Traction Force of cells adhering to the PDMS
substrate using ultra-soft polydimethylsiloxane (PDMS) substrate, mixing base to cross-
linker at 65:1 ratio and which is further treated with y-Aminopropyltrimethoxysilane
solution and Poly-D-Lysine solution tor favored surface modification for enhanced
adherence force of cell on surface, such that the exact biophysical state of cells are
determined in quantifiable terms in order to precisely ascertain the time and quantum of
drug administration in drug screening or the allied microfluidic pharmaceutical
application, avoiding any false-positive results under the conditions of either spatio-
temporal evolution of cellular traction foxes monitored for static incubation periods with
no media replenishment or, for dynanic flow conditions that inherently induce cell
deformation and detachment in microconfinement. The displacement field on a
fluorescent bead embedded substrate by Phase contrast images of the cell and the
fluorescent images of bead underneath are captured at 30 seconds interval by a cooled
monochrome charge coupled device inverted camera attached to the microscope, over
different incubation period. Images are then stored and analyzed by the combination of
an image processing software and Matlat image processing features and thus generating
the displacement field. The Corresponding Traction Force Field or temporal changes in
Maximum Traction Force (MTF), is determined by utilizing the unconstrained Fourier
Transformed Traction Cytometry (FTTC) method. The device and the method of UPTFM
according to the present invention is particularly applicable in pharmaceutical industries
gainfully with regard to in vitro drug screening, where exact biophysical state of the cell
and hence the appropriate time for administering related drug can be ascertained, thus
having wide scale application in pharmaceutical industry and R&D Laboratories.

WE CLAIM:
1. A microfludic chip/system adapted for mapping cellular traction force and/or
measuring cellular state in static or dynamic conditions comprising:
a microchannel provided with a selective UPTFM substrate;
said microchannel having an inlet and an outlet ports for passage of fluid there through;
smaller diameter of marker fluorescent beads in said microchannel adapted for enhanced
spatial resolution of TFM ;and
means adapted such as to capture phase contrast images of the cells in the channel and
also the fluorescent images of the beads
2. A microfludic chip/system adapted for mapping cellular traction force and/or
measuring cellular state in static or dynamic conditions comprising:
a microchannel provided with a selective UPTFM substrate;
said microchannel having an inlet and an outlet ports for passage of fluid there through;
smaller diameter of marker fluorescent beads in said microchannel adapted for enhanced
spatial resolution of TFM ;and
a microscope means adapted and operatively connected such as to capture phase
contrast images of the cells in the channel and also the fluorescent images of the beads.
3. A microfludic chip/system as claimed in anyone of claims 1 or 2 wherein said UPTFM
substrate is obtained of a selective Ultrasoft-PDMS ,base to cross-linker ratio 65:1,
substrate for TFM.
4. A microfludic chip/system as claimed n anyone of claims 1 to 3 wherein said smaller
diameter of marker fluorescent beads comprise fluorescent beads of 0.046±0.006 urn.
5. A microfludic chip/system as claimed in anyone of claims 1 to 4 wherein said
selective Ultrasoft-PDMS comprises cytocompatibility inducing surface modified
Ultrasoft-PDMS having Aminopropyltrimethoxysilane (APTMS) and Poly-D-Lysine.

6. A process for the manufacture of the microfludic chip/system as claimed in anyone of
claims 1 to 5 comprising:
I) preparing the said ultra-soft polydimethylsiloxane (PDMS) substrate following (a)
mixing base to cross-linker in 65:1 ratio (w/w) , adding the amine modified
fluorescent labeled polystyrene beads to the PDMS mixture, followed by stirring;(b)
placing a small drop of the mixture on one of the edges of a glass slide and casting
a approximately 10 to 100 µm thick film of PDMS-bead mixture on glass coverslip;
(c) leveling the top surface ;(d) subsequently, the PDMS coated coverslips was
incubated at 40 to 60°C preferably about 50°C for 4 to 6 Hrs preferably about 4
hours for desired cross-linking; and (e)finally, treating the same with 0.05%
solution of Y-Aminopropyltrimethoxysilane in water for 15 to 30 minutes preferably
about 15 minutes at 22 to 40 °C preferably about 37°C and with 0.1 mg/ml Poly-D-
Lysine solution for 30 to 60 minutes preferably about 30 minutes at 20 to 40°C
preferably about 37°C to obtain the substrate ;
II) providing said microchannel which is oxidized and press-bonded against the
substrate for TFM; and
III) providing the required fluidic connections .
7. A still further aspect of the present invention is directed to a process wherein the said
microchannel dimensions are 50 µm to 2 mm preferably about 2 mm (Width, W) x 50
urn to 1 mm preferably about 70 µm (Height, H) x 0.5 to 5 cm preferably about 1 cm
(Lengh, L).
8. A method for quantifying the stability of cells in micro confinement using the
microfludic chip/system as claimed in anyone of claims 1 to 5 comprising the step of
involving ultrasoft-polydimethylsolixane based traction force microscopy to identify the
physiological state of the cell surviving in a micro confinement.
9. A method as claimed in claim 8 compising the step of mapping the cellular traction
force comprising:
(i) providing the cells inside said microfluidic device;
(ii) incubating in a CO2 incubator

(iii) taking out of the incubator and setting under a fluorescence phase-contrast
inverted microscope for analysis;
(iv) replacing the cell culture medium of each microchannel by phosphate buffered
saline and subsequently adding cell culture grade trypsin-EDTA solution ;
(v) capturing the phase contrast images of the cell and the fluorescent images of
bead underneath at selective interval by a cooled monochrome charge
coupled device camera attached to the microscope.
10. A method as claimed in claim 8 wherein the images were further stored and analyzed
by the combination of an image processing software and Matlab image processing
features with the bead displacement field obtained from the analysis, the fraction force
field being determined using equation (5).
11. A method for measuring the cellular state in static condition using the microfludic
chip/system as claimed in anyone of claims 1 to 5 comprising:
i) injecting the cells into the microfluicic device and incubating for selective
time intervals;
ii) at the end of each time interval, phase contrast images of the cell and the
fluorescent images of bead distribution underneath were acquired at selected
positions of the device and the traction force field evaluated.
12. A method as claimed in claim 11 wherein the active medium was replaced with a
fresh one after every about 12 hours of ncubation whereby about 15 to 100 nl media
was available per cell on an average.
13. A method for measuring the cellular state in dynamic condition using the microfludic
chip/device as claimed in anyone of claims 1 to 5 comprising:
(i) injecting the cells inside the microchannels through the inlet port;
(ii) sealing the inlet and outlet ports of the microchannel and the set up was kept
inside a cell culture incubator;
(iii) taking out the set-up after about 4 to 24 Hrs preferably after about four hours
and was connecting to a pump means preferably a syringe pump filled with
cell compatible solution preferably PBS (pH 7.4) solution;
(iv) subjecting the adherent cells to different flow rates and
(v) capturing the resulting phase contrast and fluorescence images.

14. A method as claimed in claim 13 wherein the dynamic deformation of the cells on an
imposed pressure driven flow were grabbed at selected intervals by phase contrast
microscope.
15. A chip device adapted to quantify the biophysical state of a cell comprising:
a microchannel provided with a selective; UPTFM substrate;
said microchannel having an inlet and an outlet ports for passage of fluid there through;
smaller diameter of marker fluorescent heads in said microchannel adapted for enhanced
spatial resolution of TFM ;and
means adapted to capture phase contrast images of the cells in the channel and also the
fluorescent images of the beads.
16. A method of mapping cellular tracticn force and/or measuring cellular state in static
or dynamic conditions and a microfluid c device adapted for mapping cellular traction
force and/or measuring cellular state in static or dynamic conditions substantially as
herein described and illustrated with reference to the accompanying figures and
examples.

A microfludic chip/system adapted to measure Traction Force of cells adhering to the
substrate involving ultra-soft polydimothylsiloxane (PDMS) substrate, mixing base to
cross-linker at 65:1 ratio and further treating with γ-Aminopropyltrimethoxysilane
solution and Poly-D-Lysine solution for favored surface modification for enhanced
adherence force of cell on surface, such that the exact biophysical state of cells are
determined in quantifiable terms. The system/chip and the method of UPTFM according
to the present invention is particularly applicable in pharmaceutical industries gainfully
with regard to in vitro drug screening, where the appropriate time for administering
related drug can be ascertained, thus having wide scale application in pharmaceutical
industry and R&D Laboratories.