WO2021188084A2

WO2021188084A2 - A measurement system for tear chemicals in the tear for diagnosing diseases

Info

Publication number: WO2021188084A2
Application number: PCT/TR2021/050234
Authority: WO
Inventors: Figen VARLIBAS; Amin Tatabaei MOHSENI; Sultan ILBAY; Faruk OYTUN
Original assignee: Vsy Biyoteknoloji Ve Ilac Sanayi Anonim Sirketi
Priority date: 2020-03-18
Filing date: 2021-03-16
Publication date: 2021-09-23
Also published as: WO2021188084A3; TR202004203A2

Abstract

The measurement system (1) of the present invention comprises a detecting biosensor set (6) which changes colour according to the amount of tear chemicals, including dopamine, glucose, ascorbate, lactate and proteins, present in the tear. When the measurement process is completed, the detecting biosensor set (6) is analyzed visually, and therefore the level of various chemicals in the tear such as dopamine can be detected.

Description

A MEASUREMENT SYSTEM FOR TEAR CHEMICALS IN THE TEAR FOR DIAGNOSING DISEASES Field of the Invention

The present invention relates to a measurement system, which enables the indication of diseases to be detected through the level of chemicals available in tears. Background of the Invention

While an increase in dopamine levels in the Striatum region of our brain causes the onset of schizophrenia , a decrease in dopamine levels can lead to Parkinson's disease due to neurological degeneration and increased iron levels in the brain.² Furthermore, dopamine level imbalance is detected in drug addicts and in cases of HIV infection due to this addiction.

Unfortunately, it can be very difficult to detect level of dopamine secretion in the brain, which is one of the most important indicators for controlling the state of people with suspected or diagnosed neurological disease, especially Parkinson's disease. Scientific research has proven that the level of dopamine in tears is directly associated with changes in the brain.

Systemic diseases (diseases affecting the whole body) are diagnosed via symptoms reported by patients, symptoms observed during an examination, or via chemical analysis of blood and/or urine samples obtained from a medical history file. Patient samples are typically sent to a diagnostic laboratory to determine the levels of a wide range of markers, including ions, antibodies, hormone levels, and various disease-specific biomarkers. After a period of time ranging from minutes to days depending on the test, the laboratory report is sent back to the doctor and the results are conveyed to the patient.

In order to facilitate these analysis methods, home type test kits were developed in the 1950s . The said test kits react to the biomarkers of diseases through colour change. The colour change rate is directly proportional to the rate of chemical change.

Colour-changing strips which are used by the patient, a non-specialist relative of the patient, or a specialist are graded by means of a colour guide. The colour comparison result determines at which concentration range the analyzed biomarker is.

Blood and blood plasma or serums, which are among very important body chemicals, are not always sufficient for examining diseases. This is because the change in the concentration of chemicals in the blood is too small to be detected in some cases, which causes difficulties in measurements and/or misdiagnosis. For example, the dopamine concentration in the blood of a healthy individual is around 475x10^-9 mM. However, the concentration of dopamine in tears can be around 370x10^-3 mM. In other words, the molarity of dopamine in tears is approximately 780.000 times higher than its concentration in the blood. The concentrations of some other chemicals in the tears are also sufficient to make measurements. Therefore, tears provide a more suitable basis for being used in the diagnosis of many diseases . Likewise, the molarity of lactate in tears is between 2 and 5 mM, while it is between 0.36-0.75 mM in blood plasma (about 10 times more concentrated in tear). The molarity of ascorbate in tears is between 0.22 and 1.31 mM, while it is between 0.04-0.06 mM in blood plasma (about 15 times more concentrated in tears).

Table 1. Molarity comparison of analytes in tear and blood

In another scientific study , by means of using the ELISA kit, higher dopamine levels were found in tear fluid than in plasma:

Table 2. Comparison of dopamine concentration in tear and blood

The colorimetry and fluorescence assay for dopamine according to the present invention is carried out with the Cu-MnxOy/C-dots/TMB composite system. This sensor responds to the tear fluid comprising dopamine molecules. The colour of the sensor material coated on carbon nanoparticles and copper-manganese oxide based TMB is dark blue in its free state and it becomes transparent upon being affected by the dopamine in the tear. This colour change is related with the dopamine concentration.

In the state of the art, some methods applied in the detection of diseases - especially for Parkinson's disease- in an individual and the disadvantages experienced in them can be expressed as follows:

1. When 60% to 80% of dopamine -producing cells in the brain are lost, sufficient dopamine cannot be produced and motor symptoms of Parkinson's disease appear. Accordingly, Parkinson's disease is determined clinically by examining motor symptoms, lifestyle factors and medical history data . In this method, since the diagnosis of Parkinson's is based on clinical data, laboratory measurements to support this basis are needed, because it is likely to be confused with Parkin son's-like diseases or diseases that mimic Parkinson's.

2. Since there is no reliable way of directly measuring dopamine levels in the brain, several indirect ways are used to determine the said imbalance of the level. Doctors can measure the density of dopamine transporters which are directly related to nerve cells using dopamine. This test involves injecting a radioactive material which binds to dopamine transporters that doctors can measure using a camera . In this method, the dopamine level cannot be shown directly, as dopamine measurement is performed indirectly. On the other hand, use of the radioactive materials employed in this method is generally not preferred due to their harmful effects.

3. As another method, they can also be scanned through diffusion- weighted magnetic resonance imaging (DW-MRI) findings of Substantia Nigra in Parkinson's disease. Images resulting from molecular movements of water in the tissue are obtained with diffusion MR technique. Since this method is an expensive and limited imaging method, it cannot be applied to all patients or people with a suspected condition. Furthermore, it can take hours to obtain laboratory result of dopamine analysis with MRI and similar methods. However, monitoring the treatment process may not always require measurements having very high precision. Especially in bedside tests, taking measurements and obtaining results rapidly are more important than precision.

4. ELISA kit which quantitatively detects dopamine in urine and blood plasma by colour change is commercially available. These kits are for research purposes only and should not be used in clinical, therapeutic or diagnostic procedures. Dopamine is extracted using a cis-diol specific affinity gel and acylated to N-acyl-dopamine and then enzymatically converted to N-acyl-3-methoxytyramine. Dopamine binds to the solid phase of the microtiter plate. The sample and the dopamine acylated from dopamine bound to solid phase compete for a fixed number of antiserum binding sites. When the system is in balance, free antigen and free antigen- antiserum complexes are washed away. Antibody bound to solid phase dopamine is detected via anti-rabbit IgG/peroxidase. Substrate TMB/peroxidase reaction is monitored at 450 nm. The amount of antibody bound to solid phase dopamine is inversely proportional to the dopamine concentration of the sample.

The ELISA kit is considered to be an expensive method of measurement (current price is USD 280) and it requires technical knowledge to use. Commercially available ELISA kits are calibrated for urine and plasma and they are not suitable for tears. In addition, it takes hours to obtain the test results. The patent applications known in the state of the art are as follows:

United States patent document no US20140194706A1 discloses apparatus, systems and methods employing contact lenses having one or more sensor that sense an analyte in tear fluid and one or more recesses that collect the tear fluid. This contact lens comprises at least one sensor configured to sense the presence of an analyte in the collected tear fluid. The claims of this patent include collecting the tear sample into a microscale chamber and then analyzing it by the sensor having a microprocessor. The signals detected by the sensor are transmitted to a device connected to the internet via electronic circuitry and antennas. - Chinese patent application no CN107860805 discloses an aptamer-gold nanoparticle/reduced graphene oxide-Nile blue nanocomposite based electrochemical dopamine aptamer sensor.

South Korean patent document no KR20180103653 discloses a dopamine detection biosensor. It comprises a field effect transistor (FET) in which an electrode forms the source and which senses a flow of electrical current.

The functional layer includes a dopamine reactant which selectively reacts with dopamine.

Patent document no WO2016029139 discloses a contact lens which has hydrogel coating on its surface in order to detect biomarker in tears. Within the scope of the said application, a method is developed by means of using aptamer molecules in colorimetric analyses. No method or methodology generating numerical output to observe the colour change that occurs and to transfer analysis information is provided.

European patent document no EP3131454 discloses a functional contact lens and related systems and methods. The contact lens of the present invention is used to sense the changes caused by the detection of at least one target analyte by means of electronic and electrochemical methods and to transmit the sensed signal to an external device via an antenna integrated into the lens structure. It does not perform colorimetric detection and data transfer.

United States patent application no US2012245444 discloses a wireless powered contact lens with glucose sensor. The contact lens includes an electrochemical sensor which enables to measure the level of glucose in the tear fluid of an individual. This contact lens provides the power via RF antenna or photovoltaic device, and it can also perform data transmission over the electrochemical sensor. It does not perform colorimetric detection and data transfer.

Patent application no WO2018187693 discloses ocular devices and methods for the employment thereof. The device of the invention is placed into a lacrimal punctum or conjunctival sac of a person, and it senses the chemical composition of the person's tears with one or more sensor materials responsive to one or more components. Each sensor material transfers the medical state of the person to outside with electromagnetic signals. It does not suggest any coding method for diagnosis of a disease. This patent discloses ocular devices. The analysis method used in these devices is colorimetric, however the colour change is detected through the wave length in the detection method.

Turkish patent application document no TR 2015/17446 discloses contact lens design detecting tear glucose level. The invention is intended for the use of diabetic patients and relates to the development of a material which will detect the glucose level in the tear by means of a biosensor thereon. No method or methodology generating numerical output to observe the colour change that occurs and to transfer analysis information is provided. European patent document no EP3148435 discloses a system for monitoring health related information of individuals.

European patent document no EP2846182 discloses ophthalmic lens system capable of interfacing with an external device. United States patent document no US20170371128 discloses a disposable lens applied to electronic operation device for recognition.

Patent document no EP2569667 discloses a method for preparing an ophthalmic lens provided with a QR code. The invention described in this context suggests a printing method to print and transfer on the lens with QR codes and similar codes, but it does not mention any detection or its use as a sensor.

European patent document no EP3159693 discloses a detection device and method, and program, and it is a device comprised of pressure, temperature and motion sensors.

Patent application no WO2017178621 discloses a system which performs analysis through image processing. Mouth water (saliva) sample, which is one of the body fluids, contacts the test cell and causes a colour change. This colour change is analyzed with a digital image and the results are reported to the user in the form of a QR code.

The strips for urine analysis available in the market are used for detection of many parameters such as pH value of urine, protein, glucose and pregnancy hormone (hCG) through colour change. A few drops of urine sample are transferred onto the coloured cells on the test strips and it is waited for a colour change for a certain period of time. Results are obtained by direct comparison of the colour blocks printed on the box. Colour blocks represent pseudo values, actual values will vary close to pseudo values.

There are several problems and risks which limit the use of these strips:

There is a high probability of erroneous colour reading by eye.

Unrealistic reference colours can be presented, since the references are printed out on a paper on the box. It is difficult to determine the exact number, since a wide range of values is given.

Since it is not possible to monitor more than one cell colour at the same time, the reading period may pass and the colour scale may change.

It is difficult to follow the results, and there is a high probability of confusion.

It is difficult to record and report the results and there are possibilities of errors (trouble in taking notes manually).

Strips and cells cut in large sizes cause high material consumption.

Test cells cut in large size require more urine sample.

In the user manual of the strips, it is mentioned that they should be kept in a small glass (200 ml) of urine sample, and this amount is too much for body fluids other than urine.

In order to prevent some of these problems, there are studies which determine the result of urine analysis with image processing method using mobile phone application. For example, after a few drops of the urine sample touch the strip kit, Vivoo analyzes and determines numerical values for the user. The image processing method helps for more accurate colour analysis; however in this study the problem of sample collection and the risk of confusion in results are not eliminated.

Summary of the Invention

The objective of the present invention is to provide a measurement system for determining the level of various tear chemicals such as dopamine, which is easy to use, has high reliability, low cost, which can be recorded and does not require technical training. Detailed Description of the Invention

A contact sensing structure developed to fulfill the objectives of the present invention is illustrated in the accompanying figures, in which:

Figure 1 is the view of the measurement system of the present invention when it has a substrate in the form of a strip.

Figure 2 is the view of operation principle of the measurement system of the present invention when it has a substrate in the form of a strip.

Figure 3 is the view of the measurement system of the present invention when it has a substrate in the form of a strip that is placed under the eyelid. Figure 4 is the view of the operation principle of the measurement system of the present invention when it has a substrate in the form of a strip that is placed under the eyelid.

Figure 5 is the view of the measurement system of the present invention when it has a substrate in the form of a contact lens. Figure 6 is the view of operation principle of the measurement system of the present invention when it has a substrate in the form of a contact lens.

The components shown in the figures are each given reference numbers as follows:

1. Measurement system

2. Substrate

21. Absorbent part

22. Non-absorbent part 23. Holding area

3. Code area

31. Code

32. Reference colours

4. Sensor area 5. Part to be placed under the eyelid

6. Detecting biosensor set The measurement system (1) of the present invention is used to determine the level of tear chemicals including dopamine, glucose, ascorbate, lactate and proteins; and comprises

- at least one substrate (2) suitable for collecting tears which comprises at least one absorbent part (21) and at least one non-absorbent part (22) ,

- at least one code area (3) which is located on the non-absorbent part (22) of the substrate (2), and comprises at least one code (31) containing information about one, several or all of the information related to sensor number, sensor type, intended use and method of use of the sensor, production date and LOT number,

- at least one sensor area (4) that is located on the substrate (2) and comprises at least one detecting biosensor set (6) for each analyte desired to be detected which is suitable for interacting with an analyte, and whose colour changes as a result of this interaction.

In one embodiment of the present invention, the substrate (2) can be in the form of a strip on which a tear sample collected from the patient's eye can be dropped. In case a strip is used in this structure, local anesthetic eye drops USP solution, which does not prevent eye irritation, is dropped into the patient's eye in order to obtain basal tears for collecting the tear sample from the patient's eye (Proparacaine Hydrochloride Ophthalmic Solution 0.5%) and the sample collected from the resulting tears by means of a micropipette is dropped t onto the absorbent part (21) on the surface of the substrate (2) in the form of a strip.

In another embodiment of the present invention, the substrate (2) can be in the form of a strip which is placed under the eyelid and directly collects the tears from the eye. In another embodiment of the present invention, the substrate (2) may be in the form of a contact lens which is positioned like an ordinary contact lens placed directly on the pupil and allows collecting tears directly from the eye. The middle part (21 and 23) of the said lens has high light transmittance (90% and above) and provides the patient's vision during the time waited to collect tear samples. In addition, support will be taken from its middle part (23) as a holding point for placing the contact lens on the eye surface.

In an embodiment of the present invention, there is at least one holding area (23) which allows the user to easily hold the substrate (2), such that it will be on the surface of the substrate (2) in the form of a strip, excluding the absorbent part (21) and the non-absorbent part (22).

In an embodiment of the present invention, if the substrate (2) is in the form of a strip placed on the eyelid, there is a part to be placed under the eyelid (5), which is suitable for being placed under the eyelid in order to contact the tear directly.

In an embodiment of the present invention, the measurement system (1) of the present invention comprises at least one reference colour (32) to compare with the colour of the detecting biosensor set (6). The said reference colour (32) corresponds to the colour of said detecting biosensor set (6) at a certain concentration for a particular tear chemical such as dopamine.

In an embodiment of the present invention, the substrate (2) can be produced from any material such as paper, cellulose, acrylic, plastic or PMMA (polymethyl methacrylate).

In an embodiment of the present invention, the information contained in the code area (31) can be encoded by any method available in the art such as iQR, QR code, datamatrix, etc. or to be developed in the future. In an embodiment of the present invention, the detecting biosensor set (6) interacts with tear chemicals including dopamine, glucose, ascorbate, lactate and proteins, and as a result of this interaction, the colour of the detecting biosensor set (6) changes. Therefore, the presence and/or concentration of the said chemicals can be detected. Cu-Mn-0 micro-crystals and carbon nanoparticle for measuring dopamine level and boric acid for measuring glucose level can be given as example for the said detecting biosensor set (6), but it is not limited to these. Different detecting biosensors (6) may also comprise different concentrations of the same analyte.

In an embodiment of the invention, the detecting biosensor set (6) can be produced by methods such as printing, dip-coating, spin-coating, spray-coating, electro spinning, inkjet printing, microjet printing, nanojet printing, but it is not limited to these.

In an embodiment of the present invention, the sensor area (4) comprises a plurality of detecting biosensor sets (6) that are arranged to form a pattern. This pattern can be formed in any shape that is even such as a matrix in size of m x n, circle, ellipse, triangle, etc., or uneven. Similarly, the reference colours (32) can also be formed in any shape that is even such as a matrix in size of m x n, circle, ellipse, triangle, etc., or uneven.

The measurement system (1) of the present invention comprises at least one strip substrate (2) including at least one absorbent part (21) and at least one non absorbent part (22) suitable for collecting tears and having at least one holding area (23). There is at least one code area (3) which is preferably located on the non-absorbent part (22) of the strip substrate (2), and comprises at least one code (31) containing information about one, several or all of the information related to sensor number of the said strip substrate (2), sensor type, intended use and method of use of the sensor, production date and LOT number. There is at least one sensor area (4) on the substrate (2) that comprises at least one detecting biosensor set (6) for each analyte desired to be detected which is suitable for interacting with an analyte, and whose colour changes as a result of this interaction. In order for the tear to be absorbed by the strip substrate (2), the part to be placed under the eyelid (5) is placed directly under the eyelid.

The measurement process is carried out by contacting the measurement system (1) of the invention with tears for a predetermined period of time. At the end of the measurement process, the image of the code area (3) and the sensor field (4) is captured by an image detector such as a camera, etc.. This detecting means can be a mobile phone with a camera or a portable computer. Then, this image is processed and analyzed by the device capturing the image or a central server. Processing of the said image may include one, several or all of the processes of calibrating the said image, obtaining colour information of the detecting biosensor set (6), normalizing the said colour information in accordance with calibration data, comparing the said normalized and/or non-normalized colour information with the data on the said device and/or central server, and interpreting the information obtained as a result of this comparison according to the table or tables provided in the said device and/or the central server. Image processing can be performed by any image processing algorithm which is known in the state of the art or which may be developed in the future.

In an embodiment of the invention which can be used in combination with other embodiments, the information and/or interpretations obtained as a result of the processing of the images are added to the user's profile kept on the device and/or the central server. Therefore, the user can easily see the results of the measurements made in the past and the interpretations made in accordance with the said results.

In an embodiment of the invention which can be used in combination with other embodiments, an optical member such as a filter or a lens can be attached in front of the image sensor. The said optical member can be used to change various properties of the light to be transmitted such as colour, focus and/or polarization, but it is not limited to these.

In case the substrate (2) addressed within the scope of the invention is in the form of a strip on which a tear sample collected from the eye of the patient can be dropped, for example it can be applied as follows (Figure 2):

1- In order to obtain basal tears, an eye drop which will not prevent eye irritation is used and it is waited for about 60 seconds.

2- Then, a certain amount of sample of the basal tear fluid is collected from a location close to the patient's eyelids with the help of a micropipette (Micro Capillary Tubes (MCT)) or cellulose sponge and polyester rod.

3- A few drops of tear sample are dropped onto the sensor area (4) located on the absorbent part (21) of the measurement system (1) of the present invention.

4- Image is captured from the code area (3).

5- The captured image is sent to the developed software on the remote server

(cloud) through the internet and image analysis is performed. Thus, the colour and contrast or wavelength differences detected in the image are compared with analytical tables, and the amount of tear chemical is determined. This amount is sent to the application and the user is informed, and the reports are added to the patient's file.

In case the substrate (2) addressed within the scope of the invention is in the form of a strip which can be placed under the eyelid, for example it can be applied as follows (Figure 4):

2.1. In order to obtain basal tears, an eye drop which will not prevent eye irritation is used and it is waited for about 60 seconds.

2.2. Then the part (5) of the measurement system (1) of the present invention to be placed under the eyelid is placed under the eyelid, and it is kept for about 60 seconds. 2.3. At the end of this time, the measurement system (1) of the present invention is removed from under the eyelid.

2.4. Image is captured from the code area (3).

2.5. The captured image is sent to the developed software on the remote server

In case the substrate (2) addressed within the scope of the invention is in the form of a contact lens which is positioned like an ordinary contact lens placed directly on the pupil and allows collecting tears directly from the eye, for example it can be applied as follows (Figure 6):

3.1. In order to obtain basal tears, an eye drop which will not prevent eye irritation is used and it is waited for about 60 seconds.

3.2. Then, the measurement system (1) of the present invention having a substrate (2) in the form of contact lens is placed on the patient’s pupil like a lens, and it is waited for about 60-300 seconds.

3.3. The image is captured from the code area (3) while it is still on the user’s eye or after it is removed.

3.4. The captured image is sent to the developed software on the remote server

Claims

1. A measurement system (1) for determining the level of tear chemicals characterized by

- at least one substrate (2) suitable for collecting tears which comprises at least one absorbent part (21) and at least one non-absorbent part (22),

- at least one sensor area (4) that is located on the substrate (2) and comprises at least one detecting biosensor set (6) for each analyte desired to be detected which is suitable for interacting with the said analyte, , and whose colour changes as a result of this interaction.

2. A measurement system (1) according to claim 1, characterized by the substrate (2) which is in the form of a strip on which a sample from the tear collected from the patient’s eye can be dropped.

3. A measurement system (1) according to claim 1, characterized by the substrate (2) which is in the form of a strip which directly collects the tear from the eye by being placed under the eyelid.

4. A measurement system (1) according to claim 1, characterized by the substrate (2) which is in the form of a contact lens which is positioned like an ordinary contact lens directly placed on the pupil and allows collecting tear directly from the eye.

5. A measurement system according to claim 2 or 3, comprising at least one holding area (23) which allows the user to easily hold the substrate (2).

A measurement system (1) according to claim 3, in case it is in the form of a strip placed under the eyelid, comprising a part to be placed under the eyelid (5) which is suitable for being placed under the eyelid for directly contacting the tear.

7. A measurement system (1) according to claim 1, comprising at least one reference colour (32) for comparing with the colour of the detecting biosensor set (6).

8. A measurement system (1) according to claim 1, comprising the strip substrate (2) which is produced from any material such as paper, cellulose, acrylic, plastic, PMMA (poly methyl metacry late).

9. A measurement system (1) according to claim 1, comprising detecting biosensor set (6) which is suitable to interact with tear chemicals including dopamine, glucose, ascorbate, lactate and proteins.

10. A measurement system (1) according to claim 1, characterized by sensor area (4) which comprises a plurality of detecting biosensor sets (6) arranged to form a pattern.

11. A measurement system (1) according to claim 10, characterized by a pattern in any shape which is even such as a matrix in size of m x n, circle, ellipse, triangle, etc., or eneven.

12. A measurement system (1) according to claim 10, comprising reference colours (32) in any shape which is even such as a matrix in size of m x n, circle, ellipse, triangle, etc., or uneven.

13. A measurement method characterized by the steps of contacting a measurement system (1) according to any one of the preceding claims with the tear for a predetermined time, capturing the image of the code area (3) and the sensor field (4) by an image detector such as a camera, etc., processing and analyzing the captured image by the device capturing the image or a central server.

14. A measurement method according to claim 13, characterized in that the step of processing the image includes one, several or all of the processes of calibrating the said image, obtaining colour information of the detecting biosensor set (6), normalizing the said colour information in accordance with the calibration data, comparing the said normalized and/or non- normalized colour information with the data on the said device and/or central server, and interpreting the information obtained as a result of this comparison according to the table or tables provided in the said device and/or the central server.

15. A measurement method according to claim 13, comprising the step of adding the obtained information and/or interpretations to the user profile kept in the said device and/or central server.

16. A measurement method of a measurement system (1) according to any one of the claims 1 to 13, characterized in that the detecting biosensor set (6) is produced according to any one of the methods of printing, dip-coating, spin-coating, spray-coating, electro spinning, inkjet printing, microjet printing, nanojet printing.