WO2023113749A1

WO2023113749A1 - Biosensor system for quantitative measurement of phenylalanine from blood

Info

Publication number: WO2023113749A1
Application number: PCT/TR2022/051477
Authority: WO
Inventors: Ilayda OZHAVZALI
Original assignee: Gazi Universitesi Rektorlugu
Priority date: 2021-12-14
Filing date: 2022-12-12
Publication date: 2023-06-22
Also published as: TR2021019849A2

Abstract

The invention relates to a biosensor system that quantitatively measures phenylalanine from blood and makes dietary recommendations. Said biosensor system comprises a measuring device that makes the quantitative measurement of phenylalanine, a mobile device communicating/paired with the measuring device, and an application running on the processor in the mobile device. Said invention is used as a screening test in newborns for phenylketonuria disease, or to monitor the phenylalanine levels in the blood with the blood that patients with diagnosed phenylketonuria can easily drip from the fingertips with a lancet at home. By collecting the results obtained from the measuring device in the system that is the subject of the invention in the database, the control/follow-up of the disease is provided and since the software in the mobile device, with which the measuring device communicates, is specially shaped via machine learning, more accurate calculations and dietary recommendations are made for the patient for the amount of phenylalanine that the patient should take compared to the prior art.

Description

BIOSENSOR SYSTEM FOR QUANTITATIVE MEASUREMENT OF PHENYLALANINE FROM BLOOD

Technical Field of the Invention

The invention relates to a biosensor system that quantitatively measures phenylalanine from blood and makes dietary recommendations. Said biosensor system comprises a measuring device that makes the quantitative measurement of phenylalanine, a mobile device communicating/paired with the measuring device, and an application running on the processor in the mobile device.

State of the Art

Phenylketonuria (PKU) is an inherited disease in which the level of phenylalanine in the blood is high due to the deficiency of the phenylalanine dehydrogenase enzyme, and it occurs due to the disorder in the phenylalanine metabolism. Phenylalanine is an amino acid that is essential for our body and must be obtained from food. This amino acid needs to be converted to tyrosine by the enzyme phenylalanine hydroxylase in our body and to perform its necessary functions in the form of tyrosine. As a result of an error in the conversion to tyrosine, the level of phenylalanine increases in the blood and phenylketonuria occurs.

Phenylketonuria is an autosomal recessive metabolic disorder. The disease is transmitted from mother and father to baby through genes. PKU was first described by Norwegian Doctor Asbjdrn Folling in 1954. In PKU disease, the enzyme phenylalanine hydroxylase (phenylalanineH) in the liver, which will catalyse phenylalanine, an essential amino acid, into another amino acid, tyrosine, is inactive. Deficiency of this enzyme produces a number of disorders including classical phenylketonuria, mild phenylketonuria and mild hyperphenylalaninemia. Phenylalanine and its derivatives, which cannot be converted and accumulate in the body, pass into the cerebrospinal fluid, increasing the level of the compounds there, and if not treated, they cause profound and irreversible mental and neurological developmental retardation.

It is of great importance to diagnose PKU as early as possible. At any time that passes without a diagnosis of PKU, phenylalanine, which cannot be catalysed and accumulates in various parts of the body, causes irreversible damages in the body. If the disease is not diagnosed early, severe mental retardation, convulsions, and seizures, aggressive or autistic behavioural disorders, and skin lesions in the form of dermatitis can be seen in babies. If it is diagnosed early, the phenylalanine level in the blood is kept between 2-6mg/dl (120-360 pmol/L) with a diet devoid of phenylalanine and severe intelligence impairment is prevented. The earlier the diet treatment is started, the less the intelligence is affected.

Due to these critical reasons, screening programs are carried out at the country level so that newborn babies can start and continue their lives in a healthier way. These screening programs are carried out for diseases that are common in the society according to the conditions of the countries. The goal here is to identify and intervene in metabolic diseases at an early stage. In this context, "Phenylketonuria Screening Test", one of the screening tests in newborns, has been included in the heel blood screening program since 1994 within the scope of the National Screening Program. Screening newborns for Phenylalanine Hydroxylase deficiency is important for PKU because these screenings can prevent irreversible damage or reduce its effects as much as possible.

Newborn screening programs show that the frequency of PKU differs from country to country. It is stated that the prevalence of PKU in Europe varies between 1/3000- 1/30000. It is stated that the frequency of PKU in Europe is 1 in 10000 live births, but it is noted that it is higher in some regions. Resistant HFA is reported in 1 out of every 4000 live births in Turkey, which is attributed to the high frequency of consanguineous marriages as in Northern Ireland. According to the National Child Health and Development Association, the frequency of PKU in the UK is about 1/15000. It is noted that the highest frequency belongs to Turkey.

Initially, screening tests based on the reaction of phenylpyruvate with FeCI3 in urine were used for the diagnosis of PKU. However, since the amount of phenylalanine in the blood must exceed a certain level for the diagnosis of PKU in this test, and the necessary precise measurement could not be made, its use was abandoned in practice. Another method used for the diagnosis of PKU is the Guthrie test, which is a measurement method based on the inability of Bacillus subtilis bacteria to grow in an environment that does not contain phenylalanine. Reproduction of Bacillus subtilis spores occurs in the presence of phenylalanine in the presence of beta-2- thienylalanine, an inhibitory substance. In newborns, blood taken from the heel is absorbed into special filter paper and placed in a medium containing beta-2- thienylalanine, an antimetabolite of phenylalanine antagonist. High phenylalanine level in the blood eliminates the suppression of beta-2-thienylalanine in the medium and ensures the growth of bacteria. The area where bacteria grow around the disc is compared with the area where bacteria grow around a control disc containing a certain amount of phenylalanine. Depending on the amount of phenylalanine in the blood taken from the baby, the area around the disc where bacteria grows expands.

However, in the Guthrie test, there is a possibility that a proper measurement cannot be made in the heel blood that is not taken in accordance with the procedure. This leads to the fact that newborns are brought back to the hospital environment and subjected to the test again. Even at this stage, measurement is quite prone to human error. In addition, quantitative results cannot be obtained in this test, so the Guthrie test is not preferred much in practice.

Today, "fluorometric analysis" and "tandem mass spectrometry" are used more professionally instead of "Guthrie test" for the determination of increased phenylalanine in blood in phenylketonuria. Professional laboratory conditions are needed for these methods and heel blood taken from newborns is placed on Guthrie papers in cellulose tissue in order to be able to measure, and the papers are sent to the laboratories after drying.

Sequential mass spectrometry, also known as tandem mass spectrometry, MS/MS or MS² in use today, is an instrumental analysis technique in which two or more mass analysers are connected using an additional reaction step to increase their ability to analyse chemical samples. After the patient’s blood that is impregnated and dried on special cellulose tissue papers undergoes some chemical pre-processes, quantitative measurement is made in the device. Fluorometric analysis, which is another method used today, is an analytical method that enables the identification and characterization of very small amounts of a substance by excitation and determination of substances by emitting ultraviolet light and measurement of the characteristic wavelength of the emitted fluorescent light. In the measurement of phenylalanine with a fluorometric analysis kit, after the phenylalanine in the dried blood drop sample is eluted, it reacts with reactive mixtures to form a fluorescent compound. The fluorescent signal, which is strengthened and stabilized by the other chemical reagent added, is measured with a fluorometer adjusted to the appropriate wavelength and evaluated according to the calibrator sample signals included in the kits, and the amount of phenylalanine in the initial patient blood sample is determined in mg/dl.

Professional laboratory conditions are needed in fluorometric analysis or tandem mass spectrometry methods. The blood samples taken become readable in the devices after passing through certain chemical stages. The fact that these stages take a long time and include a large number of process steps makes these stages open to human errors, but also creates a disadvantage in terms of both cost and personnel load. Since the devices used in these methods are quite expensive, they are only found in certain parts of the country and in very few numbers. Since the instruments must be pretreated before being tested, large costs per test arise with pre-treatment. In addition, the delivery of blood samples taken from different cities to the main laboratories where the tests are carried out creates a disadvantage in terms of both transportation and time.

Phenylketonuria is treated with a special medical nutrition practice/diet applied to keep blood phenylalanine levels within normal limits for life. In the treatment of PKU, special medical nutrition therapy with limited phenylalanine should be applied for life. Since mental and developmental disorders may occur in patients who do not comply with or do not start or stop treatment, patients should learn the medical nutrition therapy/diet very well. PKU treatment is carried out in nutrition and metabolism centres related to this subject. For dietary arrangements, it is necessary to go to said nutrition and metabolism centres. The devices and methods in the prior art are insufficient in terms of easily accessing the diet to be applied by the patients and providing the most accurate control of the disease with personalized calculations. Real-time phenylalanine levels are determined with a device called “PKU Now” in the prior art, and thus the device helps to maintain optimal phenylalanine levels. “PKU Now” is a point-of-care device that will allow accurate measurement of L-phenylalanine (Phe) levels from a 20 microlitre sample taken from a finger or heel. The “PKU Now” test kit includes 20 test strips, 20 capillary tubes, a capillary stand, a PKU Now Metre and 2 level controls. However, since there is no application for regulating the patient's diet and there is no software for this in the device, the device is insufficient in terms of presenting measurement and treatment together. As it is known in the prior art, if the diagnosis is made early, severe intelligence impairment is prevented by keeping the phenylalanine level in the blood between 2-6mg/dl (120-360 pmol/L) with a diet devoid of phenylalanine, and the earlier the diet treatment is started, the less the intelligence is affected.

The prior art patent application EP1314786A1 describes a quantitative measuring device that enables simple and rapid determination of D-galactose, branched chain amino acids, L-leucine, and L-phenylalanine in a biological sample (blood). Along with checking the formazan concentration in the measurement of the device described here, a system in which optical or electrochemical methods are used together is explained. In addition, since there is no application for regulating the patient's diet and there is no software for this in the device, the device is insufficient in terms of presenting measurement and treatment together.

The prior art patent EP3039422B1 describes devices that generally measure and identify the presence of ammonia or ammonium ions in a body fluid, water or other environmental sample. Here, to detect elevated phenylalanine, the enzyme phenylalanine dehydrogenase is immobilized to at least one electrically conductive surface optionally contained within a hydrogel. However, since the separation of ammonium into ammonia and hydrogen in the blood occurs in a very short time (it is a very unstable molecule) in the measurement to be made using the concentration difference of the ammonium ions mentioned in said devices, it is thought that it may cause measurement errors.

Due to the fact that the phenylalanine measurement devices and methods in the state of the art include many process steps that take a long time, are insufficient in terms of providing measurement and treatment together as they do not include an application for regulating the diet of the patient, the devices offered not being able to make personal calculations with machine learning and prior art devices being high cost devices; there is a need to develop low-cost measurement devices that provide measurement in a short time and do not require many process steps, contain an application for regulating the patient's diet, offer measurement and treatment together, and can make individual calculations with machine learning.

Brief Description of the Invention

In the invention, a biosensor system, that makes quantitative measurement of phenylalanine from the blood, provides control/follow-up of the disease by collecting the entered measurement results in the database, and makes much more accurate calculations and dietary recommendations with machine learning compared to the prior art for the patient for the amount of phenylalanine that the patient should take, is explained. Said biosensor system comprises a measuring device that makes the quantitative measurement of phenylalanine, a mobile device communicating/paired with the measuring device, and an application running on the processor in the mobile device.

Said invention is used as a screening test in newborns for phenylketonuria disease, or to monitor the phenylalanine levels in the blood with the blood that patients with diagnosed phenylketonuria can easily drip from the fingertips with a lancet at home. In addition, the control/follow-up of the disease is ensured by collecting the measurement results entered into the device in the database, and since the software in the device is customised specifically for the person, much more accurate calculations and dietary recommendations are made for the patient-specific amount of phenylalanine that the patient should take.

The first aim of the invention is to enable the blood taken from the patient to be used directly without any pre-treatment in the detection and control of phenylketonuria disease and to present a portable device that provides the quantitative measurement of phenylalanine in the blood in a short time compared to the prior art. With the invention, bedside measurement is provided in a short time. While measurement takes hours in the prior art, with the invention, quantitative measurement of phenylalanine is provided within minutes (between 1 -3 minutes). In addition, since the blood taken from the heel in newborns and the blood taken from the finger in routine patients can be measured without putting it on Guthrie papers, which is the application of the prior art, human-induced errors that may occur during the collection of blood sample on Guthrie papers are eliminated.

Another aim of the invention is to provide the most accurate follow-up of the disease with the quantitative phenylalanine measurement results made from blood and to make a special diet recommendation for the patient by calculating the amount of phenylalanine that the patient should take. The device that is the subject of the invention is paired with mobile devices such as telephones via Bluetooth. With the collection of the measurement results taken with the invention in the database, an instant diet regulation is provided with the amount and type of food previously entered into the application in the mobile device. The system that is the subject of the invention provides many possibilities together with the application that is created to process the received data and control the disease, installed in mobile devices, and is compatible with the measuring device in the biosensor system that is the subject of the invention. All measurement results are recorded with date and time on the mobile application, along with notes added by the patient such as “before-meal”, “after-meal” or “before dinner” next to the measurement result, so that the disease is controlled together with the physician. The physician can view the phenylalanine results and notes of the patient on the system whenever s/he wishes.

When the patient using the biosensor system that is the subject of the invention inputs the food and its amount that s/he intends to eat into the mobile device before the meal, the phenylalanine level of the meal content is approximately calculated with the mobile device paired with the measuring device in the biosensor system that is the subject of the invention, so that the patient's diet is regulated. If the measurement that the patient will make after the meal is entered into the application in the mobile device, the software is shaped individually with machine learning over time and much more accurate calculations are made. The measuring device in the biosensor system only measures the level of phenylalanine in the blood and displays the result on the LCD screen. The remaining values such as “estimation of approximate post-meal phenylalanine level” are calculated and displayed on the mobile application. In this way, the invention offers both measurement and treatment together. Another object of the invention is to provide a low-cost device for measuring phenylalanine from blood. The kits and devices in the prior art are costly and require pre-treatment of blood taken from the patient and the use of chemicals. Contrary to the prior art, in the invention, there is no need for chemical pre-treatments, so the cost of the tests is reduced, and the burden of the working personnel is reduced. As the process steps are reduced, human-induced errors are eliminated, and the expense that will be incurred by re-testing due to errors is prevented, thus providing an advantage in terms of both cost and time. With the test strips used in the invention, the cost per test is reduced. Since the invention is produced at a much more affordable cost than the devices in the state of the art, it will be able to be delivered to all parts of the country, from health centres to large laboratory environments.

With the invention, a low-cost biosensor system that provides measurement in a short time, allows the blood taken from the patient to be used directly without any preprocessing, contains an application for regulating the patient's diet and offers measurement and treatment together, can make individual calculations with machine learning, measures phenylalanine from the blood and recommends a diet for patients with phenylketonuria based on their phenylalanine level is provided.

Description of Drawings

Figure 1 : Front view of the measuring device in the biosensor system.

Figure 2: Side view of the measuring device in the biosensor system.

Definition of Elements/Parts Composing the Invention

In order to better explain the biosensor system that is the subject of the invention, the parts in the figures are numbered, and the equivalent of each number is given below:

1 . Strip

2. LCD Screen

3. Bluetooth button 4. Forward button

5. On/off button

6. Back button

7. Strip inlet

Detailed Description of the Invention

The invention relates to a biosensor system that quantitatively measures phenylalanine from blood and makes dietary recommendations. Said biosensor system comprises a measuring device that makes the quantitative measurement of phenylalanine, a mobile device communicating/paired with the measuring device, and an application running on the processor in the mobile device. In said invention, a biosensor system, that makes quantitative measurement of phenylalanine from the blood, provides control/follow-up of the disease by collecting the entered measurement results in the database, and makes much more accurate calculations and dietary recommendations with machine learning compared to the prior art for the patient for the amount of phenylalanine that the patient should take, is explained.

The measuring device in the biosensor system that is the subject of the invention works with the principle of electrochemical/amperometric analysis. The biosensor system that is the subject of the invention comprises an application running on the processor in the mobile device with which the measuring device in the biosensor system communicates over any communication protocol, and in addition to this, in the measuring device in biosensor system, it comprises

• a disposable strip (1 ) configured to contain the immobilized phenylalanine dehydrogenase enzyme that will react with phenylalanine on it and containing the electrodes holding the electrons released as a result of the reaction of the immobilized phenylalanine dehydrogenase and the phenylalanine in the patient's blood, strip inlet (7) for insertion of the strip (1 ) into the measuring device in the biosensor system, connector located on the motherboard, enabling the interaction of the strip (1 ) and the measuring device in the biosensor system,

• voltage amplifiers located on the motherboard, to be able to read the low- level current formed by the reaction of the enzyme to be immobilized on the strip (1 ) and the phenylalanine in the patient's blood,

• transimpedance amplifier located on the motherboard, converting current to voltage,

• analogue to digital (A/D) converter located on the motherboard, converting voltage to digital information,

• microcontroller located on the motherboard, processing digital information,

• processor located on the motherboard,

• LCD driver located on the motherboard,

• LCD display (2) on the measuring device for reading data.

There is also a Bluetooth button (3), forward button (4), on/off button (5) and back button (6) on the measuring device for communication/pairing of the measuring device with the mobile device. The back button (6) is used to show the measurement results made earlier, and the forward button (4) is used to show the measurement results made later. In addition, all daily/monthly/yearly results can be seen regularly in the mobile application. The on/off button (5) enables the device to be switched on and off. The test strip (1 ) containing the immobilized phenylalanine dehydrogenase enzyme and electrodes is manufactured from polyvinyl chloride (PVC) or a plastic-like material.

The A/D converter in the measuring device in the biosensor system described in the invention converts the conditional analogue signals into a digital data stream so that the data acquisition system can process them for display, storage, and analysis. The A/D converter takes an analogue signal and converts it to a digital field. A Digital-to- Analogue Converter is used to convert digital information to analogue signals. According to the digital information applied to its inputs, a voltage is seen at its output. A transimpedance amplifier is a circuit element that produces voltage when a current is applied. The central processor (CPU) can be briefly expressed as the part of computers that processes data and performs software commands. The integrated circuit chip containing the CPU is the microcontroller (MCU). It can be defined as MCU=CPU+ Peripherals (like ADC) + Memory. A converter (transducer) is a device that converts different types of sensed energy into an appropriate electrical signal. The LCD Driver ensures that the LCD screen is compatible with the operating system so that it can do its work stably and properly.

The general working principle of the measuring device in the biosensor system that is the subject of the invention is as follows: The enzyme, which is immobilized with the patient's blood dripped on the strip (1 ), enters into a chemical reaction, the electrode captures the electrons released as a result of the reaction, and then converts the electrons into current according to the converter Cotrell equation. Then, the measured current is increased to values that can be read by the voltage amplifiers in the measuring device in the biosensor system via the connector, and the transimpedance amplifier converts the current into voltage, which is an analogue signal. A/D converter, on the other hand, converts analogue signals, i.e., voltage, into digital information. Then, the microcontroller (MCU) processes the digital information and transmits it to the LCD driver. The processed information is displayed on the LCD screen via the LCD driver.

The measurement and recommendation method of the measuring device in the biosensor system that is the subject of the invention running on the processor comprises the process steps of:

• starting the measuring device in the biosensor system to measure the phenylalanine value before the meal and the enzyme to be immobilized and the patient's blood dripped on the strip (1 ) chemically reacting,

• the electrode on the strip (1 ) capturing the electrons released as a result of the reaction and then the converter converting the electrons into current according to the Cotrell equation, increasing the measured current to values that can be read by the voltage amplifiers in the measuring device in the biosensor system via the connector, • transimpedance amplifier converting current to voltage,

• A/D converter converting voltage to digital information,

• as a result of the microcontroller (MCU) processing the digital information, transmitting it to the LCD driver and displaying the processed information on the LCD screen, obtaining the pre-meal phenylalanine value measured and processed in the measuring device in the biosensor system,

• Sending data to the mobile device according to the phenylalanine measurement value obtained and making dietary recommendations by calculating phenylalanine on the processor according to the data received by the mobile device.

The step of making a diet recommendation by calculating phenylalanine on the processor according to the data received by the mobile device mentioned above further comprises the process steps of:

• the user selecting the type of food to be eaten and inputting the weight of the food to be eaten via the mobile device application interface,

• Calculating, comparing, and displaying the safe phenylalanine value range for the patient, which should be 21 -137 umol/L for under 18 years old, and 35-85 umol/L for 18 years old and over, by using the phenylalanine value measured with the measuring device by the mobile device processor according to the selected food type and the value entered, and displaying it on the mobile device application interface,

• multiplying the phenylalanine value of the type of food to be eaten and the basis weight of the food to be eaten by the processor in the mobile device,

• summing the multiplication of the phenylalanine value of the type of food to be eaten and the basis weight of the food to be eaten with the phenylalanine value measured before the meal by the processor in the mobile device, • displaying a “you can safely eat” warning on the interface of the mobile device application in the case that the aforementioned total value is in the safe phenylalanine value range for the patient, which should be 21 -137 umol/L for under 18 years old, and 35-85 umol/L for 18 years old and over, or less than this range, or displaying a "please rearrange your diet" warning on the interface of the mobile device application in the case that this total value is in the safe phenylalanine value range for the patient, which should be 21 -137 umol/L for under 18 years old, and 35-85 umol/L for 18 years old and over, or more than this range.

The safe phenylalanine value range mentioned here varies according to age groups, and it is 21 -137 umol/L for under 18 years old and 35-85 umol/L for 18 years and older. Comparisons are made according to this value range.

In order for the test strips (1 ) not to deteriorate until they reach the patients, the phenylalanine dehydrogenase enzyme, which will react with the phenylalanine on the strip (1 ), is fixed on the strip (1 ) by immobilization methods. After the patient's blood is dripped onto said strip (1 ), it is inserted into the measuring device in the biosensor system through the strip inlet (7). Electrons released as a result of the reaction of phenylalanine dehydrogenase immobilized on the disposable test strip (1 ) and the phenylalanine in the patient's blood dripped onto the strip (1 ) are captured by the electrodes. Phenylalanine in the patient's blood and the immobilized enzyme phenylalanine dehydrogenase on the test strip enters into a chemical reaction according to the following Reaction 1 .

L-Phenylalanine + H2O + NAD⁺ — Phenylpyruvate + NH3+ NADH + H⁺

Reaction 1

The H⁺ produced by this reaction is measured. The flow of electrons corresponds to the flow of current through the working electrode and reference electrode. These electrons, i.e. , the charge passing through the electrode, are measured with the device that is the subject of the invention and this measured charge correlates with the phenylalanine level in the blood. The measured load is calculated in terms of current with the Cottrell equation and made readable on the device of the invention, which includes a transimpedance amplifier (current-voltage converter) and an analogue-to- digital converter (ADC).

Cotrell Equation is shown as

wherein; i: current to be measured, n: number of electrons transferred, F: Faraday's constant (96,487 C per equation), A: electrode area, Co: analyte concentration, D: diffusion constant, t: time passed after potential is applied. The Cotrell equation is an equation used to measure the level of phenylalanine in the blood and to calculate the level of phenylalanine in a mobile device.

The measured current is increased to values that can be read by the voltage amplifiers in the measuring device in the biosensor system via the connector.

On the LCD screen, the phenylalanine level in the patient's blood can be seen quantitatively.

By pressing the Bluetooth button (3) on the device that is the subject of the invention, the device becomes able to pair with mobile devices such as telephones. The mobile device mentioned here can be a phone, computer, or tablet. The measuring device in the biosensor system that is the subject of the invention paired with the mobile device, provides many possibilities together with the application written for the mobile device it is paired with. All measurement results are recorded with date and time on the mobile application, along with the notes (such as before I after the meal) that the patient will write next to them. In the mobile application database, there are both the phenylalanine levels in foods and the patient's measurement results in the form of date I time as the patient enters his own measurement results. The database consists of the results from the measuring device in the biosensor system and the mobile application and is stored on the servers. The relevant database is hosted on server management platforms.

When the patient using the device that is the subject of the invention inputs the food and its amount that s/he intends to eat into the mobile device before the meal, the phenylalanine level of the meal content is approximately calculated with the mobile device paired with the measuring device in the biosensor system that is the subject of the invention, so that the patient's diet is regulated. In the application installed in the mobile device, there are phenylalanine amounts of all consumed foods. Considering the diet the phenylketonuria patients follow, the variety of foods consumed is decreases slightly. If the measurement to be made by the patient after the meal is input into the application in the mobile device, the software is shaped individually with the machine learning over time, and when the patient eats the food, the phenylalanine level value in the patient's blood after the meal is calculated more accurately.

For example, if the post-meal phenylalanine value was estimated as 0.3 mg/dl when the patient ate an apple, but this value was found as 0.5 mg/dl after the measurement, the device will report the phenylalanine level as 0.5 mg/dl after eating an apple in its next estimate. By this way, the invention offers both measurement and dietary advice, in other words, the treatment.

The phenylalanine value range that should be found in the blood varies according to age groups. The patient will add age information when entering the application written for mobile devices. The patient will measure the phenylalanine value before the meal, this measurement result will be transferred to the application in the mobile device paired over the Bluetooth, after the measurement result is transferred, the patient will write a note such as "my pre-meal measurement" next to the measurement result in the application.

Then, when the patient inputs the food and its amount (for example, 200 grams of rice dish) to be eaten at the meal, the phenylalanine value in the food content will be calculated in the application (for example, “contains approximately 0.02 mg/dl phenylalanine”) and guidance will be made on the application, taking into account the previous phenylalanine level of the patient (e.g.: “Estimated post-meal phenylalanine level with the entered food: 0.3 mg/dl - Safe range” or “Estimated post-meal phenylalanine level with the entered food: 1.5 mg/dl- Out-of-range value! Make nutritional adjustments”). As the patient inputs data, the margin of error of the measurement results will gradually decrease and a completely patient-specific device will be provided. These measurements are made with a mobile application that is compatible with the measuring device in the biosensor system and is programmed for the mobile device.

The purpose of the mobile application is to enable the patient to be able to monitor the phenylalanine values before and after the meal as well as to track the phenylalanine values, and to determine the estimated phenylalanine level after the meal if the meal content is selected. It is the creation of a personalized device that can make more accurate “post-meal phenylalanine value estimation” with machine learning by adding the post-meal phenylalanine level to the post-meal phenylalanine estimate s/he chose.

The phenylalanine value contained in each gram of food is readily available in the mobile application database. With the patient's choice of basis weight, the phenylalanine value in 1 gram of food is multiplied by the gram and the phenylalanine value that s/he will receive from that meal is calculated. The “estimated post-meal phenylalanine value” is calculated by adding the measurement made by the patient before the meal (the phenylalanine value in his blood) and the phenylalanine value he will get from the meal. If this value is within the range of phenylalanine that should be in the blood the patient is provided with a warning of “estimated phenylalanine value after meal, you can eat with confidence”, and if this value is out of range, the patient is provided with a warning of “estimated phenylalanine value after meal, value out of safe range, please rearrange your diet”, and the patient is provided to rearrange his diet.

As known, every person's metabolism is different. The mobile application compatible with the measuring device in the biosensor system that is the subject of the invention and developed with machine learning in order to be able to calculate more accurate post-meal phenylalanine (FA) value every day makes an inference by comparing the prediction it makes with the patient's result and makes predictions for it in the next measurement. For example: The current patient's FA level was measured as 0.5 mg/dl, the patient input to the application that s/he will eat 100 gr apple, the FA level to be taken from 100 gr apples is calculated as 0.2 mg/dl, and after receiving the warning "estimated FA level after meal = 0.7mg/dl, you can eat your meal within safe ranges", s/he consumed 100 gr apples, and measured the post-measurement value as 1 mg/dl from the measuring device in the biosensor system. If the patient inputs this result in the part where he inputs the meal calculation in the mobile application, the FA level that will be obtained as a result of the next 100 g apple eating of this patient will now be stated as 1 mg/dl, not 0.7mg/dl (the values given are random). Therefore, a device specific to the patient will be provided.

Claims

1. A biosensor system in which blood is used directly without any pre-treatment, makes quantitative measurement of phenylalanine from the blood by electrochemical analysis and provides a dietary recommendation, comprising a measuring device that makes the quantitative measurement of phenylalanine, a mobile device that communicates/pairs with the measuring device over any communication protocol, and an application running on the processor in the mobile device, wherein said measuring device comprises

• a disposable strip (1 ) configured to contain the immobilized phenylalanine dehydrogenase enzyme that will react with phenylalanine on it and containing the electrodes holding the electrons released as a result of the reaction of the immobilized phenylalanine dehydrogenase and the phenylalanine in the patient's blood,

• strip inlet (7) for insertion of the strip (1 ) into the measuring device in the biosensor system,

• connector located on the motherboard, enabling the interaction of the strip (1 ) and the measuring device in the biosensor system,

• voltage amplifiers located on the motherboard, to read the low-level current formed by the reaction of the enzyme to be immobilized on the strip (1 ) and the phenylalanine in the patient's blood,

• microcontroller located on the motherboard, processing digital information,

• processor located on the motherboard,

• LCD driver located on the motherboard,

• LCD display (2) on the measuring device for reading data.

2. A biosensor system according to Claim 1 , wherein said mobile device is a telephone, computer, or tablet.

3. The measurement and recommendation method of the measuring device in a biosensor system according to claim 1 running on the processor, comprising the process steps of:

• the electrode on the strip (1 ) capturing the electrons released as a result of the reaction and then the converter converting the electrons into current according to the Cotrell equation,

• increasing the measured current to values that can be read by the voltage amplifiers in the measuring device in the biosensor system via the connector,

• transimpedance amplifier converting current to voltage,

• A/D converter converting voltage to digital information,

4. A method according to claim 3, comprising, in the step of making dietary recommendations by calculating phenylalanine on the processor according to the data received by the mobile device, the process steps of:

• calculating, comparing and displaying the safe phenylalanine value range for the patient, which should be 21 -137 umol/L for under 18 years old, and 35-85 umol/L for 18 years old and over, by using the phenylalanine value measured with the measuring device by the mobile device processor according to the selected food type and the value entered, and displaying it on the mobile device application interface, multiplying the phenylalanine value of the type of food to be eaten and the basis weight of the food to be eaten by the processor in the mobile device, summing the multiplication of the phenylalanine value of the type of food to be eaten and the basis weight of the food to be eaten with the phenylalanine value measured before the meal by the processor in the mobile device, displaying a “you can safely eat” warning on the interface of the mobile device application in the case that the aforementioned total value is in the safe phenylalanine value range for the patient, which should be 21 -137 umol/L for under 18 years old, and 35-85 umol/L for 18 years old and over, or less than this range, or displaying a "please rearrange your diet" warning on the interface of the mobile device application in the case that this total value is in the safe phenylalanine value range for the patient, which should be 21 -137 umol/L for under 18 years old, and 35-85 umol/L for 18 years old and over, or more than this range.

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