Field of the Invention:
The invention generall~r,e lates to a lab-on-a-chip (LOC) device. Particularly, the Invention
provides an Electrolyte Insulator Semiconductor (EIS) based microfluidic iinmunosensor
useful for the detection of antigens. In addition, this device also provides a generalized
platform for biofunctionalization of the microfluidic devices which consists of microchannels
used not only for immunosensor applications but for various other applications too.
Background of the invention:
Numerous clinical trials and intensive research efforts have indicated that continuous
metabolic monitoring holds great potential to provide an early indication of various body
disorders and diseases. In view of this, the development of biosensors for the measurement of
metabolites has become an area of significant scientific and technological study for various
research groups across the world.
In recent years, attention has been drawn to a small biochip used to efficiently analyze small
amounts of sample in a short period of time. Such a microchip is generally obtained by
fabricating a pattern and having a width of several tens to several hundreds of micrometers
and a depth of several tens of micrometers onto a substrate, such as a glass or silicon
substrate, using a photolithographic technique heretofore known as semiconductor
technology.
Within the context of research and diagnostics of any analyte at ~nolecular level, initial steps
have been taken to develop a lab on a chip device by combining the coinponents like bonding
of a glass/silicon substrate with the other required components for performing any analytical
test.
US201 1298455 discloses a magnetostrictive sensor with driving and detecting elements into
a microfluidic chip to detect a chemical, biochemical or biomedical species. The whole
system can be referred to lab-on-a-chip (LOC) or micro-total-analysis-systems. However, it
has been seen that magnetostrictive sensors do not work efficiently sometimes as the
generated magnetic fields are weak, which therefore leads into the unreliable detection of the
anal ye.
W096104547 discloses a chip based restriction enzyme treatment of DNA and the subsequent
separation of enzyme digests by capillary electrophoresis and the amplification of DNA
sequences by application of the polymerase chain reaction (PCR) with subsequent
electroplioretic separation.
While these standard laboratory processes have been demonstrated in a miniaturized format,
they have not been used to form a complete system. A complete system requires additional
manipulation such as front-end sample processing, binding and functional assays and the
detection of small signals followed by infonnation processing. The true challenge is that of
complete functional integration because it is here that system architecture and design
constraints on individual components will manifest themselves. For example, a fluidic
process is required to concatenate analytical steps that require the spatial separation, and
subsequent transport to new locations, of sets of analyte. Several possibilities have been
considered including electroosmotic pumping and transport of droplets by temperatureinduced
gradients in local surface tension. While feasible in demonstration experiments, these
techniques place rather severe requirements on the overall systerns lay-out to handle the very
considerable DC voltages required for efficient electroosmotic mixing or to restrict substrate
heating when generating thermally generated surface tension gradients so as to avoid adverse
effects on protein and other samples.
The integrated chemical sensor based on ion-selective field effect transistors is known in the
art (A. Sibbald, Recent advances in field-effect chemical microsensors, J. Mol. Electron. 2
(1986), 51 to 83). But, this process has certain problems like the lack of long term stability
and poor adhesion of the chemically sensitive layer.
The Conventional pH sensors include glass electrodes and chenlical indicators (e.g. liquids or
paper that changes color depending on the actual pH value), but because of size andlor
detection principle they can hardly be miniaturized and integrated into a silicon chip. Yet
miniaturization and integration would enable a substantial cost reduction paving the way for a
range of new applications. The currently existing microchips suffer with the problem of cross
contamination which generally occurs while the detection and analysis of samples. Further,
one of the prominent problems faced by these existing microchip is the unstable temperature
of the substrate due to which the reliability of the tests is not certain.
The Electrolyte-insulator-Se~niconductor (EIS) is a capacitive sensor and the changes in
surface potential between the electrolyte and the sensing insulator could be measured
.
according to the shift of capacitance-voltage (C-V) curves. Over the past years, for higher
pH-sensitivity and better stability, most of the metal oxide insulators as the ion sensitive
layers on EIS were proposed to detect H+ ion concentration. Currently, researches are going
on to miniaturize and integrate Electrolyte-Insulator-Semiconductor sensor in a chip which
could be used for the analysis of a test sample in order to achieve the fast response time and
possibility of a multi-analyte detection etc.
However, there is a need to provide a miniaturized and integrated Electrolyte-Insulator-
Semiconductor sensor based biochips which are efficient in analyzing multiple test samples.
Also, the biochip obviates the possibility of cross contamination of samples.
Objective of the Invention:
The primary object of the invention is to overcome the drawbacks mentioned in the prior art.
An objective of the present invention is to provide an Electrolyte Insulator Semiconductor
(EIS) based microfluidic Imlnunosensor for the detection of antigens.
Another objective of the present invention is to provide an EIS based microfluidic
Immunosensor which help in maintaining the constant temperature of the chips (i.e. substrate)
and the flowing liquids/analytes during the analysis of a test sample.
Another objective of the present invention is to provide an Electrolyte Insulator
Semiconductor (EIS) based microfluidic immunosensor with the provision to carry out
antibody immobilization and detection of antigens.
Another objective of the present invention is to provide an Electrolyte Insulator
Semiconductor (EIS) based microfluidic Immunosensor with the provision for
biofunctionalization, thereby obviating the need for the processing of the test samples
elsewhere.
Another objective of the present invention is to provide an Electrolyte Insulator
Semiconductor (EIS) based microfluidic immunosensor which helps in preventing the cross
contamination of the test samples by provision of replacement of the flexible tubes.
3
Another objective of the present invention is to provide a precise co~itrollingo f the analytes
by non contact mode vaive operation.
Another objective of the present invention provides a possibility of complete automation of
the device by operating the syringes. valves etc by precision stepper motors operated by
microcontrollers.
Another objective of the present invention is to provide a generalized platform for
biofunctionalization of the microfluidic devices which consists of microchannels used not
only for immunosensor applications but for various other applications too.
These and other advantages of the present invention will be more apparent from the foregoing
description in conjunction with the accompanying drawings.
Brief description of the accompanying drawings:
Other features as well as the advantages of the invention will be clear from the following
description.
In the appended drawing:
Figure 1: An integrated microfluidic lab on a chip device containing different parts to
perform the desired operation.
Figure 2: Pumping station (syringe holder assembly with stepper motor and gearbox
arrangement).
Figure 3: Syringe piston holding assembly (clamping system).
Figure 4: Combined drawing of Syringe holder assembly with clamping system.
Figure 5: Microchannel positioning system on fluidic ports.
Figure 6: Electronic circuit system to drive the motors to operate the microvalves.
Figure 7: EIS based microfluidic im~nunosensorp rototype.
Description of the invention:
Accordingly, the invention relates to the lab on chip devices. Specifically, the Invention
provides an Electrolyte Insulator Semiconductor (EIS) based microfluidic immunosensor
chip.
e As used herein. the terins "inicrofluidic", "inicrochannel." and "inicrofluidic channel" refers
to a structure or channel having at least one dimension that may most conveniently be
expressed in terms of micrometers. For example. the term "microfluidic channel" may refer to
a channel having at least one dimension of approximately 500 [mu]m or less. approxin~ately
100 [muln~ or less, approxinlately 50 [tnu]m or less, approximately 20-50 [mu]m,
approximately 10-20 [mulm, approximately 5-1 0 [inu]m, approximately 1-5 [mulm,
approximately 1 [mulm. or between 0.1 and 1 [mulm. One or ordinary skill in the art will
recognize that the dimensions of such channels may run into the millimeters, but that most
dimensions are in the micrometer range.
The integrated microfluidic EIS based immunosensor comprises following components:
a) a base sheet;
b) another sheet essentially vertical to said base sheet;
c) a pumping unit connected to said essentially vertical sheet for pumping desired fluid
to plurality of micro syringe(s) of desired volume, said micro syringe(s) being fixed at
its proximal end onto said vertical sheet and in communication with said pumping
unit through a clamping unit and at the distal end communicating with plurality of
replaceable inicrotube(s),
d) said micro tube(s) carrying said fluid from said micro syringe(s) to a pre-mixing
chamber to form a fluid mixture;
e) a microchip located on said base sheet whereby said microchip is in fluid
colnmunication with said pre-mixing chamber to receive said fluid mixture for
performing test(s) for the detection of said analyte; and
f) a sensor means located on said base sheet in cominunication with said microchip.
The sensor means is an electrolyte insulator semiconductor sensor or ISFET sensor or like.
The complete device has been shown in figure 1 while figure(s) 2 to 7 exhibits major
components of the device.
Particularly, the device described in Figure 1 has following components. The numbers
present in the brackets represents the respective components in the device.
Teflon or any other sheet of similar nature (1):
... 111.
iv.
v.
vi.
vii.
. . . v111.
ix.
X.
xi.
xii.
xiii.
xiv.
Micro valve slots (2):
Chip (3)
Input and output ports (4);
Microchannel (5)
Flexible tubings (6);
Side opening of the ~nicrovalve( 7);
Vertical Teflon assembly (8);
Stepper motor (9):
Premixing chamber (1 0);
EIS sensor (1 1);
Pumping station (1 2);
Syringe clamping system (I 3);
Digital display (1 4).
The pumping unit (as shown in Figure 2) comprises:
i. Ball bearing (I 5);
ii. Gear box (I 6);
iii. Syringe holding slot (I 7);
The clamping system (as shown in Figure 3) comprises:
i. Threaded shaft connector (I 8);
ii. Clamping system (1 9);
iii. Spring lock system in clamp (20);
iv. Syringe piston handle (2 1);
The syringe holder assembly is shown in Figure 4 while the microchannel positioning system
(as shown in Figure 5) comprises O-rings (22).
The Teflon sheet (I) is micromachined to create a desired pattern while the microvalve slots
(2), input and output ports (4) are arranged in the Teflon sheet as per the requirement. Later
the chip (3) containing desired number of microchannels (5) with different patterns was
placed on to the Teflon sheet by aligning the input and output ports and tightened with the
metal screws (not shown here) in to the base sheet. The flexible tubings (6) of desired size
6
C and dimensions were inserted through the side opening of the microvalve (7). Both the
tubings are aligned with the patterns created in the base sheet and is fixed on it.
Further, the desired volume of microsyringes was fixed on to pun~ping station (Figure 2) of
the vertical Teflon assembly (8) and connected to the microtubes. The microsyringe piston
rod was clamped (Figure 3) to the threaded shaft (I 8) having spring lock system (20) to hold
the syringe piston handle (21). The Clamping system (Figure 3) holding the syringes, further
connected to the lead screw of the stepper motor (9) by a spring mechanism placed on the top
of the Teflon vertical assembly fonning gear box (1 5) type arrangement.
As the lead screw rotates, any of the syringe piston assembly is then conveniently connected
to it by a gear arrangement and the piston rod is further vertically moved up and down as
desired during the operation.
According to the process protocol's requirement, any number of syringe(s), microvalve(s)
and tubing(s) can be arranged to the Teflon sheet by making equal number of patterns on it.
The chip maintains the process liquid temperature by placing the number of heating elements
within the patterns on the Teflon sheet and over which the tubings can be fixed in the
patterns. The chip also has a premixing chamber (10) in which the process liquids will be
heated continuously with metal electrodes/copper strips before they enter into the
microchannel/microchip.
The microfluidic chip substrate is made of siliconlglass (3). Other materials for the chip
include plasticlpolymers. The substrate is patterned for fabricating microchannels.
Specifically, a silicon chip can be patterned using well known anisotropic etching methods
and is further bonded to Pyrex glass which is further pre-functionalized till the crosslinker
stage and it was then kept on to the Teflon sheet by matching inlet and out ports (4).
The antibody immobilization within the microchannel will be carried out as per the
established protocols while rinsing and washing of the channels will be carried out by
operating the microsyringes and valves as per the requirement of the test.
The specific antibody is immobilized in the microchannels according to the requirement of
the sample to be tested. The binding of antigen having specificity to the immobilized
antibody causes the enzyme-substrate reaction which consequently allows the pH change of
C the solution. The pH change is further detected by allowing the liquid drop flowing out of
microchannel on to EIS sensor (I 1).
The reaction of the liquid drop with the EIS sensor gives results in terms of capacitance vs.
voltage. which in turn indicates the presence of the analyte in the test sample.
The microvalves are to be operated in ON OFF mode by special valve mechanism run by a
stepper motor which is fitted on to the Teflon vertical assembly. The microvalve in OFF
position presses the flexible tube inserted through horizontal slot provided in the valve while
in ON position it unpress the tube.
In another aspect of the Invention, there is provided a method of immobilization of different
antibodies on the chip and use of the bonded chip for the capturing and detection of antigens
in microchannels.
Specification of the device:
The mechanical accessories of the present invention can be movedldriven by tiny stepper
motors controlled by a simple electronic circuit. The specifications of the basic electronic
components used in the prototype instrument are presented here and the complete
automation of the device can be possible by using advanced microcontroller based
programming. In addition, the device can be made compact by using tiny electronic
components, valves and other accessories.
The stepper motor driver circuit used in this instrument is programmed in a
Microcontroller in such a way that, it generates pulse at a variable frequency as the
required speed. The programming has been written to provide a clock wise and anti clock
wise switching rotation to the motor by switching on its winding design with clock wise
and anti clock wise rotation.
Specifications of the basic electronic components used in prototype. This is not limiting.
IC 7805: Used to regulate 5 volt dc for niicrocontroller
(PIC 1 6F630)
Darlington Transistor (Tip 122) used to switch the
stepper motor winding to complete the circuit during
each pulse generated by ~nicroprocessor
PIC 16F630, 14-Pin, Flash-based 8-Bit CMOS
Microcontroller used as a variable pulse generator for
controlling motor movement (clockwise and
anticlockwise) and speed.
Electrolytic capacitor (1000 pFI25 volt) used to filter
DC voltage across circuit.
Electrolytic capacitor (1 pF16.3 volt) used to maintain
5 volt DC across Microcontroller.
Resistor 1 k-ohm used to reduce the base current at the
base terminal of the transistor.
Diode 4148 used to protect transistor from reverse
voltage.
Micro-switch used to provide command to
microntroller.
Ceramic capacitor used for maintaining voltage and
spike protection.
Trimmer: It is a variable resistance used to control the
speed of the motor or pulse frequency in
Microcontroller.
d. Advantages of the Invention:
The Electrolyte Insulator Selniconductor based microfluidic immunosensor chip helps
in maintaining the constant temperature of the reagentlsubstrate during the analysis of
the test sample, which is critical for reliability of results.
The chip possesses provision for biofunctionalization. which consequently helps in
obviating the need for processing the test sample elsewhere.
The chip also has the provision of replacement of the flexible tubes which helps in
avoiding the cross contamination of the samples during the reactions.
I) A microfluidic lab-on-a-chip immunosensor device for the detection of an analyte
present in a sample, said device comprising:
a) a base sheet;
b) another sheet essentially vertical to said base sheet;
c) plurality of micro syringe(s) being fixed at its proximal end onto a pumping unit
through a clamping unit and at the distal end communicating with plurality of
replaceable microtube(s), said pumping unit being in communication with said
vertical sheet;
d) said micro tube(s) carrying said fluid from said micro syringe(s) to a pre-mixing
chamber to form a fluid mixture;
e) plurality of microchip(s) located on said base sheet whereby said microchip is in fluid
communication with said pre-mixing chamber to receive said fluid mixture for
perfor~ningte st(s) for the detection of said analyte; and
f ) plurality of sensor mean(s) located on said base sheet in communication with said
microchip.
2) Device as claimed in claim 1, wherein said sensor means is selected from EIS sensor,
ISFET sensor or like.
3) Device as claimed in claim 1, further comprising plurality of essentially motorized
microvalves.
4) Device as claimed in claim 1 , wherein said pumping unit is controlled by a
microcontroller.
5) Device as claimed in claim 1, wherein said base sheet is made of a polymeric
material.
6) Device as claimed in claim 5, wherein said polymeric material is Teflon or the like.
7) Device as claimed in claim I , wherein said vertical sheet is made of a polymeric
material.
8) Device as claimed in claim 7, wherein said polymeric material is Teflon or the like.
9) Device as claimed in claim 1, wherein said clamping unit comprises threaded shaft
connector, a clamp means with spring lock system and a syringe piston handle.
10) Device as claimed in claim 1. \vlierein said pumping unit comprising gears assembly
for in co-operation with said syringe piston handles for facilitating up and dbwn
movement of the said piston handles.
11) Device as claimed in claim 1, \\,herein said pumping unit further comprising syringe
holding slots.
12) Device as claimed in claim I , wherein said microchip comprising plural ports for inlet
and outlet of said fluid mixture.
13) Device as claimed in claim 1, further comprising a heating element for maintaining
constant temperature of said reagents/microchip.
14) Device as claimed in clailn 1, further comprising a digital display in communication
with said sensor means.
15)Device as claimed in claim I, wherein said gear assembly comprising a stepper
motor(s) and ball bearing(s).
16)Device as claimed in any of the preceding claims wherein said base sheet is
micromachined to contain desired pattern.
17) Device as claimed in any of the preceding claims adapted to be used for performing
functions like biohnctionalization, biochemical test, analytical test for multiple test
samples.
18) Device as claimed in any of the preceding claims wherein said fluid is any reagent for
performing tests.