DESC:FIELD OF THE INVENTION:
The present invention relates to a device and method for the quantitative determination of the Total Serum Bilirubin (TSB) concentration in whole blood. Particularly, the present invention relates to a device which uses the principle of reflectance spectroscopy for determination of the total serum bilirubin concentration in a rapid, easy, portable and cost-effective manner from a very small amount of sample.

BACKGROUND OF THE INVENTION:

Bilirubin is a naturally occurring water-soluble, tetrapyrrolic yellow pigment that occurs in the normal catabolic pathway. Bilirubin is divided in two types, i.e., conjugated and unconjugated bilirubin. It is formed due to breakdown of heme from hemoglobin, myoglobin, peroxidases, and cytochromes in vertebrates. Normally, it is a waste product which is cleared by certain enzymes like glucuronosyl transferase in the liver. However, when the liver is improperly functioning, the bilirubin gets accumulated in the bodydue to improper breakdown of erythrocytes, liver failure, etc. This elevated Bilirubin can cause various diseases, and if not treated, can also lead to death. Elevated bilirubin may be due to genetic diseases, anaemia, liver issues, etc. The structure of bilirubin contains four pyrolle rings. It is of yellow – orange color in appearance. The chemical formula of Bilirubin is C 33 H 36 N 4 O 6. Bilirubin is also called as Neonatal Bilirubin, Direct Bilirubin, Conjugated Bilirubin, Indirect Bilirubin and Unconjugated Bilirubin. Normal values of direct bilirubin range from 0 to 0.4 mg/dL High bilirubin levels in a newborn means that the neonate is not processing, red cell breakdown effectively or an underlying cause is responsible High levels of Bilirubin can cause hepatitis, cirrhosis, gallstones. Symptoms of high bilirubin levels in newborns are skin and/or jaundice. High bilirubin levels in a newborn means that the neonate is not processing red cell breakdown effectively or an underlying cause is responsible.
Thus, there is a need to detect the bilirubin as early as possible.

Neonatal hyperbilirubinemia is one of the most frequent problems encountered in newborns. Highly elevated levels of bilirubin (degraded product of erythrocytes) can lead to kernicterus, jaundice, liver failure etc. and if delayed can also lead to death. The excessive amount of bilirubin within the body may be due to excessive hemolysis or liver failure.

There are various approaches available to measure bilirubin, such as Virtual assessment, which is a non-invasive method obeyingkramer’s rule, it describes the exponential relationship between the serum bilirubin level and the progression opacity of the skin discoloration. However, this method is not reliable, as it is operator-dependent and is affected by the newborn’s skin colour.

Yet another approach is the Diazo method, which is based on different solubility properties of conjugated and unconjugated bilirubin. As also disclosed in US4030885A, bilirubin is reacted with diazo reagent in the presence of caffeine, benzoate, and acetate as accelerators to form azobilirubin (coloured compound) which acts as the indicator. However,this method may underestimate a low level of bilirubin in a given sample, requires multiple chemicals, reagents, accelerators and it is time consuming.

To overcome the complex process disclosed above, one may consider High Pressure Liquid chromatography, which is a rapid and precise method to determine the total bilirubin. However, the drawback here is the high cost of operation. Non-invasive methods also involve the use of image capturing, electronic device or gadgets, etc.

Therefore, there is a needfor a device that overcomesat least the above mentioned problems. There is a need for a rapid, precise, non-invasive and cost-effective device and method for the measurement of total bilirubin concentration. Moreover, there is a need for quantitatively measuring bilirubin concentration using whole blood.

OBJECTS OF THE PRESENT INVENTION:
The principal object of the present invention is to provide a device and method to measure the Total Serum Bilirubin (TSB) concentration in whole blood

Another object of the present invention is to provide a device which measures the Total Serum Bilirubin (TSB) concentration in whole blood within 2-3 minutes.

Yet another object of the present invention is to provide a device which measures the Total Serum Bilirubin (TSB) concentration in whole blood using very low quantity of sample

Yet another object of the present invention is to provide a device which measures the Total Serum Bilirubin (TSB) concentration in whole blood using normal light instead of laser light.

Yet another object of the present invention is to provide a device which measures the Total Serum Bilirubin (TSB) concentration in whole blood using minimum components.

Yet another object of the present invention is to provide a device wherein the Total Serum Bilirubin (TSB) concentration in whole blood is measured accurately despite any hemolysis occurrence.

Yet another object of the present invention is to provide a device wherein the Total Serum Bilirubin (TSB) concentration in whole blood is measured without the use of any gasses like krypton for calibration beam.

Yet another object of the present invention is to provide a portable device wherein the Total Serum Bilirubin (TSB) concentration in whole blood is measured.

SUMMARY OF THE PRESENT INVENTION:
An aspect of the present invention is to provide a device to determine the Total Serum Bilirubin (TSB) concentration comprising:
a. at least a lateral flow strip having:
i. at least a nitrocellulose membrane;
ii. at least a cell separator; and
iii. at least anabsorption pad;
Another aspect of the present invention is to provide a device to determine the Total Serum Bilirubin (TSB) concentration comprising:
a. at least a lateral flow strip;
b. at least a color sensor; and
c. at least two LEDs.

Yet another aspect of the present invention is to provide a device to determine the Total Serum Bilirubin (TSB) concentration comprising:
a. at least a lateral flow strip having:
i. at least a nitrocellulose membrane;
ii. at least a cell separator; and
iii. at least aabsorption pad;
b. at least a color sensor; and
c. at least two LEDs.
d. a lateral flow strip holder having adjustable dual housing positions for the lateral flow strip;
e. A display screen; and
f. Onboard interface(s), all connected to a portable user interface by means of a wireless communication

A further aspect of the present invention is to provide a method of determining the Total Serum Bilirubin (TSB) concentration in whole blood.

BRIEF DISCRIPTION OF DRAWINGS:
Figure 1 Shows an exploded view of the cassette sandwiching the lateral flow strip according to an embodiment of the present invention.
Figure 2 Shows an expanded view of the lateral flow strip according to an embodiment of the present invention.
Figure 3 Shows an external view of the device according to an embodiment of the present invention.
Figure 4 shows the graph to the experimental results found after conducting the tests.

DEFINITIONS:
The definitions listed below are described toprovide clarity, by no means are the definitions of these terms to be considered to limit the scope of this disclosure.
Fluid: Means any material that cannot sustain a tangential, or shearing, force when at rest and that undergoes a continuous change. It may include whole blood, blood, serum etc.
Serum: The clear liquid part of the blood that remains after blood cells and clotting proteins have been removed/separated.
Bilirubin: a yellowish pigment that is made during the normal breakdown of red blood cells. Higher than normal levels of bilirubin may indicate different types of liver or bile duct problems
Whole Blood: Blood in its unaltered form as collected from a donor. Typically, Whole blood is made up of red blood cells, white blood cells, platelets, and blood plasma amongst other ingredients.
Nitrocellulose membrane: A type of matrix employed to capture fluid for testing
Cell separator: a filter which allows the serum component of blood to flow past but holds back the blood cells.
Grey Pad: A reference pad used for calibrating and for noise cancellation
Absorbance pad: A matrix which collects the sample before dissipating/releasing it

DETAILED DESCRIPTION OF THE INVENTION:

Various embodiments of the present invention providea device to determine the Total Serum Bilirubin (TSB) concentration.

An embodiment of the present invention is to provide a device to determine the Total Serum Bilirubin (TSB) concentration comprising:
a. at least a lateral flow strip (2) having:
i. at least a nitrocellulose membrane (8);
ii. at least a cell separator (9); and
iii. at least anabsorption pad (10);
wherein, the lateral flow strip maintains a continuous fluid flow by in-built pressure points which generate a capillary action for fluid mobility.
The lateral flow strip has a flat configuration and the nitrocellulose membrane is connected to the absorption pad by the cell separator such that the three components are positioned adjacent to each other void of any gap or overlap of components.
In an embodiment of the present invention, the lateral flow strip containsa cell separator placed between theabsorption pad (sample port) and a nitrocellulose membrane (whitepad) to measure the total serum bilirubin concentration, additionally a grey pad (reference pad) is provided for noise cancellation, calibration and to avoid faulty readings. As the sample whole blood is collected into the absorbing pad, the blood spreads through the absorbing pad, onto the cell separator which traps the blood cells permitting only the blood serum to flow onto the nitrocellulose membrane where it is exposed to at least two LEDs of specific wavelength. The continuous flow from the absorbing pad to the nitrocellulose membrane is ensured by in-built pressure points which generate a capillary action for fluid mobility. This technique of generating capillary action in a horizontal geometry and using a cell separator in the process enables the invention to conduct measurements using whole blood while overcoming the requirement of sample treatment using chemicals and complex processes.

In an embodiment of the present invention, the lateral flow strip has a flat configuration and the nitrocellulose membrane is connected to the absorption pad by the cell separator such that the three components are positioned adjacent to each other void of any gap or overlap of elements, ensuring mobility of fluid from the absorption pad to the nitrocellulose membrane.

An embodiment of the present invention is to provide a device to determine the Total Serum Bilirubin (TSB) concentration in whole blood comprising:
a. at least a lateral flow strip having:
i. at least a nitrocellulose membrane;
ii. at least a cell separator; and
iii. at least anabsorption pad;
b. at least a color sensor; and
c. at least two LEDs.
Wherein the at least two LEDs have a wavelength ranging from 400-500nm and 550-650nm respectively, and the color sensor has a photodiode which is optically aligned with the transmitter, and grey pad (7). The intensity of the light source may be adjusted based on the absorbance capacity of the fluid within the lateral flow strip.

A further embodiment of the present invention the device comprises of a color sensor for quantitatively measuring bilirubin concentration in the serum sample using the principle of reflectance spectroscopy. Once the LED wavelengths are emitted onto the grey pad / the white pad. The spectral composition of surface-reflected radiation with respect to its angularly dependent intensity and the composition of the incident primary radiation, is investigated to measure the color intensity of the sample. For bilirubin measurement, darker the “yellow” more is the bilirubin concentration in the sample. This intensity of yellow is measured to calculate the Total Serum Bilirubin concentration.

Accordingly, the embodiment discloses a device to determine the Total Serum Bilirubin (TSB) concentration comprising:
a. at least a lateral flow strip including:
i. at least a nitrocellulose membrane;
ii. at least a cell separator;
iii. at least an absorption pad; and
iv. at least a grey pad
b. at least a color sensor including:
i. Transmitter;
ii. Receiver;
iii. Photodiode; and
iv. A pre-programed, self-calibrating algorithm;
c. at least two LEDs;
d. a lateral flow strip holder having adjustable dual housing positions for the lateral flow strip;
e. A display screen; and
f. Onboard interface(s), all connected to a portable user interface by means of a wireless communication

Wherein the photodiode of the color sensor, senses light having a wavelength ranging from 400-500nm for measuring Bilirubin levels at isobestic point and a wavelength ranging from 550-650nm for Hemolysis correction.

In an embodiment of the present invention, the portable user interface connected to the onboard surfaces may be selected from but not limited to a mobile phone, a tablet, a computer, inter alia and the wireless communication may be selected from but not limited to Bluetooth, WiFi connection, internet connection, or such other wireless communication.

In an embodiment of the present invention, two LEDs preferably have wavelengths each emitted alternatively for a defined period of time i.e. ~10ms such that the first wavelength measures the absorbance of the fluid component while the second wavelength measures the presence of any hemolysis. Preferably, the two LEDs are emitted onto the grey pad one after the other, for calibration followed by emission on the nitrocellulose membrane (containing serum) for a reading. Using the specific range of LED in the wavelength of 400-650 nm enables the invention to overcome the need for laser or such other sources.

In a further embodiment of the present invention, the lateral flow strip is cased inside a cassette made of materials selected from but not limited to plastic, or another such industrially acceptable material and measures a maximum of 93mm x 141mm, the cuvette measures measuring 2.1mm and the housing cells measure 16mm x 11mm / 30mm x 9mm;

In an embodiment of the present invention, the lateral flow strip contains a nitrocellulose membrane, a grey pad for reference reading and a white pad to measure the total serum bilirubin level. The device of the present invention is capable of measuring component levels in sample fluid being whole blood and/or serum by accommodating an appropriate wavelength of LED according to the selected sample to be measured

A further embodiment of the present invention is to provide a method of determining the Total Serum Bilirubin (TSB) concentration in whole blood having the following steps:
i. The cassette housing the lateral flow strip is positioned to P1 aligning the grey pad in the optical path of the LEDs, the at least two LEDs are emitted on the reference pad (grey pad) void of any bilirubin/serum, which absorbs and transmits the emissions onto the color sensor based on which the device is calibrated;
ii. The cassette housing the lateral flow strip is shifted to P2 position, aligning the nitrocellulose membrane in the optical path of the LEDs, the at least two LEDs are emitted on the nitrocellulose membrane containing serum sample to receive at least two LED emissions;
iii. The at least two LED emissions are absorbed and transmitted from the nitrocellulose membrane to the color sensor, based on which the sample serum is quantified for Total Serum Bilirubin concentration;

A further embodiment of the present invention, discloses a method to quantitatively measure the total serum bilirubin concentration in whole blood by employing the device, the method comprising following steps:
i. Device 500 is turned on and lateral flow strip 2 is placed on holder’s 1st position (P1) to initiate calibration;
ii. At least two LEDs emitted from transmitter onto grey pad (7);
iii. Light absorbance of the two wavelengths measured by photodiode according to a preprogrammed algorithm, to complete calibration;
iv. Once calibrated, lateral flow stripshifted to holder’s 2nd position (P2) where the white pad (nitrocellulose membrane) (8) containing fluid sample is exposed to two LED wavelengths alternatively for a period of ~10ms each;
v. Light absorbance by the fluid sample is measured by the photodiode according to a preprogrammed algorithm;
vi. The fluid concentration is calculated by the algorithm based on the output of the photodiodes and the same is communicated to user by means of a user interface

Wherein the absorbance readings of the wavelength are detected by the photodiode and considered by the algorithm in calculating the true absorbance of the fluid after calibrating the hemolysis count amongst other deviation/scattering components, to accurately quantify the total serum bilirubin concentration in whole blood.

In an embodiment of the present invention, the device uses normal light source of the spectral range selected from but not limited to 400-650 to analyze the sample as well as for calibration.

An embodiment of the present invention discloses a device (500) which comprises a lateral flow strip (2) requiring very low quantity of sample.

The present invention works on the principle of reflectance spectroscopy to measure the total serum bilirubin present in whole blood. The present invention provides connectivity to the device by wireless means including Bluetooth, Wi-Fi, etc.

In a further embodiment of the present invention, the device comprises of a temperature sensor.

UTILITY
The present invention has at least the following advantages:
• Use of lateral flow strip instead of cuvette to enable using low sample quantity
• Use of normal light instead of lasers
• Use of low components
• Independent of diffusers, prisms, COOx analyzer systems, K-OPLS mapping function, etc.
• Enables using whole blood without faulty readings caused by variables such as hemolysis, turbidity etc.
• No requirement of any gases like krypton for calibration beam
• Measurement / testing lime required is as low as 2-3 minutes
• Present invention uses wireless connectivity such as Bluetooth or WiFi instead of optical transmission
• Present invention enables determination of the total serum bilirubin concentration in a rapid, easy, portable and cost-effective manner from a very small amount of sample.

The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to a person skilled in the art, the invention should be construed to include everything within the scope of the disclosure.

DETAILED DESCRIPTION OF DRAWINGS
Figure 1 of the present invention shows an exploded view of the cassette 100, wherein the cassette 100 comprises of a top portion 1, a lateral flow strip 2 and a bottom portion 3. The top portion 1 comprises openings to enable sample collection, absorption and transmission of LEDs.

Figure 2 of the present invention shows an isometric view of the lateral flow strip 102, comprising absorption pad 10, cell separator 09, nitro cellulose membrane 08, grey pad 07.
Figure 3 Shows an external view of the device 500 according to an embodiment of the present invention, comprising a display screen (105), a tray (104), which displays dual housing positions (P1) & (P2), for the cassette (100).

Figure 4 Shows a graph illustrating the performance evaluation of the present invention according to an embodiment of the present invention.

EXAMPLES
EXAMPLE 1
The device (500) of the present invention was switched on, the cassette (100) housing the lateral flow strip (2) was positioned to (P1) position the grey pad in the optical path for reading.
Calibration was initiated and the LED of wavelength 560nm was emitted onto the grey pad (7) for 10ms and absorbance reading calculated for calibration.
After calibration, the cassette housing the lateral flow strip (2) was shifted to position (P2) the nitrocellulose membrane in the optical path for reading.
The device tray (104) was opened to allow sample collection.
A whole blood sample volume of 25 µl was collected from an infant aged 15 days. The sample whole blood was dropped into the absorption pad (10) of the lateral flow strip (2).
The tray (104) was closed and LED of wavelength 450nm was emitted onto the nitrocellulose membrane (8) containing the sample for a period of ~10ms.

Readings from grey pad (7) and readings from nitrocellulose membrane (8) were collected, calculated and a final reading of the total serum bilirubin concentration in the sample being 15.01 mg/dl, was displayed on the screen (105).

EXAMPLE 2
The device (500) of the present invention was switched on, the cassette (100) housing the lateral flow strip (2) was positioned to (P1) position the grey pad in the optical path for reading.
Calibration was initiated and the LED of wavelength 570nm was emitted onto the grey pad (7) for 10ms and absorbance reading calculated for calibration.
After calibration, the cassette housing the lateral flow strip (2) was shifted to position (P2) the nitrocellulose membrane in the optical path for reading.
The device tray (104) was opened to allow sample collection.
A whole blood sample volume of 26µl was collected from an infant aged 15 days. The sample whole blood was dropped into the absorption pad (10) of the lateral flow strip (2).
The tray (104) was closed and LED of wavelength 460nm was emitted onto the nitrocellulose membrane (8) containing the sample for a period of ~10ms.
Readings from grey pad (7) and readings from nitrocellulose membrane (8) were collected, calculated and a final reading of the total serum bilirubin concentration in the sample being 10.06 mg/dl, was displayed on the screen (105).

EXPERIMENTAL DATA
Performance evaluation of the present invention was conducted. Particularly, a comparative analysis of Total Bilirubin device using the present invention (MiniLab) and standard prior art (Lab) was conducted.
After testing for Total 99 clinical samples, it was observed that Total Bilirubin values quantified using the present invention was found to be of standard quality with sensitivity of 96.88% for the measuring range from 1-40 mg/dl and Coefficient value of R = 0.9868. The present invention was therefore concluded to be well comparative with the prior art as required despite measuring Bilirubin concentration in whole blood without the need of any chemicals to treat the blood sample.
Please see Figure 4 for the corresponding graph to the experimental results found after conducting the above mentioned tests.

The foregoing description of the invention and the definitions mentioned above have been set merely to illustrate the invention and are not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to a person skilled in the art, the invention should be construed to include everything within the scope of the disclosure.
,CLAIMS:
Claim 1 : A device to quantify total serum Bilirubin concentration comprising:
a. at least a lateral flow strip (2) having:
i. at least a nitrocellulose membrane (8);
ii. at least a cell separator (9); and
iii. at least an absorption pad (10);

Claim 2 : The device to quantify total serum Bilirubin concentration as claimed in claim 1, additionally comprising:
a. at least a color sensor; and
b. at least two LEDs.

Claim 3 : The device to quantify total serum Bilirubin concentration as claimed in claim 1 wherein, the lateral flow strip maintains a continuous fluid flow by in-built pressure points which generate a capillary action for fluid mobility.

Claim 4 : The device to quantify total serum bilirubin concentration as claimed in claim 1 wherein, the lateral flow strip has a flat configuration and the nitrocellulose membrane is connected to the absorption pad by the cell separator such that the three components are positioned adjacent to each other void of any gap or overlap of elements, ensuring mobility of fluid from the absorption pad to the nitrocellulose membrane.
Claim 5 : The device to quantify total serum Bilirubin content as claimed in claim 1 comprises of:
a. at least a lateral flow strip including:
i. at least a nitrocellulose membrane;
ii. at least a cell separator;
iii. at least an absorption pad; and
iv. at least a grey pad
b. at least a color sensor including:
i. Transmitter;
ii. Receiver;
iii. Photodiode; and
iv. A pre-programed, self-calibrating algorithm;
c. at least two LEDs;
d. a lateral flow strip holder having adjustable dual housing positions for the lateral flow strip;
e. A display screen; and
f. Onboard interface(s), all connected to a portable user interface by means of a wireless communication

Claim 6 : The device as claimed in claim 1, wherein the at least two LEDs preferably has wavelengths each emitted alternatively for a defined period of time i.e. ~10ms such that the first wavelength measures the absorbance of the fluid component while the second wavelength measures the presence of any hemolysis.

Claim 7 : The device as claimed in claim 1, wherein the at least two LEDs have a wavelength ranging from 400nm to 500nm and 550-650nm respectively, and the color sensor has a photodiode which is optically aligned with the transmitter, and grey pad.

Claim 8 : The device as claimed in claim 1, wherein the photodiode of the color sensor, senses light having a wavelength ranging from 400-500nm for measuring Bilirubin levels at isobestic point and a wavelength ranging from 550-650nm for Hemolysis correction.

Claim 9 : The device as claimed in claim 1, wherein the lateral flow strip is cased inside a cassette made of materials selected from but not limited to plastic, or another such industrially acceptable material and measures a maximum of 93mm x 141mm, the cuvette measures measuring 2.1mm and the housing cells measure 16mm x 11mm / 30mm x 9mm;

Claim 10 : The device as claimed in claim 1, wherein the device is capable of measuring component level in whole blood and serum by accommodating an appropriate wavelength of LED according to the selected sample to be measured

Claim 11 : The device as claimed in claim 5, wherein the portable user interface may be selected from but not limited to a mobile phone, a tablet, a computer, inter alia and the wireless communication may be selected from but not limited to Bluetooth, WiFi connection, internet connection, or such other wireless communication.

Claim 12 : The device as claimed in claim 1, wherein the intensity of the light source may be adjusted based on the absorbance capacity of sample fluid within the lateral flow strip.

Claim 13 : A method to quantitatively measure the total serum bilirubin concentration in whole blood by employing the device as claimed in claim 1, the method comprising following steps:
i. Device turned on and lateral flow strip placed on holder’s 1st position to initiate calibration;
ii. At least two LEDs emitted from transmitter onto grey pad;
iii. Light absorbance of the two wavelengths measured by photodiode according to a preprogrammed algorithm, to complete calibration;
iv. Once calibrated, lateral flow stripshifted to holder’s 2nd position where the white pad (nitrocellulose membrane) containing fluid sample is exposed to two LED wavelengths alternatively for a period of ~10ms each;
v. Light absorbance by the fluid sample is measured by the photodiode according to a preprogrammed algorithm;
vi. The fluid concentration is calculated by the algorithm based on the output of the photodiodes and the same is communicated to user by means of a user interface

Claim 14 : The method as claimed in claim 13, wherein the absorbance readings of the wavelength is detected by the photodiode and considered by the algorithm in calculating the true absorbance of the fluid after calibrating the hemolysis count amongst other deviation/scattering components, to accurately quantify the fluid component value.