EP3977097A1

EP3977097A1 - Compact device for non-invasive measurement of markers in physiological fluids

Info

Publication number: EP3977097A1
Application number: EP19742649.7A
Authority: EP
Inventors: Viktor Tomasov; Oleg ADAMCHUK
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-05-29
Filing date: 2019-05-29
Publication date: 2022-04-06
Also published as: WO2020240258A1

Abstract

A compact device for non-invasive measurement of markers in physiological fluids of a mammal including human is disclosed. The device comprises an optical module comprising a source which is emitting electromagnetic radiation for exitating fluorescent substance; a sensor module at the output of optical module for receiving electromagnetic radiation from optical module; a processor for data processing; a controller; display and an interface. The fluorescent substance is in a capillary, the capillary is inserted into removable strip and the removable strip is inserted into the optical module. The sensor module comprises compact photon counter equipped with thermal compensation circuit and active avalanche quenching system. The optical module further comprises electromagnetic waves trap for absorbing of unnecessary electromagnetic radiation in the optical module.

Description

Title of Invention:

COMPACT DEVICE FOR NON-INVASIVE MEASUREMENT OF MARKERS IN

PHYSIOLOGICAL FLUIDS

[0001]

Technical field

The present invention provides a device for the non-invasive monitoring and measuring of different metabolite levels in body fluids of mammals, including human.

[0002]

Background Art

The efficient testing of low molecular weight markers in body fluids is one of the most complex and difficult tasks in modern medicine. This task is mainly carried out using expensive laboratory testing requiring specific preparation of the test sample.

The quality of the diagnosis depends on the accuracy of the measurements. At present, many laboratory methods are in use utilizing complex equipment and multi-stage pre-treatment of the test samples. These kinds of analyses require trained personnel and expensive test equipment. This severely limits the number of analyses carried out which reduces the quality of the diagnosis. In the case of certain diseases, it is necessary to continuously monitor the patient’s condition through the analysis of the levels of specific markers. Most often, such analyses use blood samples, which is associated with painful sampling procedures. The most common of such procedures is the continuous monitoring of blood sugar levels in patients suffering from or predisposed to diabetes.

Blood sugar level (glycaemia) is one of the diagnostic markers indicative of a person’s general health. The number of people suffering from blood sugar disorders is increasing every year and specifically more younger people suffering from blood sugar disorders. Continuous measurement and control of the body’s response and fluctuations in blood sugar levels in response to the amount of sugar found in food is vital both at home and away to provide timely diagnostics and medical assistance for glycaemia. People with diabetes are required to take blood sugar monitoring multiple times per day. The main method used for express testing in domestic conditions is the measurement of blood sugar levels in capillary blood from a finger puncture. This is an invasive, and usually painful, procedure. Finger punctures are extremely inconvenient for the patient and can take several days to heal. As a result, continuous blood sugar testing becomes an inconvenient and even hazardous procedure. This problem can be resolved through non-invasive testing methods using readily available body fluids, such as saliva, tears or urine. The simplest tests can be performed using saliva. No complex equipment is required for such tests and they can be quickly performed in any circumstances.

Blood sugar measurement based on saliva glucose levels has been widely tested in the laboratories of various medical clinics and institutions around the world. As a result, a correlation has been established between the levels of sugar in blood and saliva, enabling the use of saliva as an indicator of blood sugar levels.

A number of different methods exist for the measurement of glucose levels in fluids using spectrometry and amperometry. All of these methods utilize a specific property of the test fluid, as well as multiple reactions for inducing the required property in one of the components of the test solution.

It must be noted here that spectrometric and fluorometric evaluation methods based on recording changes in the spectral characteristics of the analyzed substance in relation to the concentration of the test compound have seen broad-scale development in recent years.

A number of versions of spectrometric gadgets currently exist for various purposes. The most compact glucometers are semi-portable in size. A reduction in the size of the device usually results in a decrease in sensitivity or stability. To perform a test“on the go”, such readers usually require extreme precision in operation and a trained user. At the same time, home testing using the same method can be extremely difficult. These problems result from the direct transfer of optics into a smaller gadget without any modifications made to account for changes in the conditions of their use.

W02006/044973 discloses a device for sensing the concentration of a target analyte in a sample with a sensing element attached to an optical conduit. The sensing element comprises a reporter group that undergoes a luminescence change with changing analyte concentrations.

US2008/0101986 describes an analytical test strip for determining different markers in bodily fluids. The strip comprises a substrate layer with an electroluminescent module for generating a signal.

EP2989975 discloses a system for determining glucose levels comprising reference and excitation light sources. The system involves the excitation of a chemical indicator, the intensity of which is related to blood glucose levels. The invention also pertains to an optical measurement system comprising a fiber optic sensor with a corresponding module and system for processing the data.

US2013/0060107 describes a glucose sensor for detecting glucose levels in subcutaneous tissue. The device measures the viability of fluorophores in the presence of various glucose concentrations. A non-consumptive optical sensor is proposed for fluorescence immobilized on a substrate in hydrogel. Also provided is an inexpensive device employing widely-used components, such as light-emitting diodes, photodiode detectors, phase fluorometry, etc. US2017/0215775 discloses a system for the continuous monitoring of glucose levels. The system comprises a hand-held monitor with a display, an external transmitter, an insulin pump, and a glucose sensor. The glucose sensor comprises an optical glucose sensor and an electrochemical glucose sensor.

US2018/0070866 discloses a device with ultra-low power consumption and a non-invasive senor system for measuring blood analytes in vivo comprising several sensors. The device measures the blood hydrogen peroxide levels, pH, and/or glucose levels (as well as other analytes) in body secretions (e.g. tears, saliva, sweat). The device comprises a number of chemoreceptor sensors, a microprocessor, a signal amplifier, signal filters, error correction algorithms, an analog-digital converter, and a wireless unit for transmitting electromagnetic data to a remote device for processing and/or storage (e.g. on a server or a cloud-based storage system) and/or visual presentation using software. The method involves applying a sensor matrix to the skin, with the resulting electrical impulses corresponding to the glucose levels of fluids, such as tears, saliva, blood, etc. US8385998 provides contact lens with integrated biosensor for the continuous, non-invasive monitoring of physiological glucose by employing biocompatible nanostructure-laden lens materials. These contact lenses can be worn by diabetics who can colorimetrically see changes in their contact lens color or other fluorescence-based properties, giving an indication of tear and blood glucose levels. This invention for the glucose biosensor based on the new disposal contact lens provides a safe, convenient and non-expensive glucose sensing device. The sensing device disclosed provides an efficient and non-invasive solution for monitoring blood glucose.

Another device is described in US20120177576. The invention is directed to an optical device comprising a contact lens having a glucose-sensing optical pattern imprinted, marked, coated or otherwise disposed on or incorporated within the contact lens. The indicator pattern is further comprised of a glucose-sensing coating containing a boronic acid derivative, which reacts in the presence of glucose to create a readable pattern, which can then be correlated to a pre-determined or pre-calibrated blood glucose level.

A large number of different transdermal systems, devices and optical analysis methods for monitoring glucose levels have been developed over the past decade. These systems can be used without collecting samples and can potentially be used continuous monitoring day after day.

The device disclosed in W02001079818 uses attenuated total reflection (ATR) infrared spectroscopy. Preferably, the device is used on a fingertip or another body part and compares two specific regions of a measured infrared spectrum to determine the blood glucose level of the user. The device and procedure may also be used for other materials which exhibit unique mid-IR signatures of the type described herein and that are found in appropriate regions of the outer skin.

The invention disclosed in US6574490 relates to a quantitative near-infrared spectroscopy system, incorporating multiple subsystems in combination, providing precision and accuracy to measure analytes, such as glucose at clinically relevant levels in human tissue. The invention overcomes the challenges posed by the spectral characteristics of tissue by incorporating a design which includes, in preferred embodiments, six highly optimized subsystems. [0003]

Summary of the invention

The persent invention provides a device with improved characteristics enabling the measurement of the levels of various markers in physiological fluids. High measurement sensitivity enables the analysis of samples containing extremely low amounts of the analyzed marker. The ease of use and small dimensions of the device built based on the claimed invention enable monitoring the patient’s condition at home using non-invasive physiological fluids (saliva, tears, sweat, urine). This result is achieved through the utilization of a new detection system and the position of the subsystems of the device. One of the key innovations is related to a specially-designed compact photon counter, the method of positioning the photon counter in the device, and the algorithm for the operation of the device. The use of the optical design described here eliminates the need for additional optical elements that would significantly contribute to measurement errors.

The invention disclosed herein possesses a number of advantages over the currently known devices. The hand-held dimensions and ease of use of the device enable using the device of the invention as a home testing device or a portable unit for field use. The positioning methods and special technical solutions used in the electronic and optical design of the device enable reducing the dimensions of the device while still retaining high sensitivity. The device has no moving parts, making it resistant to mechanical influences and temperature effects. The novel design of the optical subsystem eliminates strict requirements for the alignment of the sensor in relation to the emission of the beam of light (12). The emission and signal detection systems are resistant to temperature effects and mechanical influences, including mechanical shocks. The algorithm of operation and the design solutions employed allow achieving very low power consumption and resistance to fluctuations in supply voltage and the draining of batteries. The algorithm includes automatic adjustment of the device to changes in operating conditions. The high level of stability in the operation of the device, in turn, ensures the high repeatability of the measurements. Deviations in repeated measurements performed using the device amount to no more than 5%. The high sensitivity and broad spectrum of measured concentrations along with low measurement errors make it possible to measure extremely low concentrations of markers in various physiological fluids.

[0004]

Brief description of the drawings

[0005] [Figure 1] Block-scheme of signal processing.

[0006] [Figure 2]. Detailed scheme of device with APD type of SPC and T sensor on SPC crystall.

[0007] [Figure 3]. Detailed scheme of device with APD type of SPC and T sensor on SPC on box.

[0008] [Figure 4] . Detailed scheme of device with PMT type of SPC.

[0009] [Figure 5]. Design of optical module with standing apart focusing lens for APD type of SPC.

[0010] [Figure 6]. Design of optical module with focusing lens on filter for APD type of SPC.

[0011] [Figure 7]. Design of optical module for PMT type of SPC.

[0012] [Figure 8]. Design of strip.

[0013] [Figure 9] . General view and size of the deivce.

[0014] [Figure 10]. Spectrums of excitation (A) and emission (B) of fluorophore in glucose solution.

[0015] [Figure 11]. Calibration plot of signal detection and glucose concentration in solution.

[0016]

Description of Embodiments

The device operates on the principle of excitation and registration of fluorescent radiation from a fluorescent dye bound to the marker. The core technology underlying the device is presented in Fig. 1. The device comprises a number of units: an optical module (101) with a chemical receptor, a sensor module (102), a data processor and controller (2), and an output module (1), e.g., a screen or a printer.

The optical module (101) generates the fluorescence signal which is transmitted to the sensor module (102). In the sensor module, the signal is converted into the form of an electrical signal and this information transmitted to the data processor (2). Marker levels are calculated in the data processor (2) based on formulas saved in the data processor (2) and calibration. The controller is combined with the data processor (2) and it monitors the operating mode of the subsystems of the device. The resulting marker levels are displayed on the display (1) or any other output device.

Sensor module

The sensor module comprises a detector device comprising Single Photon Counters (SPCs), pulse shaper (3), and tuning systems (5, 8, 7, 6, 21). The deviation of the geometric center of the SPC sensors from the center of the body of the device does not exceed 30 pm, eliminating the need for additional alignment and focusing of the optical system. The detector is located inside a standard TO-46 package with a transparent window cap (a cylinder with a diameter of 4.7 mm and height of 4 mm).

Different types of Single Photon Counters (SPCs) based on two types of sensors - Avalanche Photodiode (APD) (4) and Photomultiplier Tube (PMT) (22) - can be employed in the device of the invention. The configuration of the sensor module depends on the type of sensor used in the SPC, i.e. either APD or PMT.

The operation of the APD (4) and PMT (22) is based on the principle of cascading generation of an electron avalanche upon a single photon hitting the window of the sensor, creating an electric pulse at the output of the SPC sensor. The electric pulse is an analog signal, which is transformed by the pulse shaper (3) into a transistor-transistor logic (TTL) pulse corresponding to the digital format consisting of a logical zero (0) or a logical one (1). The TTL pulse is received by the logic integration socket of the data processor (2). Low logical voltage or a logical zero is registered in the absence of or at a low voltage, such as in the case of the absence of a photon. High logical voltage or a logical one is registered at a sufficient voltage level resulting from the collision of a photon with the detector. The TTL pulse is transmitted to the data processor (2), where it is received as a logical signal, i.e. 0 or 1.

The duration of the avalanche in an APD sensor is relatively long, resulting in a long delay until the detection of the next photon, or dead time. Active Avalanche Quenching System (AAQS) technology (5) is employed to accelerate the operation of the sensor to avoid the loss of any photons. AAQS (5) stops the avalanche immediately after it has been registered by the pulse shaper (3). The operation of the AAQS unit (5) is initiated by the front line of the TTL pulse. Decreasing the reaction time of the APD increases the dynamic range of the photon counter (the rate of counting the number of avalanches caused by the collision of photons with the detector).

The APD detector (4) operates at the base voltage Vbias, which takes the diode to a state close to inducing a spontaneous avalanche. Upon hitting the detector, energetically saturated photons knock out excited electrons (by adding a portion of energy); these, in turn, knock out further electrons from the following layers of the diode. The APD (4) is temperature- dependent. Optimal operating conditions are controlled by the T compensator (6) that receives temperature data from the T sensor (21 or 8) and adjusts the voltage level Vbias (7). The T sensor (8) can be located on the sensor on the chip and record the temperature of the detector itself as shown in Fig. 2. At the same time, some detector designs are manufactured without internal temperature sensors or lack the possibility of installing the sensor on the chip. In such case, we foresee that the temperature sensor can be located on the body of the detector, as shown in Fig. 3.

The exact correlation between Vbias and temperature is recorded by the T compensator (6) in accordance with the specific temperature measurement method used, i.e. either on the chip (8) or on the body (21).

The SPC unit can also employ a Photomultiplier Tube (PMT) detector (22). The PMT does not require auxiliary voltage Vbias and is only weakly dependent on fluctuations in temperature. Accordingly, no Vbias (7) adjustment, temperature sensor (8, 21) or T compensator (6) is required. Fig. 4 is a version of the invention utilizing PMT.

Data Processor and Controller

The functions of processing data and synchronization of the SPC (22, 4) with the light source (16) are combined in the data processor and controller subsystem (2).

Power to the light source (16) is provided through the light source power driver (15), which receives activation and deactivation commands from the controller subsystem (2). The light source ( 16) is a LED-type light source that goes into stable intensity illumination made a few seconds after activation. In order to prevent the registration of signals in unstable LED mode, the SPC (22, 4) is activated only after the light source (16) has been activated and stabilized. Photon counting at the data processor (2) is commenced with the activation of the SPC. Photon registration time is established in accordance with the marker analysis method and is set by the data processor (2), e.g. 10 seconds. The SPC and light source are deactivated simultaneously; upon their deactivation, the data processor stops recording signals from the SPC and initiates their processing. The sufficiency of signal statistics for calculating the concentration, as well as the optimal power consumption of the device is taken into account in establishing operating time.

At the end of the registration period, the obtained data are processed and converted into marker concentration on the basis of calibration functions.

In a calibrated volume of solution, the number of reemitted photons per unit of time is proportional to the number of glucose molecules present in the solution. The concentration of the detected marker (C) in the physiological fluid is determined by the formula:

C=(N :niint~ base) ' K

The sensor also registers the general background photon flow in the detector and has its own dark count generated by internal processes inside the sensor. These effects are persistent in character, are recorded as the base value of the sensor ( Nbase ) and taken into account in calculations of marker concentrations. The difference between the number of photons recorded on the sensor ( N count ) and the base value of the sensor (Nbase) is multiplied by the calibration coefficient ( K ). The calibration coefficient (K) correlates the number of photons hitting the sensor with the concentration of the marker in the analyzed volume. The coefficient is determined based on calibration tests carried out using a known concentration of the analyzed marker.

Display

The result obtained is displayed on the display (1) quantitatively as the concentration of the marker per unit of volume of the sample and/or qualitatively as an indication of the level of the marker on a colored scale from green (normal) to red (dangerous). Chemical Components

The capillary (18) contains a chemical substance capable of forming complexes with the analyzed substance, also known as a chemical receptor. This chemical receptor is selective for the marker and exhibits fluorophore properties only in the form of a complex with the marker. In the absence of the marker, the intensity of the fluorescence is either close to zero or the spectrum of the fluorescence is located in a band distant from the detection interval. All components of the sensor and the optical module are configured to excite and detect a specific spectrum of fluorescence corresponding to the specific marker and chemical receptor complex. A specific chemical receptor is used and the subsystems of the device specifically configured for each new task.

The chemical receptor is loaded onto the capillary strip in a dry form. A liquid sample (19) fed into the capillary (18) dissolves the chemical receptor and reacts with the receptor to form a complex. The light source (16) excites the fluorescence of the complex (20), transmitting fluorescent radiation (12) to the sensor.

Optical module

The optical module involves the excitement of the chemical receptor-marker complex and the transmission of the signal received to the sensor module with additional processing of the data.

Light source (16) is a LED-type light source which is selected based on the function of the device and emits a specific spectrum of light with high intensity (10). The source of light (11) generates a beam of light exciting the fluorophore found in the capillary (18). The LED emits photons with a wavelength of Ex in stable mode of the LED. The intensity of emission is stabilized by eliminating the possibility of overheating in the LED and fluctuations in the efficiency of emission. The absence of overheating problems eliminates the need for a high- power cooling system for the LED and reduces the power consumption of the entire emission module. The diaphragm (40) contracts the emitted beam (10) to correspond to the dimensions of the capillary (18). The sample located in the capillary is illuminated at an angle of 90°±5° in relation to the optical axis of the sensor. The diameter of the beam is determined by the dimensions of the optical channel, 1.5 mm. The optical channel abuts the capillary containing the test sample.

The material used for the capillary (18, 27) is chosen based on the emission parameters of the light source and the fluorescent signal. The material should not produce a noisy spectrum and absorb the useful signal, i.e. it must be spectrally transparent. Mechanical and thermal stability are also important. Glass, silicone, or plastic capillaries can be used. The test sample is located in an optically transparent capillary. The spot of illumination of the sample is located at the intersection of the optical axis of the excitation beam and the optical axis of the sensor in the middle of the capillary strip. The excitation beam (10) hits the specific complex (20) of the chemical receptor and the analyzed marker in the capillary (18) and is re-emitted with a shifted wavelength as fluorescent radiation (12).

Internal reflections from the capillary walls and redirection of the excitation beam to the sensor may occur as the beam passes through the capillary. The purpose of the light trap (17) is to reduce this kind of noise caused by the excitation radiation by allowing a portion (11) of the excitation radiation to pass through the capillary and then absorbing this radiation. The light trap (17) may be covered in an absorbing material. A portion of the useful signal can also fall onto the light trap, but the reduction in the noise effect significantly improves the characteristics of the fluorescent beam (12).

The emission light passes through the collimator (41), then through a narrow-band optical filter (9) with a central wavelength of transmission equal to Em, and then to the sensor.

Different methods for generating the beam are used depending on the type of the sensor. An APD (4) sensor has a small surface area which necessitates focusing the beam on the sensor. The configuration with a focusing lens (42) between the filter (9) and the sensor (4) shown in Fig. 5 is used for this purpose. As the filtered parallel beam (13) hits the lens (42), it is focused on the APD sensor (4). Another variant of this configuration has the lens (42) lens installed on the filter (9) itself as shown in Fig. 6, allowing the distance between the filter and the sensor to be reduced. The focused and filtered beam (14) is then directed to the APD sensor (4). The use of a focusing lens is not required in the case of a PMT sensor (22), as the surface area of the sensor is bigger than the light spot. Fig. 7 depicts an optical module using a PMT sensor.

Strip design

The sample is applied to a special strip (23) connected to the capillary (18, 27).

The internal walls of the strip are covered in a dry form of the fluorophore. The fluorophore is a special sensor substance that is maximally selective for the specific marker. The fluorophore in the capillary is dissolved by the liquid test sample and reacts with the marker, which changes its spectrometric characteristics. The fluorophore is excited by Ex and emits radiation at the characteristic wavelength Em. This radiation is captured by the sensor.

A schematic diagram of the strip is shown in Fig. 8. The base of the strip is made from materials efficient at absorbing photons, such as black polypropylene or polyoxymethylene (POM). The main requirement for the material used for the capillary is to have a minimal level of absorption in the 350-700 nm range. A round (28) or square (24) channel is cut into the strip material for affixing the capillaries (18, 27). The form and dimensions of the capillary are chosen in accordance with the requirements of the method and the necessity of the calibration of the internal diameter and constant wall thickness. Calibrating the internal diameter of the capillary and the diameter of the beam Ex ensures that the volume of the analyzed solution is fixed.

An optical channel (25) for excitation radiation (10) from the light source and an optical channel (26) for transmitting fluorescent radiation (12) to the sensor are cut inside the strip. A light trap (17) is cut out on the opposite side of the optical channel (25) to absorb excitation radiation passing through the capillary (11). This leads to a significant reduction in internal reflections of the excitation beam (10) inside the capillary and in the penetration of light into the sensor’s optical channel (12) where it would then hit the sensor.

General view and size of the device

Implemented inside a single device, the innovations listed above result in a significant reduction in the dimensions of the entire device. All the components and subsystems of the device fit inside a modest package (32). Fig. 9 shows the relative size of the device placed in the palm of a person’s hand. Control buttons are located on the front panel of the device: an ON/OFF button (31) for activating and deactivating the device and a START button (30) for initiating measurements. The buttons can be illuminated. The strip (23) is removably inserted into an opening on the side of the case. The display (1) is located on the top of the case.

[0017]

Example

Non-invasive measurements of glucose concentration in saliva

A direct method of glucose detection in a liquid requires performing a laboratory test. One solution to this problem is to use a substance which can bind to glucose in order to perform a spectrometric analysis. In this test, we used Glucose Sensitive Fluorophore (GSF) (20), which displays fluorescent properties only when bound to glucose in liquids. GSF molecules selectively bind to glucose molecules, creating a stable structure. GSF forms weaker bonds with other types of sugar (such as fructose, maltose, mannose, etc.). The relative concentration of such structures is ten times smaller than in the case of glucose and, as such, does not affect the test results.

After the chemical reaction, the complex molecule formed by GSF and glucose gains the ability to absorb photons with a specific wavelength and reemit these with another definite wavelength. The individual molecules of GSF and glucose lack these qualities prior to the chemical reaction.

The technology is based on a special substance, fluorophore, the molecules of which selectively bind to the glucose molecule. Prior to dissolution in physiological fluids, fluorophore is in a dry form and can remain stable for long periods of time under normal conditions. After the formation of the fluorophore-glucose complex, it acquires the ability to absorb photons with the wavelength Ex = 377nm and to re-emit them with another, significantly changed wavelength Em = 427 nm. The individual molecules of GSF and glucose lack these qualities prior to the chemical reaction. In a calibrated volume of solution, the number of photons re-emitted per unit of time is strictly correlated to the number of glucose molecules found in the solution. Fluorophore synthesis

The compound 9, 10-Bis[[N-methyl-N-(o-boronobenzyl)amino]methyl]anthracene, characterized by fluorescent properties when bound to glucose in solutions, was synthesized for the measurements. The synthesis was carried out in accordance with the procedure reported in Eggert, et ah, 1999 (Eggert, H., Frederiksen, J., Morin, C., Norrild, J.C. (1999). A New Glucose-Selective Fluorescent Bisboronic Acid. First Report of Strong a-Furanose Complexation in Aqueous Solution at Physiological pH. J. Org. Chem., 64 (11), pp 3846- 3852). The final yield of the product was about 77%.

The obtained substance was found to be about 90% pure by HPLC analysis. The substance was also analyzed by NMR and the spectra described in literature and measured in the laboratory coincided with each other down to hydrogen and carbon signals. An IR analysis was also carried out; the analysis showed the presence of all required bonds in the substance. The melting point of the substance was determined to be around 225 °C during the decomposition of the product.

Next, the fluorescent properties of the product upon binding to glucose were determined. One solution containing no glucose and two solutions containing different concentrations of glucose (with a 10-fold difference) were prepared for the spectrometric analysis and the synthesized sensor substance GSF added to all solutions.

The spectra measured for the synthesized sensor substance in the absence of glucose in the solution showed a signal close to the background level. This demonstrates that the fluorescent properties of GSF are only manifested in the presence of glucose in the solution. An excitation spectrum scan (excitation screen) was carried out using the solution containing 500 mM of glucose to determine the excitation band of the GSF-glucose complex in the UV range (Fig. 10 A). The excitation screen showed a maximum at the 330 nm line which corresponds to the values published in literature.

An emission spectrum scan (emission screen) was also carried out in the UV range between 380-500 nm (Fig. 10 B). The resulting spectrum showed an emission band between 400 and 460 nm. The spectrum showed a maximum of emission at the Em = 427 nm line, which completely coincided with the expected result. The difference in intensity between the two concentrations was more than 80% (highest concentration = 100%). Significant differences in fluorescence signal intensity were also observed for the two concentrations of glucose The stability of the fluorophore-GSF complex was determined in a series of experiments. No significant degradation in the signal was observed for 1 hour after the mixing of the solutions.

Measurement of glucose concentration using the device

Flurophore was dissolved in 50mM phosphate buffer, pH 7.5, containing 1 0m M of NaCl. This solution was introduced into the capillary and then dried.

The device was configured to measure glucose in the GSF complex using the following parameters: light source (16) emission at 370 nm and filter (9) with a bandwidth of 430±20nm.

Stock solutions of glucose with concentrations ranging from 10 mM to 1200 mM were prepared in a model solution imitating the composition of saliva. Zero concentration of glucose in the solution indicated the background value of the model solution.

Signal intensity demonstrated a strong dependence between the sugar content of the solution and the correlation parameter r² > 0.95 (Fig. 11). Based on the correlation graph, the minimum reliable measured concentration is about 5 mM with a measurement error of no more than 5%. The resulting correlation between the intensity of the signal and glucose concentration in standard solutions can be used for calculating the sugar content of saliva samples.

Claims

[Claim 1] [Compact device for non-invasive measurement of markers in physiological fluids of a mammal including human consists of an optical module (101) comprising a source emitting electromagnetic radiation for exitating fluorescent substance obtained by reacting of physiological fluid and chemical receptor, sensor module (102) at the output of optical module, processor for data processing, and a controller (2), display (1), interface, characterized in that the optical module (101) comprises (i) removable strip (23), where a capillary (18, 27) comprising fluorescent substance is removably inserted, (ii) a periodically operating source (16) of narrow band electromagnetic radiation for generating a beam of predetermined frequency, (iii) optics for forming of the beam, (iv) electromagnetic waves trap (17) for absorbing of unnecessary electromagnetic radiation in the optical module, the sensor module (102) comprises compact photon counter (4, 22) equipped with thermal compensation circuit and active avalanche quenching system.

[Claim 2] Device according to the claim 1, characterized in that the beam forming optics of the optical module (101) comprises first collimator (40) located between the source (16) of the electromagnetic radiation and capillary (18, 27); and the second collimator (41), bandpass filter (9) and lens (42) located between capillary (18, 27) and compact photon counter (4, 22).

[Claim 3] Device according to any one of previous claims, characterized in that the removable strip (23) has length, width and thickness, wherein the thickness is essentially smaller compared to the length and width, and the thickness is substantially equal to the diameter of the capillary (8, 27) inserted into the removable strip (23).

[Claim 4] Device according to any one of previous claims, characterized in that the electromagnetic radiation trap (17) is the inner surface of the optical module absorbing electromagnetic radiation.

[Claim 5] Device according to any one of previous claims, characterized in that the device is configured to measure the level of glucose or blood sugar content in physiological fluids of a subject, for example in saliva, wherein based on the level of glucose or blood sugar content in saliva it is calculated the blood glucose level or blood sugar content.

[Claim 6] Device according claim 5, characterized in that the fluorescent substance obtained by reacting of physiological fluid and chemical receptor absorbs electromagnetic radiation in the range of wavelengths 300-400 nm, for example at the wavelength 377 nm and emits electromagnetic radiation in the range of wavelengths 400-460 nm, for example at the wavelength 427 nm.

[Claim 7] Device according to any one of previous claims, characterized in that the amount of the fluorescent substance in the capillary (18, 27) is calibrated.

[Claim 8] Device according to any one of previous claims, characterized in that the chemical receptor (fluorophore) in the capillary (18, 27) is applied on the wall of the capillary.

[Claim 9] Device according to any one of previous claims, characterized in that the case of the removable strip (23) has an aperture for entering of the excitating radiation, an aperture for exiting of the fluorescent radiation, and an aperture for inserting the capillary.

[Claim 10] Device according to any one of previous claims, characterized in that the removable strip (23) is manufactured from a material effectively absorbing photons, for example from black polypropylene or polyoxymethylene (POM).

[Claim 11] Device according to any one of previous claims, characterized in that the width of the end of the removable strip (23) adjacent to the aperture for inserting the capillary is decreasing.

[Claim 12] Device according to any one of previous claims, characterized in that the sensor module (102) comprises pulse shaper (3) connected into the output of the single photon counter (4, 22), the output signal from the pulse shaper (3) is fed to the active avalanche quenching system (AAQS) (5) and back to the single photon counter (4, 22).

[Claim 13] Device according to any one of previous claims, characterized in that the single photon counter (4, 22) is an avalanche photodiode (APD) (4) or photomultiplier tube (PMT) (22).

[Claim 14] Device according to any one of previous claims, characterized in that the offset between geometric axis of the single photon counter (4, 22) and the geometric axis of the aperture for exiting of the fluorescent radiation of the removable strip (23) is less than 30 pm.

]