Determination of the time response of an analyte in a liquid.

Abstract

An apparatus is presented for determining one or more time response values ​​of an analyte or group of analytes (96) in a liquid (99), the apparatus comprising: an optically transmissive element including holes (6), the holes (6) being blind holes (6) extending into the optically transmissive element from respective openings (7) in the optically transmissive element, the cross-sectional dimensions of the openings (7) of the holes (6) being dimensioned to prevent larger particles or debris from entering the holes (6) while allowing the analyte or group of analytes in the liquid (99) to enter the holes (6) by diffusion; and one optically transmissive element (2) adapted to illuminate at least the holes (6) in the optically transmissive element (2). or a plurality of light sources (10) and a photodetector (20) adapted to receive light (21) emerging from the hole (6) in response to illumination (11) by the one or more light sources at each of a plurality of time points, the photodetector (20) being further adapted to generate one or more signals based on the received light, each of the one or more signals being temporally resolved and representative of at least a portion of the received light, the apparatus further comprising a data processing device comprising a processor configured to determine one or more time response values ​​based on the one or more signals.

Description

[Technical Field]

[0001] The present invention relates to an apparatus for determining a difference scale indicating the difference in concentration between two or more predetermined analytes in a liquid, and more particularly to an apparatus comprising a translucent porous element for determining a difference scale indicating the difference in concentration between two or more predetermined analytes in a liquid, as well as a corresponding method and computer program. [Background technology]

[0002] Obtaining information about an analyte in a liquid can generally be advantageous for one or more reasons. For example, knowledge of parameters associated with an analyte can provide insights into the analyte, whether known or unknown. Otherwise, in the case of a liquid containing two analytes whose concentration differences are unknown, it may allow for drawing (new) conclusions about the liquid, for example, regarding whether hemolysis occurred in vivo or in vitro, which consequently affects the interpretation of the measurement of the concentration of a particular analyte.

[0003] For specific instruments and methods, the possibility of obtaining additional parameters of an analyte, such as a difference scale indicating the difference in concentration between two or more predefined analytes in a liquid, can be particularly important if the information complements information provided separately by the instrument, especially if the additional parameters enable the identification of analytes that would otherwise be indistinguishable based on one or more parameters provided by the instrument when the additional parameters are absent. Alternatively, if the additional parameters are cumulative with respect to separately provided information, this may enable a better determination of the values ​​of the additional parameters, i.e., a determination of values ​​closer to the true values.

[0004] Therefore, improved apparatus, methods, and computer programs are needed, as well as improved apparatus, methods, and computer programs for determining difference measures that indicate the difference in concentration between two or more predetermined analytes in a liquid. [Overview of the project]

[0005] The object of the present invention is to provide improved apparatus, methods, and computer programs, and in particular, improved apparatus, methods, and computer programs for determining a difference scale that indicates the difference in concentration between two or more predetermined analytes in a liquid.

[0006] According to a first aspect, the present invention relates to a method for determining a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in a liquid, for example, in whole blood, for example, in a whole blood sample, i. A light-transmitting element containing pores, wherein the pores are non-penetrating holes extending into the light-transmitting element from each opening within the light-transmitting element, and the cross-sectional dimensions of the openings of the pores are determined to allow two or more predetermined analytes in a liquid to enter the pores by diffusion, while preventing larger particles or fragments from entering the pores. ii. One or more light sources, and iii. Photodetectors The steps to provide, The steps include bringing the pores of the light-transmitting element into contact with the liquid, The steps include illuminating at least one pore in the light-transmitting element using one or more light sources, The steps include receiving light emitted from the hole in response to illumination at each of several time points, A step of generating one or more signals based on received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light; A step of determining one or more time response values ​​based on one or more signals, The present invention provides a method comprising the step of determining a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in a liquid, based on one or more time response values.

[0007] A possible advantage of the present invention is that it makes it possible to obtain information about the difference scale, which may be useful in deriving information about whether a particular analyte is present and, optionally, at what concentration it is present (for another analyte or in absolute terms).

[0008] This could, for example, enable the identification of analytes or groups of analytes that would otherwise be indistinguishable. For instance, in the case of optically similar analytes or groups of analytes, it may be difficult or impossible to distinguish them without the present invention. However, if they differ in parameters affecting the diffusion coefficient (e.g., molecular weight, shape, and / or spread), these parameters affect the diffusion coefficient, which in turn affects or can determine the time response value. This time response value, when determined, allows for the drawing of conclusions regarding the (qualitative) presence of a particular analyte or group of analytes (such as presence at concentrations above a predetermined absolute or relative threshold), and perhaps even further, regarding a (quantitative) measure of the absolute or relative concentration of that particular analyte or group of analytes.

[0009] The present invention may further benefit from providing the possibility of obtaining a difference measure indicating a difference, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in the pores of the translucent element, since filtration is performed, or can be performed, by diffusion that does not require external energy. Another possible advantage is that, because diffusion is fast, measurements of a liquid diffused into the pores of the translucent element can be performed immediately after (or within a short time thereafter) the liquid arrives at the porous translucent element, such as immediately after the liquid is introduced into the measuring chamber equipped with the porous translucent element through the liquid inlet. Another possible advantage is that the apparatus may be simple, with few parts and no parts that need to be moved or changed in position during filtration and measurement. Another possible advantage is that the apparatus may be kept small in size, and the volume required for measurement is very small compared to apparatus with conventional filtration devices.

[0010] Another possible advantage is that the device allows for the determination of capture information in addition to one or more time response values. For example, information may be obtained in one or more other forms of optical measurements, such as absorbance and / or spectroscopic measurements, making it possible to derive knowledge about the concentration and / or type of analyte(s) in a liquid (similar to the previous comments on identifiability, it may be possible to identify analytes or groups of analytes that have similar or identical time response values).

[0011] Generally, when referring to distinguishability, such as optical distinguishability, it can be understood in the context of the claimed apparatus. For example, even if it is not possible to optically distinguish two analytes based on absorbance within, for example, the apparatus (embodiment) according to the present invention, they can be considered optically distinguishable if, in fact, another apparatus, such as a more advanced apparatus (e.g., having higher light intensity and / or better spectral resolution), can actually enable the optical identification of the same analytes.

[0012] A 'difference scale' is understood as a measure that indicates the difference, such as the absolute or relative difference, in the concentration between two or more predetermined analytes in a liquid. A 'difference scale' is, for example, a relationship such as the ratio between two analytes, e.g., the ratio between two (possibly optically indistinguishable) analytes (concentration). analyte1 / Concentration analyte2 It is possible. Alternatively, the 'difference scale' is Concentration analyte1 From Concentration analyte2 The difference after subtracting a certain factor can be an absolute value. A possible advantage of this embodiment is that it enables the provision of information regarding the concentration of the analyte, which may be particularly important when the analyte is not optically distinguishable.

[0013] 'Each of one or more signals is temporally resolved' can be understood to mean that each of one or more signals contains data corresponding to or representing different time points, such as each of one or more signals being acquired at a series of time points or time intervals, different clearly defined time points, etc.

[0014] 'Time response value' can be understood to mean a value indicating this, such as quantifying the time scale of the transient response of a system containing an analyte or group of analytes in a liquid. For example, according to an embodiment, the time response value can be a 'time constant' as used in physics and engineering, typically denoted by the Greek letter τ (tau), which is a parameter characterizing the response of a first-order linear time-invariant system to a step input. For example, the rate of change dC p in the concentration C of the analyte in the pore as a function of time t p / dt is directly proportional to the difference C o between the concentration Cp in the pore and the concentration C p -C o at the opening of the pore with a proportionality constant of 1 / τ. According to one example, the concentration Cp of the analyte in the pore and the concentration C o of the analyte at the opening of the pore are each zero until the time t = 0 (see the step function or Heaviside function H(t)) when the concentration of the analyte at the opening of the pore immediately becomes the concentration K0, which can be explained as follows. o dC

[0015] / dt + τ p C -1 = K0H(t) p This has the following solution. C (t) = K0(1 - e p ) -t / r Therefore, the concentration in the pore that is zero at time t = 0 becomes K0(1 - e ) (≈0.63K0) at t = τ and approaches K0 as t approaches infinity (t → ∞). -1

[0016] According to another example or embodiment, the time response value may be represented by one or more constants in another functional representation. According to another example or embodiment, the time response value may be expressed as the rate of change at a particular time point, such as for signals sampled at time-separated intervals, and the time response value may be the difference between two signal values, such as two adjacent signal values.

[0017] However, as other examples show, the time response can take other forms, including more advanced forms, such as scenarios where diffusion cannot be adequately explained by the response to a step input of a first-order linear time-invariant system, for example, multiple phases (e.g., in the case of cell-free hemoglobin, cfHb, the osmotic plasma phase of a whole blood sample and the impossible phase inside the red blood cells of a whole blood sample). Another example is a signal that includes contributions from both gradual and rapid diffusion of the analyte. Alternatively, if, for whatever reason, the acquired signal is similar to or identical to the under-attenuated step response, one or more time response values ​​may include one or more values ​​representing one or more of the rise time, time to the first peak, settling time, and duration.

[0018] 'One or more time response values' is understood to mean that several time response values ​​can be determined for an analyte or group of analytes in a liquid. Firstly, for example, multiple wavelengths may be used, each of which may provide one or more time response values ​​due to each wavelength yielding a signal representing a particular analyte (or subgroup of analytes) within the group of analytes. Secondly, for example, even in the case of a single wavelength, the time response may be the result of several parameters, which inevitably involves the time response being explainable, or most accurately explained, with respect to several corresponding time response values ​​(e.g., a first time response value showing the time response of a rapidly diffusing analyte, and a second time response value showing the time response of a slowly diffusing analyte).

[0019] 'Transient response' is understood to be common in the art as a response to changes from and / or toward equilibrium (or from a particular configuration toward equilibrium). For example, when a liquid containing one or more analytes is placed at the opening of a pore, and the pore changes from a situation where it contains only the corresponding liquid without the one or more analytes, a transient response occurs, for example, at this location, where the one or more analytes diffuse into the pore until equilibrium is reached.

[0020] 'Analyte' is understood to be any entity, substance, or composition, which may in particular be an atom, ion, and / or molecule. 'Group of analytes' may optionally be understood to be a group of analytes that share one or more properties, such as chemical properties or structural, optical, or physical properties.

[0021] 'Default (analyte)' can be understood as a prior-known analyte, such as, optionally, another default analyte among two or more default analytes, or an analyte for which one or more optical and / or diffusive properties are known with respect to another (default) analyte. In alternative aspects and / or embodiments, analytes are not default in that they are unknown or not designed to be measured prior to, and therefore, 'default(plural) analytes' may be replaced with 'analytes' or 'analytes(plural) to be (further) analyzed' throughout this application.

[0022] 'Two or more (analytes)' can be understood as two or more sets of analytes, each set may or may consist of a single analyte or a group of analytes (such as analytes that share one or more properties).

[0023] The term “liquid” refers to any liquid, including whole blood, plasma fractions of whole blood, cerebrospinal fluid, urine, pleura, ascites, wastewater, pre-prepared liquids for any type of injection, and liquids having components detectable by spectroscopy. A liquid can be understood to have a refractive index (real part of such refractive index) of 1.50 or less, e.g., 1.45 or less, e.g., 1.40 or less, e.g., 1.38 or less, e.g., 1.36 or less, for example, at 416 nm or about 416 nm or 455 nm or about 455 nm.

[0024] In embodiments, the liquid is a liquid sample. The term “sample” refers to the portion of liquid used or required in the analysis using the apparatus of the present invention. The term “whole blood” refers to blood composed of plasma and cellular components. Plasma accounts for approximately 50%–60% of the volume, while cellular components account for approximately 40%–50%. The cellular components are erythrocytes (red blood cells), leucocytes (white blood cells), and thrombocytes (platelets). Preferably, the term “whole blood” refers to the whole blood of a human subject, but it can also refer to the whole blood of an animal. Red blood cells make up approximately 90%–99% of the total number of blood cells. They are formed as biconcave discs with a diameter of approximately 7 μm and a thickness of approximately 2 μm when undeformed. Red blood cells are highly flexible, which allows them to reduce their diameter to approximately 1.5 μm and pass through very narrow capillaries. One core component of red blood cells is hemoglobin, which binds to oxygen for transport to tissues, and then binds to carbon dioxide, which releases the oxygen and is delivered to the lungs as waste. Hemoglobin is responsible for the red color of red blood cells, and therefore the overall red color of blood. White blood cells make up less than 1% of the total number of blood cells. They have a diameter of about 6 to 20 μm. White blood cells are involved in the body's immune system, for example, against bacterial or viral invasions. Platelets are the smallest blood cells, with a length of about 2 to 4 μm and a thickness of about 0.9 to 1.3 μm. They are cellular fragments containing enzymes and other substances important for coagulation. In particular, they form temporary platelet thrombi that help seal up damage within blood vessels.

[0025] The term "blood plasma (or plasma)" refers to the liquid portion of blood and lymph fluid, which makes up about half of the volume of blood (e.g., about 50% to 60% by volume). Plasma does not contain cells. It contains all clotting factors, especially fibrinogen, and about 90% to 95% by volume of water. Plasma components include electrolytes, lipid metabolites, markers for infection or tumors, enzymes, substrates, proteins, and further molecular components.

[0026] The term “wastewater” refers to water used in relation to rinsing, flushing, or manufacturing processes, and therefore contains waste products and / or particles, and is therefore unsuitable for drinking and food preparation.

[0027] 'Determining one or more time response values ​​of an analyte or group of analytes' can be understood as both qualitatively determining whether a time response value is above / below a certain threshold or within / out of a certain interval (YES / NO), and quantitatively determining a time response value, for example, on an ordinal, interval, or ratio type scale.

[0028] Determining one or more time response values, and more specifically, acquiring data for determining one or more time response values, may be understood to rely on an 'optical probe' as understood to be common in the art, such as irradiating at least a portion of a liquid (e.g., a portion of the liquid inside a pore) with light, receiving at least a portion of the light, and allowing the received light to (possibly) derive information about the analyte within it.

[0029] In one embodiment, the apparatus may be configured to automatically determine one or more time response values ​​of an analyte or group of analytes in a liquid. 'Apparatus for automatically determining one or more time response values ​​of an analyte or group of analytes in a liquid' can be understood as any apparatus capable of automatically determining one or more time response values ​​of an analyte or group of analytes in a liquid, such as in a liquid sample, without requiring human intervention after the liquid (sample) has been provided to the apparatus.

[0030] The term "translucency" refers to the property of a material that allows light to pass through. The term "transparency" refers to the property of a material that allows light to pass through without being scattered. Therefore, the term "transparency" can be considered a subset of the term "translucency."

[0031] Preferably, one or more layers of film exhibit reflectivity of more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 50%, for example more than 75%, for example more than 90%, and even more than 99%, in the spectral range of detection when tested with an integrating sphere, i.e., in the spectral range in which signals representing relevant plasma components unfold, for example, in the case of perpendicularly incident light, for example, in the range of 380 nm to 750 nm, 400 to 525 nm, or 416 nm or about 416 nm, or 455 nm or about 455 nm (for example, at the interface between the translucent element and one or more layers).

[0032] Techniques applied to measure the reflectance from an interface (often diffuse, or containing diffuse light) or transmittance through an interface or through the length of a (bulk) material may involve using an integrating sphere, such as relying on Fourier transform infrared (FTIR) analysis. Light strikes a sample (such as an interface or portion of a bulk material), such as the interface between a translucent element and one or more layers, at a normal 90° angle to one or more layers. The reflected and / or transmitted light is scattered as it interacts with the sample. An integrating sphere is a device that collects scattered transmitted and / or reflected light from a diffuse sample, using a highly reflective surface of a spherical wall that 'bounces' the light until it reaches the detector. In this way, accurate results can be achieved from surfaces that would normally yield low reflectance or transmittance due to scattering.

[0033] A 'transparent (element)' can generally be understood as an element containing a translucent material, for example, the above material (such as the translucent material and / or the material of the translucent element) has an attenuation coefficient, and as a result, the transmittance coefficient of light passing through the material (ignoring any interfacial effects, for example) (with selective partial or total diffusion) is at least 50% for a length of 100 micrometers of material, for example, the percentage of light that does not pass through the length of material is less than 50% of 100 micrometers, for example less than 40% of 100 micrometers, for example less than 20% of 100 micrometers, for example less than 10% of 100 micrometers, for example less than 5% of 100 micrometers, for example less than 416 nm or 455 nm or 455 nm. The advantage of this may be that it allows photons to be taken into and / or taken out of the translucent element. The expression 'translucent element' can be understood and used synonymously with 'element containing a translucent material'. In one embodiment, neglecting any interfacial effects, for example, the transmittance coefficient of light passing through the translucent element from front to back in a direction perpendicular to the front and / or back is at least 10%, for example at least 25%, for example at least 50%, for example at least 75%, for example at least 90%, for example at least 95%, for example at least 99%, for example at least 99%, for example at least 99%, for example at least 90%, for example at least 90%, for example at least 955%, for example at least 99%, for example at least 90%, for example at least 95%, for example at least 95%, for example at least 99%, for example at least 90%, for example at least 95%, for example at least 95%, for example at least 99%, for example at least 95%, for example at least 95%, for example at least 95%, for example at least 99%, for example at least 95%, for example at least 95%, for example at least 95%, for example at least 9

[0034] The terms 'back side' and 'backside' are used synonymously and interchangeably. The 'attenuation coefficient' can be understood as the Napierian attenuation coefficient u. For example, the transmission T through a material is obtained as follows: T = exp(-int(u(z)dz)) In the formula, 'exp' represents the exponential function, 'int' represents the integral (through the length of the material), and z represents the corresponding axis passing through the material and the corresponding coordinate system.

[0035] The attenuation coefficient can be obtained in a manner common to the art, for example, by measurement in a standard spectrophotometer measuring absorbance through a 1 cm cuvette. The measured absorbance, denoted by A (or Abs), is determined in a standard instrument as A = log(I0 / I), where log is a base--10 logarithm, I0 is the intensity before the cuvette, and I is the intensity after the cuvette. Thus, the measured absorbance is related to the Napierian attenuation coefficient as A = log(e)int(u(z)dz), where e = 2.71828, which represents the fundamental number for the natural logarithm.

[0036] In general, when referring to optical properties (e.g., transmittance, absorption, internal reflection, reflection) throughout this application, it can be understood that this is done in general with reference to electromagnetic radiation (or light) having wavelengths in the range of 380 nm to 750 nm, for example 400 to 520 nm, for example 400 to 460 nm (or 415 to 420 nm), for example 415 nm or about 415 nm, or 416 nm or about 416 nm, or 450 nm or about 450 nm, or 455 nm or about 455 nm, for example, at least one wavelength.

[0037] The light-transmitting element has a front side and a rear side facing the opposite direction from the front side, and the front side is It can be adapted to be in direct contact with the liquid (with one or more layers not in front of the translucent layer), or The light-transmitting element may be adapted to be separated from the liquid, such as being exclusively separated from the liquid by one or more layers in front of the light-transmitting element, and one or more layers are The light-transmitting element is adapted to be non-reflective to light reaching one or more layers at at least one angle of incidence, for example, at least normal incidence. The layers are adapted to be reflective (one or more of the above layers are metals and / or materials having a dissipation coefficient that ignores their light transmission properties), and the refractive index of one or more of the layers is equal to or higher than that of the light-transmitting element, and / or At interfaces such as external interfaces, the light-transmitting element is adapted to enable internal reflection, such as total internal reflection, of light reaching the interface. Non-through holes extend into the translucent element from the respective openings that connect the non-through holes to the liquid and fluid on the front side (through one or more layers, if present).

[0038] 'Directly in contact with the liquid' can be understood as the front surface of the light-transmitting element being a solid-liquid interface, where, for example, one or more layers do not separate the light-transmitting element from external elements such as the liquid. 'Separated from the liquid by one or more layers in front of the light-transmitting element' can be understood as one or more layers, such as thin film layers (thin film layers of 100 micrometers or less), being present at the solid-liquid interface in front of the light-transmitting element. 'Exclusively separated' can be understood as no other layers separating the light-transmitting element from the liquid.

[0039] 'Adapted to be non-reflective to light reaching one or more layers at at least one angle of incidence' can be understood as meaning that at at least one angle of incidence (such as normal incidence), when incident light (at 416 nm or about 416 nm, or 455 nm or about 455 nm) enters the light-transmitting element, the light is reflected little or no from one or more layers (for example, the reflection coefficient is less than 0.95, e.g. less than 0.9, e.g. less than 0.8, e.g. less than 0.7, e.g. less than 0.6, e.g. less than 0.5, e.g. less than 0.4, e.g. less than 0.3, e.g. less than 0.1, e.g. less than 0.01).

[0040] For example, non-reflectivity may be due to absorption and / or transmission. At least one angle of incidence may be normal incidence. According to one embodiment, a light-transmitting element is presented, wherein the front (side) of the light-transmitting element is separated from the liquid by one or more layers on the front side of the light-transmitting element, and the one or more layers are adapted to be light-transmitting to light that reaches the front side by normal incidence from the light-transmitting element.

[0041] According to one embodiment, a light-transmitting element is presented, wherein the front (side) of the light-transmitting element is separated from the liquid by one or more layers on the front side of the light-transmitting element, and the one or more layers are adapted to be absorbent to light that reaches the front side of the light-transmitting element by normal incidence.

[0042] 'Absorbing' can be understood as meaning that at at least one angle of incidence (such as normal incidence), more than 1%, e.g., more than 10%, e.g., more than 25%, e.g., more than 40%, e.g., more than 50%, e.g., more than 60%, e.g., more than 75%, e.g., more than 90% of the incident light (at 416 nm or approximately 416 nm, or at 455 nm or approximately 455 nm) is neither reflected into the light-transmitting element from one or more layers nor transmitted through one or more layers.

[0043] 'Adapted to be reflective to light reaching one or more layers at at least one angle of incidence' can be understood as meaning that, when incident light enters in the direction passing through the light-transmitting element, at least one angle of incidence, the light is reflected from one or more layers (for example, the reflection coefficient is at least 0.25, e.g., at least 0.4, e.g., at least 0.5, e.g., at least 0.6, e.g., at least 0.75, e.g., at least 0.90, 0.95, e.g., at least 0.99, e.g., at 416 nm or about 416 nm, or 455 nm or about 455 nm, and / or normal incidence), and the refractive index of one or more layers is equal to or higher than the refractive index of the light-transmitting element. According to such a 'reflective' embodiment, one or more layers may be or may include a metallic layer (such as a layer comprising platinum, palladium, an alloy containing platinum or palladium as a main component, silver, and / or aluminum) and / or a layer of material having a dissipation coefficient that ignores the above layer as light transmittance.

[0044] According to one embodiment, a translucent element is presented, wherein one or more layers that separate the translucent element and / or the front side of the translucent element from the liquid, for example, one or more layers that exclusively separate the translucent element, are arranged at the interface between the translucent element and / or one or more layers on one side and the liquid on the other side to enable internal reflection, such as total internal reflection.

[0045] 'Adapted to allow internal reflection' can be understood as internal reflection at the interface between media where the medium containing the incident and reflected light is traveling through a medium with a higher refractive index compared to the medium on the opposite side of the interface, for example, the reflection coefficient is at least 0.25, e.g., at least 0.4, e.g., at least 0.5, e.g., at least 0.6, e.g., at least 0.75, e.g., at least 0.90, e.g., at least 0.95, e.g., at least 0.99) for normal or non-normal incidence, e.g., at an angle of 45° with respect to the normal. In embodiments, the dissipation coefficients of both media (i.e., each medium on each side of the interface) are such that each material has a sufficiently low dissipation coefficient (or attenuation coefficient) for it to meet the requirements as translucent.

[0046] According to one embodiment, the translucent element is a translucent slab, and for example, the slab is understood to be monolithic. Each of the small holes has an opening through which it can communicate with the liquid space at the front of the light-transmitting element. Thus, the holes penetrate one or more layers (if any) to allow liquid communication between the hole and the liquid space. The holes extend into the light-transmitting element from their respective openings at the front in a direction toward the rear. The holes are “non-penetrating,” meaning that the holes terminate within the light-transmitting element. The holes do not continue through the entire light-transmitting element to the rear or to any common container or receptacle inside the element. The holes are in liquid communication only with the liquid space at the front of the light-transmitting element. Note that in some embodiments, non-penetrating holes may be cross-shaped, and therefore at least some of the holes may be connected to each other, forming an X-shape, Y-shape, V-shape, or similar interconnected shape. Such configurations are considered equivalent to non-penetrating because, even though the holes are filled only from the front and the holes intersect each other, no significant net mass transport through the holes occurs under operation. By appropriately sizing the opening of the pore on the front side, it is possible to allow relevant components in the plasma fraction or liquid of a whole blood sample to enter the pore, while preventing red blood cells or fragments in the liquid of a whole blood sample from entering the pore on the front side of the translucent element. The relevant components are substances present in the plasma fraction of a whole blood sample and that will be measured / detected using the sensor. In particular, bilirubin and carbon dioxide are relevant components.

[0047] Under operation, the front side of the translucent element is in contact with the whole blood sample or liquid. Small pores within the translucent element communicate with the whole blood sample or liquid through openings within the front side. The pore openings are sized selectively to extract a subsample of the plasma phase of the whole blood sample or a subsample of the liquid containing the analyte. Red blood cells cannot enter the pores through the openings on the front side of the translucent element. Anything larger than the pore diameter cannot enter the pore, for example, to exclude any fragments contained in the liquid. As stated, the pores are non-penetrating and communicate only with the front side of the translucent element; that is, the subsample is extracted for the optical probe inside the pore and, after measurement, released again through the same openings within the front side of the translucent element. The subsample volume corresponds to the total internal volume of the pores. Filtration and net mass transport of any filtrate do not occur through the pore-containing layer to any common filtrate receiver or any filtrate outlet. Photodetection is then performed only on the subsample contained within the pores.

[0048] Small subsamples containing representative contents of the relevant components can be moved into the pore in any preferred manner. The small non-penetrating pore allows for very efficient and rapid extraction of subsamples for the optical probe from the whole blood sample or liquid through the opening in the front side by capillary force and / or diffusion.

[0049] In a typical operating mode, the front surface of the translucent element is brought into contact with a rinse solution before the front surface comes into contact with the whole blood sample or liquid to be analyzed. This 'primes' the pores by pre-filling with a liquid that is compatible with the whole blood sample or liquid, and in particular, an aqueous solution commonly used in blood analyzers for rinsing, calibration, and / or quality control purposes, or a liquid compatible with the plasma phase if the liquid is whole blood. For example, a typical rinse solution used for rinsing in a whole blood analyzer system could be used as such a liquid. The rinse solution is K at a concentration corresponding to human plasma. + kaNa + Cl - Ca 2+ O2, pH, CO2, and HCO3 -It is an aqueous solution containing [the substance]. A non-limiting example of a suitable solution commonly used for rinsing, calibration, and / or quality control purposes is further listed below. When a whole blood sample or liquid is brought into contact with a front surface that is subsequently primed with a plasma-affinity liquid, a representative subsample of the plasma phase components of the whole blood sample, or of the liquid, is extracted and transferred in a very efficient and gentle manner by diffusion of the relevant components into the pre-filled well. In particular, any concentration gradient in the analyte content between the liquid in the well and the reference solution results in diffusive transfer, thereby producing a subsample in the well having an analyte concentration representing the analyte concentration in the liquid.

[0050] According to one embodiment, a light-transmitting element is presented in which the pores are arranged to be rinsed by diffusion, for example, solely by diffusion. In other operating modes (such as for use in embodiments where the concentration of an analyte is measured), it may also be conceivable to bring the dry front side of the sensor into direct contact with a whole blood sample or liquid. More preferably, in this operating mode, the inner surface of the pores is hydrophilic, thereby drawing a subsample from the whole blood sample or liquid in front of the translucent element into the pores by capillary force. When operating the translucent element in this mode, calibration occurs via batch calibration, as translucent elements produced from the same batch of porous membrane material tend to have equal sensitivity (equal absorbance when measuring against the same liquid using translucent elements produced from different pieces of porous membrane material from the same batch forming the translucent elements). Alternatively, the pores of the translucent element may contain a calibration dye having different absorbance characteristics from the analyte. The calibration dye is useful for normalizing / calibrating the optical probe signal while being spectroscopically distinguishable from the substance in the plasma sample to be detected / measured, e.g., bilirubin. Since the calibration dye is not present in the actual liquid, it diffuses out of the sensor during measurement, while the analyte diffuses into the sensor's pore. By optically probing the pore before and after acquiring the liquid, a quantitative measure of the substance to be detected (e.g., bilirubin) can be developed by comparing the calibration reference signal with the liquid substance signal.

[0051] The contents of the pore can be easily optically probed from the rear of the light-transmitting element, or more generally, from the front / front side and / or from one or more layers (if any) facing the light-transmitting element, and one or more layers (if any, including an absorbent layer) optically separate the optical probe region containing the pore from the liquid in contact with the front side of the light-transmitting element, thereby preventing the probe light from reaching and interacting with the liquid at the front side of the unit or light-transmitting element. Therefore, optical probing is performed selectively only on the subsample inside the pore. 'Probing (optically) from the rear (of the light-transmitting element)' can generally be understood as the incident probe light into the pore traveling in a direction from rear to front (e.g., entering the light-transmitting element via the rear in a direction from rear to front), and the light emitted from the pore to a receiving unit such as a photodetector being emitted in a direction from front to back, such as being emitted from the rear in a direction away from the front.

[0052] 'Bringing the pores of a light-transmitting element into contact with a liquid' can be understood as placing a liquid into the opening of the pore, for example, through the liquid inlet of the device, and introducing the liquid and / or analytes in the liquid into the pore, for example, by diffusion, so that the liquid is inspected in contact with the pore.

[0053] 'Illuminating at least holes in a translucent element with one or more light sources' is understood to be common in the art and may be done, for example, with 'one or more light sources adapted to illuminate at least holes in a translucent element', which is understood to be any light source capable of providing sufficient light (or more specifically, sufficient spectral flux within the relevant wavelength range or enabling optical probe of the analyte). One or more light sources may include, for example, incandescent light sources (such as tungsten bulbs), fluorescent light sources (such as mercury lamps), LED light sources, or laser light sources (such as argon-ion gas lasers).

[0054] 'Receiving light emitted from a hole in response to illumination at each of several time points' is understood to be common in the art and may be done, for example, using a 'photodetector adapted to receive light emitted from a hole in response to illumination by one or more light sources at each of several time points', which is understood to be common in the art and in particular, 'each time point' may be understood to refer to a time stamp associated with intervals (such as bins), such as a corresponding time point corresponding to a finite time interval. 'Photodetector' is understood to be common in the art, for example, a motorized photodetector that outputs a signal electrically and / or digitally. 'Photodetector' is generally understood to be used synonymously and interchangeably with 'detector' in this context. 'Photodetector' may further be understood to include, or encompass, multiple (subordinate) photodetectors.

[0055] 'Generating one or more signals based on received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light' is understood to be common in the art. 'A photodetector may be further adapted to generate one or more signals based on received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light', for example, the photodetector may be arranged to provide a temporally decomposed signal, such as a digital or analog signal, including at least two pairs (such as 3, 5, 10, 100, 1000, or 10000) of corresponding values ​​of the received light (such as the total received intensity of the light, or the received intensity within a particular wavelength interval which may be only a portion of the received light) and time.

[0056] The incident light is guided / directed into the optical probe region (including the pore) to ensure that the light traverses the pore and interacts with the (sub-sample) liquid within it. Preferably, the probe light is directed into the probe region obliquely to the surface normal of the front surface and / or the plane of one or more layers (if any) of the translucent element to ensure that the light traverses the pore filled with the liquid to be probed, thereby ensuring the maximum optical interaction path.

[0057] Light emitted from a pore in response to illumination interacts with the subsample within the pore and therefore contains information about the subsample. The emitted light, and / or the signal representing the emitted light, e.g., one or more signals, may then be analyzed using a data processing device to determine one or more time response values ​​and, optionally additionally, values ​​representing the analyte content in the whole blood sample or liquid (e.g., the concentration of the stable state within the pore). The analysis may include spectroscopic analysis of the emitted / detected light and / or signal / data processing, e.g., for noise filtering, for applying corrections, and for removing artifacts, to compare the acquired signal with a signal acquired for a calibration / reference sample. Spectroscopic analysis may be performed as known in the art, such as by any method including means for resolving multiple wavelengths in the incident light and wavelengths in the detected signal (e.g., frequency modulation or time or spatial separation of incident light of different wavelengths, and / or wavelength-sensitive detection).

[0058] According to one embodiment, a method is presented in which two or more predetermined analytes are haptoglobin-avoiding cell-free hemoglobin and haptoglobin-conjugated hemoglobin such as hemoglobin-haptoglobin complexes. A possible advantage of determining haptoglobin-avoiding cell-free hemoglobin and / or haptoglobin-conjugated hemoglobin such as hemoglobin-haptoglobin complexes is that it makes it possible to determine whether hemolysis in the sample occurred in vivo or in vitro, which in turn may make it possible to determine whether the concentration of a particular entity (such as potassium ions) in the sample represents a true patient value, and / or, optionally, to predict a true patient value from the measurement.

[0059] According to another embodiment, a method is presented in which two or more predetermined analytes are bilirubin such as free bilirubin and human serum albumin-bound bilirubin such as HSA-bilirubin.

[0060] According to a further embodiment, a method, A step of determining the presence of a hemoglobin-haptoglobin complex based on a difference scale, and / or A method is presented that further includes a step of determining the absence of the hemoglobin-haptoglobin complex based on a difference scale.

[0061] This method may include, for example, a step of qualitatively determining the presence and / or absence of hemoglobin-haptoglobin complexes by comparing a difference scale, and / or values ​​derived therefrom, such as the relative concentration of hemoglobin-haptoglobin complexes to haptoglobin-unbound hemoglobin and / or the absolute concentration of hemoglobin-haptoglobin complexes, to a predetermined threshold. The advantage of determining the presence and / or absence of hemoglobin-haptoglobin complexes may be that it provides information on whether hemolysis occurred in vivo or ex vivo, such as in vitro.

[0062] According to one embodiment, a method is presented which further comprises the steps of bringing the apparatus into contact with a reference solution so as to fill the pore with the reference solution, and / or waiting for a diffusion time to allow the diffusion of two or more predetermined analytes in the liquid into the pore to reach a stable state. When the reference solution initially fills the pore, applying the liquid to be tested results in the analytes in that liquid being able to diffuse into the pore, which is in contrast to having the pore filled with the analytes by the analytes, for example, via the capillary action of the liquid to be tested, which is likely to make it more difficult to obtain data reflecting the diffusive nature of the analytes. Once a stable state is reached, it is assumed that the concentration of the analyte(s) in the pore is equal to the concentration of the analyte(s) in the liquid to be tested.

[0063] According to one embodiment, a method The further step includes providing a data processing device equipped with a processor, the data processing device comprising one or more of the following steps: A step of controlling one or more light sources to illuminate at least a hole in a light-transmitting element, A step of controlling the detector to receive light emitted from the hole in response to illumination at each of several time points, A step of receiving one or more signals based on received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light, A step of determining one or more time response values ​​based on one or more signals, and It is configured to perform the step of determining a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in a liquid, based on one or more time response values, For example, a method is presented in which two or more default analytes are cell-free hemoglobin that does not bind to haptoglobin, and haptoglobin-binding hemoglobin such as a hemoglobin-haptoglobin complex.

[0064] 'Data processing device' is understood, as is common in the art, and in particular, as any device capable of receiving, processing, and outputting information such as digital information.

[0065] 'Processor' is understood, as is common in the art, and in particular, as an electronic circuit capable of executing computer programs, such as instructions that constitute a processing unit, such as a central processing unit (CPU).

[0066] By configuring a data processing device to determine one or more time response values ​​based on one or more signals, it is understood that one or more time response values ​​may change with changes in the value(s) within one or more signals.

[0067] The data processing device may also be configured to output a signal (output signal) based on one or more time response values. 'Outputting a signal' is understood to be common in the art to provide the data processing device with information from an external source indicating one or more time response values ​​(e.g., '211 milliseconds') and / or parameters based thereon (such as 'haptoglobin-bound hemoglobin present in the liquid (sample),' which may be further derived based on one or more time response values ​​in which such a complex exists).

[0068] The content and format of the (output) signal can take different forms; for example, the format may be a digital signal or an analog signal. For example, the output signal may be digital information. In another example, the output of the signal may be a visual and / or audible signal. The content may be, for example, a qualitative or quantitative value.

[0069] A data processing device may include, or may have, access to default instructions (e.g., via a digital storage device operably contained within and / or connected to a processor), such as default instructions that enable the data processing device to take one or more signals as input and determine one or more time response values ​​based on these one or more signals. The default instructions may be implemented, for example, as an algorithm or lookup table, or based thereon. The default instructions may be implemented, for example, as a function or algorithm including a model, such as a mathematical model, or based thereon, where data points of one or more signals are fitted to the model, for example, using regression analysis, to obtain inferences of unknown model parameters (such as τ (tau)). According to one embodiment, the method is as follows: A method is presented which involves acquiring multiple signals over different wavelength intervals, for example, each signal in the multiple signals being acquired over a specific wavelength interval relative to the wavelength intervals for the rest of the signals in the multiple signals.

[0070] One possible advantage is that multiple signals may reflect different analytes, allowing information about multiple analytes to be obtained in parallel. Another possible advantage is that at least one signal may be used as a reference or background signal, allowing background to be taken into account in another signal. For example, before determining the time response value, a reference signal (such as a signal obtained at a wavelength or wavelength interval where the analyte of interest is not optically active, or is not very optically active (e.g., less than 30% of the activity at the probe light wavelength at the highest activity wavelength, less than 20%, less than 10%)) may be subtracted from another signal (such as a signal obtained at a wavelength or wavelength interval where the analyte of interest is optically active), so that the resulting signal (ideally, or in principle) represents the analyte without overlapping background signals. 'Different wavelengths' are understood as non-identical intervals, where such intervals are partially or completely overlapping or non-overlapping.

[0071] In embodiments, for example, a time response obtained for a single wavelength (e.g., WL1) in a clearly defined sample (such as a diluted sample) may be analyzed to determine, for example, which of several analytes (which may be indistinguishable without the present invention) is present.

[0072] According to one embodiment, the method is as follows: A method is presented for determining a difference measure that indicates the difference in concentration between two or more predetermined analytes, based on multiple time response values ​​obtained for different, e.g., intrinsic, wavelength intervals, where, for example, one response value serves as a reference for another response value.

[0073] A possible advantage is that the influence of background contributions in the signal, including analyte contributions, is reduced or minimized. For example, multiple time response values ​​may be obtained at different wavelengths (where the analytes are optically active and optically inactive, respectively). In this example, the adjusted time response value is obtained as the difference between two time response values, where, for example, one time response value serves as a reference for the other, and the two time response values ​​are obtained for signals acquired over different, e.g., unique, wavelength intervals, and the difference measure is based on the adjusted time response value above. In another example, the difference measure is based on the ratio between two time response values. 'Different wavelength intervals' can be understood as non-identical, such as substantially non-overlapping wavelength intervals, perhaps overlapping wavelength intervals, etc.

[0074] According to one embodiment, the method is as follows: The step of determining a difference measure that shows the difference in concentration between two or more predetermined analytes is based on multiple time response values, and more specifically, The time response obtained for a first wavelength interval, such as a single wavelength of 415 nm, The time response obtained for a second wavelength interval, such as a single wavelength of 450 nm, and A method based on the ratio of is presented.

[0075] A possible advantage is that the influence of background contributions in the signal, including the analyte contribution, is reduced or minimized. The ratio is applied because it utilizes another signal as an internal reference, which may be the most precise method. For example, at least two time response values ​​can be obtained at different wavelengths (e.g., one with an optically active analyte and one without).

[0076] According to one embodiment, the method is as follows: The first wavelength interval is a single wavelength of 415 nm. A method is presented in which the second wavelength interval is a single wavelength of 450 nm.

[0077] The selected wavelengths may be advantageous due to their relevance to the hemoglobin absorption spectrum, and more specifically, 415 nm may be advantageous due to its identity or proximity to the hemoglobin peak wavelength at 415 nm or 416 nm, and 450 nm may be advantageous due to its distance from the hemoglobin peak wavelength at 415 nm or 416 nm, thereby making 450 nm suitable for use as a reference wavelength.

[0078] The wavelength spacing is generally understood to be a 'single wavelength', and thus, as those skilled in the art recognize, for practical purposes, it is understood to be a narrow spacing that effectively corresponds to a single (center) wavelength, for example, a Gaussian function with a peak centered around the periphery of the center wavelength, for example, less than 20 nm, for example less than 10 nm, for example less than 5 nm, for example less than 2 nm, for example 1 nm FWHM (full width at half maximum).

[0079] According to one embodiment, the method comprises one or more time response values, Each of the one or more time response values ​​is the result of subtracting the signal value at the first time point from the signal value at the second time point, and so on, based on one or more differences in the signal values ​​within each of the one or more signals, the above signal values ​​are obtained at different time points. and / or, A method is presented in which one or more characteristic time points represent one or more characteristic time points, such as the duration of change in a parameter or a specific amount of change.

[0080] 'Based on one or more differences in signal values' can be understood, for example, as the time response value being calculated based on differences in signal values, for instance, the time response value being equal to the time difference between data points having a specific difference in signal values, for example, the time difference between a first point having a signal value exceeding a certain deviation from the baseline signal value and a second point having a signal value exceeding a certain threshold, for example, the threshold being expressed as a percentage, such as 63% of the (subsequent) stable state or saturated signal value, such as the 'rise time'. The time response value calculated in such a manner may be independent of (model) assumptions about the temporal evolution of the signal (except that it increases over time and gradually stabilizes).

[0081] Alternatively, 'based on one or more differences in signal values' can be understood as the difference between signal values ​​acquired at different times, such as the rate of change in signal values. For example, the rate of change in signal values ​​for two different wavelengths may be acquired and used to determine the difference measure.

[0082] 'Characteristic time' can be understood as a measure of the system's response time. For example, for a system that is a first-order linear time-invariant system, or a system that is modeled as a first-order linear time-invariant system, the characteristic time may be given as τ (tau), as previously explained.

[0083] According to one embodiment, the method is as follows: translucent element, One or more light sources, and The detector is A method is presented in which one or more signals generated at each of multiple time points are arranged to represent the concentrations of two or more predetermined analytes in the well.

[0084] Those skilled in the art can easily conceive of a device such that, for at least a specific concentration, one or more signals generated at each of several time points represent the concentration of an analyte or group of analytes in a well. For example, the device may be configured so that the signal values ​​reflect any one or more of the following: absorbance at the wavelength absorbed by a high-demand analyte, or fluorescence intensity (a light source excites the fluorescent dye molecules of the analyte at a first wavelength, and a detector detects the emitted fluorescent dye molecules at another wavelength).

[0085] According to one embodiment, the method is as follows: A method is presented which further includes the step of determining, based on one or more signals, the concentration of cell-free hemoglobin that is not bound to haptoglobin, and / or the concentration of haptoglobin-bound hemoglobin, such as a hemoglobin-haptoglobin complex analyte, in a liquid.

[0086] A possible advantage is that the absolute concentration of one analyte or other substance can be important, for example, for research and / or clinical purposes, as it allows for the determination (with greater certainty, for example) whether hemolysis occurred in vivo or ex vivo / in vitro.

[0087] According to one embodiment, a method is presented in which a determined concentration of an analyte in a liquid is adjusted based on one or more time response values. The concentration of an analyte or group of analytes in a liquid may not be determined solely by signal values, such as the saturation signal value. This is especially true when multiple optically indistinguishable analytes are present. For example, if two optically indistinguishable analytes are present, the saturation signal value—otherwise the concentration can be directly derived—is the result of contributions from both analytes, such as a (weighted) sum. However, time-response values ​​can make it possible to determine which of the analytes are present and / or their ratio, thereby allowing us to unravel their distinct contributions to the signal value and derive the respective concentrations of one or both analytes.

[0088] Alternatively, or in addition, the determined concentration of an analyte may be adjusted relative to the (directly) measured concentration of the analyte based on one or more time response values. For example, if a specific potassium ion concentration and a specific concentration of haptoglobin-bound hemoglobin are measured, the determined concentration of potassium may be obtained by subtracting an estimated interference value from the measured value, and the determined concentration aims to estimate the true patient potassium value, i.e., exclude the potassium contribution from in vitro hemolysis (such as that determined by the concentration of haptoglobin-bound hemoglobin).

[0089] More specifically, for example, in the case of potassium ions, if haptoglobin is present, the impact factor or interference effect from cfHb is estimated by multiplying the proportional factor (0.3 mM / (100 mg / dL cfHb)) by the measured cfHb concentration, regardless of whether cfHb is bound to haptoglobin or not. The estimate of the true patient-value potassium ion concentration is estimated by subtracting the above impact factor from the measured potassium concentration (the presence of haptoglobin is an indicator that hemolysis occurred in vitro, and therefore potassium ions from red blood cells are due to the assumption that the potassium concentration in the sample has increased relative to the potassium concentration in the patient). Similar principles are applicable to other analytes, and according to embodiments, this method is used for determined in vitro / ex vivo hemolysis, K + Ca++ , and / or Na + The process further includes steps to correct hemolysis sensitivity parameters such as those mentioned above.

[0090] According to one embodiment, the method further includes the step of determining whether the concentration of cell-free hemoglobin in a liquid is above a first predetermined concentration value, such as 100 mg / dL, and / or below a second predetermined concentration value, such as 330 mg / dL, within a predetermined interval, for example, [100 mg / dL; 300 mg / dL]. Possible advantages of this may be that distinguishing analytes from one another based on time response values ​​may work particularly well within a specific concentration range, for example, only within a specific concentration range, and that by checking whether the concentration is within such a range, information can be obtained regarding the possibility or validity of distinguishing analytes based on time response values.

[0091] According to one embodiment, the method is as follows: A step of determining whether the difference scale is within one or more default difference scale ranges, such as exceeding a default difference scale threshold, and A method is presented which further includes the step of providing a signal indicating the difference scale, such as providing a visual signal to an instrument, if the difference scale is within one or more predetermined difference scale ranges.

[0092] Possible advantages include, for example, that an alarm (in the provided signal embodiment) could notify / warn the user that, for example, in vitro hemolysis has occurred and consequently, a particular measured analyte concentration is unreliable.

[0093] According to a second aspect, an apparatus for determining a difference scale, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in a liquid, for example, in whole blood, for example, in a whole blood sample, A light-transmitting element containing pores, wherein the pores are non-penetrating holes extending into the light-transmitting element from each opening within the light-transmitting element, and the cross-sectional dimensions of the openings of the pores are determined to allow two or more predetermined analytes in a liquid to enter the pores by diffusion, while preventing larger particles or fragments from entering the pores. One or more light sources adapted to illuminate at least the holes in the light-transmitting element, and Each of the multiple time points includes a detector adapted to receive light emitted from a hole in response to illumination by one or more light sources, The photodetector is further adapted to generate one or more signals based on the received light, and each of the one or more signals is temporally decomposed and represents at least a portion of the received light. This device The data processing device further comprises a processor, and the data processing device is Determining one or more time response values ​​based on one or more signals, Determining a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in a liquid, based on one or more time response values. A device is provided that is configured to perform the following.

[0094] According to one embodiment, an apparatus is presented in which two or more predetermined analytes are cell-free hemoglobin that does not bind to haptoglobin, and haptoglobin-binding hemoglobin such as a hemoglobin-haptoglobin complex.

[0095] According to one embodiment, the apparatus, for example, a blood gas analyzer, is as follows: Carbon dioxide, for example, CO2, Oxygen, for example, O2, and pH An apparatus is presented which is further arranged to measure the concentration in one, more, or all of the liquid samples.

[0096] The advantage of having such a (blood gas analyzer) device may be that it allows for the provision of further relevant liquid (blood) sample parameters, for example, via output, which may inform the user (even a non-expert user) whether one or more analytes may be associated with a high (too high) cell-free hemoglobin interference critical, such as whether retesting may be necessary. The advantages may be, for example, that it provides a fast response time, relevant output for non-expert users, and relevant solutions for point-of-care testing where one, several, or all of multiple parameters may be particularly relevant.

[0097] According to one embodiment, the apparatus is configured such that one or more light sources and / or photodetectors are operably coupled to a data processing device comprising a processor, and the data processing device comprising the processor is The acquisition of multiple signals over different wavelength intervals, for example, each signal in the multiple signals being acquired over a specific wavelength interval relative to the wavelength intervals for the rest of the multiple signals. Determining multiple time response values ​​by determining the time response value for each of the signals in a group of signals, for example, each time response value is determined based on signals obtained for different wavelength signals, such as being obtained for wavelength intervals that are unique to the wavelength intervals of the remaining signals in the group of signals, and Based on multiple time response values, determine a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes (96) in a liquid (99). A device is presented that is further arranged to perform the following action.

[0098] The advantage of this is that the device may therefore be able to determine multiple time response values ​​obtained from different wavelength signals that could represent, for example, the analyte and the background, which can then be used to determine a difference measure based on this (for example, via a time response value adjusted for the background), thus possibly providing a more accurate estimate of the difference.

[0099] 'Operationally coupled' can be understood as a data processing device having a processor being able to operate with, for example, one or more light sources and / or photodetectors, for controlling them and / or receiving data from them.

[0100] 'Different wavelength intervals' can be understood as non-identical wavelength intervals, such as overlapping wavelength intervals, substantially non-overlapping wavelength intervals, or non-overlapping wavelength intervals. According to one embodiment, the apparatus is a data processing device, Determining a tuned time response value, wherein the tuned time response value is determined based on at least two time response values ​​among a plurality of time response values, for example, one time response value serving as a reference for another response value, and for example, the two time response values ​​are obtained for signals obtained for different, e.g., intrinsic, wavelength intervals, and Based on adjusted time response values, determine a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes (96) in a liquid (99). A device is presented that is further configured to perform the following:

[0101] The advantage of this is that the device may therefore be able to determine a time response value adjusted to take background into account (see above), and thus provide a more accurate estimate of the difference, for example.

[0102] One or more light sources and / or photodetectors are operably coupled to a data processing device having a processor, and the data processing device having a processor The first signal is obtained at a first wavelength interval, such as a first wavelength interval centered at 415 nm. The method involves obtaining a second signal at a second wavelength interval, where the second wavelength interval is different from, for example, unique to, the first wavelength interval, and for example, the second wavelength interval is substantially centered at 450 nm. The purpose is to determine the ratio, The time response obtained for the first wavelength interval, The second time response obtained for the second wavelength interval and To determine the ratio of, Based on the ratio, determine the difference scale which indicates the difference, such as the absolute or relative difference, in the concentrations between two or more predetermined analytes (96) in the liquid (99). The apparatus according to any of the preceding claims, further arranged to perform the following.

[0103] The advantage of this is that the instrument may therefore be able to determine time response values ​​obtained from signals of different wavelengths and to determine ratios, which may allow the influence of background contributions in signals including analyte contributions to be reduced or minimized, which may therefore allow the instrument to take background into account (see above) and thus determine ratios to provide, for example, a more accurate estimate of the difference. Ratios are applied because they utilize another signal as an internal reference, which may be the most precise method. For example, at least two time response values ​​may be obtained at different wavelengths (e.g., one with an optically active analyte and one without).

[0104] According to one embodiment, the apparatus is such that the light-transmitting element has a transmittance coefficient that selectively partially or entirely diffuses light passing through the material, such as ignoring any interface effects, and the percentage of light that does not pass through the material is at least 50% for a length of 100 micrometers, for example, in the range of 380 nm to 750 nm, such as 400 to 520 nm, for example, in the range of 400 to 460 nm, for example, in the range of 415 to 420 nm, for example, 415 nm or about 415 nm, or An apparatus is presented comprising, for example, mainly comprising, for example, 50 w / w% or more comprising, for example, a material having an attenuation coefficient such that, for one wavelength of 416 nm or approximately 416 nm, 450 nm or approximately 450 nm, or 455 nm or approximately 455 nm, is 50% or less of 100 micrometers, for example, 40% or less of 100 micrometers, for example, 20% or less of 100 micrometers, for example, 10% or less of 100 micrometers, for example, 5% or less of 100 micrometers, for example, mainly comprising, for example, 50 w / w% or more comprising, for example, derived from. This may make it possible to obtain the light-transmitting properties of a light-transmitting element in a simple manner.

[0105] According to one embodiment, an apparatus comprising a porous unit further comprises an optical assembly comprising a light-transmitting element and a light-guide core, the light-guide core comprising an input branch, an output branch, and a coupling interface positioned to contact the rear side (4) of the light-transmitting element opposite the front side, for example, the input branch and the output branch being positioned on a common waveguide positioned perpendicular to the front surface. An optical assembly optionally coupled to the rear of a porous unit, as disclosed elsewhere in the Text, may enable the performance of optical measurements (e.g., in an efficient, simple, and / or well-controlled manner) on a fluid, such as a liquid, in a hole, from the rear of the porous unit (e.g., incident probe light entering the hole in a direction from rear to front, and light emitted from the hole to a photodetector in a direction from front to rear). Possible advantages of bonding, for example rigidly bonding, an optical assembly to the rear of a porous unit are that the porous unit and the optical assembly may together form a unit or cassette, such as a sensor unit or sensor cassette, which can be inserted into and removed from a (sensor) system or device, forming a consumable that can efficiently overcome problems associated with wear and / or contamination (such as contamination of the pores) of the porous unit by replacement, and that integration, for example, effective integration with optical peripherals, such as a light source and / or receiving unit such as a photodetector, can be enabled and / or facilitated via the optical assembly. In one embodiment, the optical assembly is such as that described in International Patent Application No. WO2021123441A1 (which is referred to as an optical subassembly), for example, as described in Figures 1 to 9 and the appendix to the above application, which are further specifically incorporated herein by reference. Input and output branching can be directed towards the coupling interface between the optical assembly and the translucent element, such as on the back of the translucent element.

[0106] According to one embodiment, an apparatus is presented comprising a porous unit, the apparatus further comprising a transparent element porous unit and a housing through which a channel defining the axial direction is passed, the channel comprising a sample space, and the porous unit with a front side is positioned to define a sensor surface for contact with the liquid, such as when the liquid is in the sample space, the sensor surface facing the sample space, and for example, the holes are configured with respect to the analyte in the liquid for diffusive liquid communication with the sample space. Possible advantages of having a housing that is optionally rigidly coupled to a porous unit, such as on the front of the porous unit, are that the porous unit and housing (and optionally further, an optical assembly) may together form a unit or cassette, such as a sensor unit or sensor cassette, which can be inserted into and removed from a (sensor) system or device, and may form a consumable that can efficiently overcome problems associated with wear and / or contamination of the porous unit (such as contamination of the pores) by replacement, for example, and may enable and / or facilitate integration with one or more peripheral devices, such as a (micro)fluidic system (and optical peripherals in the case of an optical assembly), for example, effective integration via the optical assembly. In one embodiment, the housing (and optionally, the optical assembly) is such as that described in International Patent Application No. WO2021123441A1, which is incorporated herein by reference in whole (the optical assembly may be referred to as an optical subassembly), for example, as described in Figures 1 to 9 and the appendix to the above application, which are further specifically incorporated herein by reference.

[0107] According to one embodiment, a device is presented comprising a porous unit, the device comprising a translucent element and an optical assembly and optionally a housing, wherein the porous unit forms a cassette, such as a coherent unit that can form a part of the device, and the cassette can be connected to the rest of the device operably and reversibly (optionally in a non-destructive manner). In a further embodiment, the cassette and the rest of the device can be connected by an intermediate fit, such as a reversible friction fit. An 'intermediate fit' is understood to be a fit that holds parts together tightly, but is not so tight that it cannot be disassembled, for example, without tools, for example, by human hands, such as an ordinary person. In further embodiments, different parts of the equipment are held together by mechanical locking members, such as one, more, or all of the following: pins (such as split pins or spring pins), click locks (such as locks, in which a spring-loaded engaging member located in one part engages with a cavity or edge of another part during assembly, so that the spring force must be weakened before disassembly), detent balls, hand-operated screws such as Tommy screws or wing screws. It may be understood that any of the mechanical locking members may perform the function of holding the parts together, however, any of the mechanical locking members may optionally be weakened or removed without tools, such as by human hands, such as by an ordinary person.

[0108] According to one embodiment, an apparatus is presented comprising a porous unit, the apparatus comprising a light-transmitting element and an optical assembly and optionally a housing, for example, the porous unit being a cassette operably and reversibly connectable to the rest of the apparatus.

[0109] According to one embodiment, a device is presented which is positioned to optically probe a liquid disposed inside a hole from the front, opposite the rear side. A possible advantage is that it may be possible to avoid the need for light to traverse the liquid outside the hole (e.g., in front of the front side) on its way to and from the hole, which could result in contributions to the optical probe signal from components in the liquid outside the hole (such as contamination). (Note that the hole itself may be beneficial in effectively filtering the liquid for the purpose of allowing signals to be obtained only from components small enough to enter the hole.)

[0110] According to one embodiment, a device is presented comprising one or more light sources, such as, and at least one photodetector, such as, one or more light sources and one or more photodetectors, wherein each of the one or more light sources and the photodetector is positioned on the front, opposite to the rear side, such as outside a translucent element on the same side as the rear side. This may be advantageous in promoting a simple and / or efficient device, such as optically probing a liquid disposed inside a hole from the front, opposite to the rear side.

[0111] According to one embodiment, the device is, One or more light sources are adapted to illuminate at least a hole in the light-transmitting element from the front, opposite the rear, and / or A device is presented in which a photodetector is arranged to receive light emitted from a hole, such as light emitted in response to illumination by one or more light sources, and the photodetector is adapted to generate a signal representing the received light, for example, primarily emitted, emitted from the hole in a direction away from the front in a direction facing the rear.

[0112] This may be advantageous in facilitating simple and / or efficient devices, such as optically probing a liquid disposed inside a hole from the front, opposite the rear side. According to one embodiment, the device is, One or more light sources are adapted to illuminate at least one hole in the light-transmitting element, for example, from the front, opposite the rear side, and the light from one or more light sources reaching the hole does not need to cross the volume outside the light-transmitting element, such as from the front, opposite the rear side, and / or A photodetector is presented which is positioned to receive light emanating from a hole, such as light emitted in response to illumination by one or more light sources, and the photodetector is adapted to generate a signal representing the received light, and the light emanating from the hole and reaching the photodetector does not need to traverse a volume outside the light-transmitting element, such as on the front side, opposite the back side, etc., as the hole is fluidly connected to it. A possible advantage is that by avoiding the light having to traverse a liquid outside the hole (such as in front of the front side) on its path to and from the hole, the contribution of components in the liquid outside the hole to the optical probe signal (such as its contamination) can be reduced, minimized, or eliminated (it should be noted that the hole itself may be beneficial in effectively filtering the liquid for the purpose of allowing signals to be obtained only from components small enough to enter the hole).

[0113] According to one embodiment, a device is presented which is configured to measure absorbance (optionally spectrally decomposed), such as the absorbance of a liquid in a well. The advantage of this may be that it allows information about the analyte in the liquid in the well, such as concentration information, to be obtained in a simple manner.

[0114] According to one embodiment, an apparatus is presented in which a data processing device is further configured to determine the concentration of an analyte or group of analytes in a liquid based on one or more signals.

[0115] Possible advantages include obtaining additional information, such as concentrations, in addition to obtaining one or more time response values. The concentration in the liquid can be determined as the stable or saturated concentration within the well. The concentration within the well can be determined, for example, based on modeling, calculation, and / or calibration.

[0116] According to one embodiment, a device is presented in which a pore is functionalized, for example, by one or more bioreceptors such as human serum albumin. 'Functionalized' can be understood as adding elements to the pore with a view to increasing the functionality of the pore for a particular (sensor) purpose, such as chemical or biological functionalization, which includes immobilizing chemical or biorecognition elements such as enzymes, antibodies, and aptamers within the pore (on both sides or in a matrix) to increase specificity or sensitivity.

[0117] According to one embodiment, an apparatus is presented in which a data processing device is configured to detect an analyte or group of analytes, such as by identifying an analyte from one or more other analytes having different molecular weights and optionally similar optical properties, based on one or more time response values ​​and the concentrations of one or more analytes in a liquid. 'Detecting an analyte or group of analytes' can be understood as both qualitatively detecting the presence of an analyte (YES / NO) and quantitatively determining its concentration, such as on an order, interval, or ratio type scale.

[0118] According to one embodiment, an apparatus is presented in which two default analytes are human serum albumin-bound bilirubin, such as HSA-bilirubin, and human serum albumin-unbound bilirubin, such as free bilirubin.

[0119] In particularly advantageous embodiments, plasma coloration by optically probed bilirubin is performed, for example, by using spectrally decomposed absorbance measurements, or by measuring spectrally integrated absorbance over a spectral range indicating the presence of bilirubin in a liquid subsample, for example, a spectral range of wavelengths from 380 nm to 750 nm, for example, a spectral range of wavelengths from 400 nm to 520 nm, or a predetermined bandwidth of approximately 455 nm.

[0120] According to one embodiment, a method is presented which further includes the step of identifying two or more default analytes, for example, that the two or more default analytes have different molecular weights and optionally similar optical properties based on one or more time response values, such as one or more time response values, and the concentration of one or more analytes in a liquid.

[0121] According to one embodiment, a light-transmitting element is presented in which the cross-sectional dimensions of the opening of the hole are 1 μm or less, for example 800 nm or less, for example 500 nm or less, for example 400 nm or less, and / or the length of the hole in the axial direction along the hole is less than 100 μm and optionally greater than 5 μm, for example less than 50 μm, for example less than 30 μm, for example 25 μm.

[0122] By using a pore having an opening on the front plane of a translucent element having a maximum cross-sectional dimension of approximately 1 μm or less, or preferably within the submicron range, for example, approximately 800 nm or less, for example, approximately 500 nm or less, or approximately 400 nm or less, any cellular components, including red blood cells, white blood cells, and platelets (thrombocytes / platelets), are prevented from entering the pore.

[0123] Even more surprisingly, pores with openings having a cross-sectional dimension of approximately 500 nm or less exhibit increased sensitivity compared to larger pores, such as pores with openings having a cross-sectional dimension of approximately 800 nm or more, but with the same total pore volume / volume porosity.

[0124] Most preferably, the pores have minimum apertures with their respective minimum pore volumes to allow efficient extraction of sufficiently large subsamples that can still be probed with an acceptable signal-to-noise ratio. Advantageously, the pores have apertures of about 30 nm or more, or 50 nm or more, or 100 nm or more, or about 200 nm or more.

[0125] Suitable pores can be produced from transparent polymer films having so-called track-etched pores, similar to those commercially available from IT4IP (IT4IP sa / avenue Jean-Etienne Lenoir1 / 1348 Louvain-la-Neuve / Belgium), with improvements such as the pores being closed at one end. Through-pores within the film can be closed, for example, by laminating a backsheet to the rear of the porous film, or by slowing ions through ion-impact tracks, and therefore the etched pores following these tracks, so that they stop within the transparent polymer film and form non-through-pores. The film is typically backed with a rigid transparent element to provide adequate mechanical strength for the light-transmitting element.

[0126] According to one embodiment, a porous material is presented in which the light-transmitting element is made of a transparent polymer. According to one embodiment, a porous material is presented in which the pores are track-etched in the light-transmitting element and, optionally, in one or more layers if present.

[0127] Transparent elements should preferably be made of a material that does not absorb light, and at the same time, it must be possible to produce non-penetrating holes within the material, for example, by track etching the material. Suitable materials for this purpose are polyethylene terephthalate (PET or PETE), or PET analogues (polyethylene terephthalate polyester (PETP or PET-P)), or polycarbonate (PC). Transparent elements may include a hydrophilic coating, for example polyethylene glycol (PEG), to increase diffusion into the pores. The hydrophilic coating may be selected according to the use of the transparent element. In some applications, the transparent element never dries out once used, and therefore it only needs to be hydrophilic at startup. In other uses of transparent elements, the transparent element requires a coating that keeps it permanently hydrophilic so that it remains usable when it dries out and is then re-wetted for further use.

[0128] According to one embodiment, the light-transmitting element is A translucent element is presented in which the porosity of a given volume of the translucent element containing pores is 50% to 5% by volume, for example, 30% to 10% by volume, for example, 15% by volume.

[0129] A pore creates porosity within the translucent element (or within a given region of the translucent element), accompanied by a corresponding front surface area where the pore openings are distributed. Porosity can be characterized with respect to the volume of voids created within the translucent element by the pore, i.e., the pore volume, which refers to the volume of the translucent element through which the pore penetrates. This volume is defined here as the volume between the front region where the pore is distributed and the same parallel region shifted into the translucent element by the maximum depth of penetration of the pore into the translucent element, as seen in the axial direction perpendicular to the front of the translucent element.

[0130] In addition, porosity can be further characterized with respect to the integrated pore volume, which is equal to the subsample volume to which the optical probe is available. The pore volume can be simply expressed as the equivalent pore volume depth DELTA, which is the pore volume referred to with respect to the corresponding front region in which the pore openings are distributed. Thus, the porosity of a translucent element can be converted to the equivalent pore volume depth DELTA as follows: A pore having an opening within a given front region A has a total pore volume V. The equivalent pore volume depth is then calculated as the total pore volume divided by the given front region: DELTA = V / A.

[0131] According to one embodiment, the light-transmitting element is A translucent element is presented in which the equivalent pore volume depth (DELTA) is less than 20 μm, for example less than 10 μm, for example 5 μm or less, and the equivalent pore volume depth (DELTA) is obtained by dividing the total volume of pores (V) by the front region (A) in which the pore openings are distributed.

[0132] This yields small subsamples with the respective concentrations of the relevant components. Small subsample volumes are desirable to facilitate rapid subsample exchange, thereby reducing the response time of the light-transmitting element and the cycle time of measurements using the light-transmitting element. Small subsample volumes are even more desirable to avoid the attenuation effect of the boundary layer of the plasma fraction in the whole blood sample near the front of the light-transmitting element. Such attenuation effects can otherwise occur in small, static samples, where, for example, if the equivalent pore volume depth exceeds a critical value, red blood cells may interfere with the efficient diffusion exchange of the relevant components from the whole blood sample volume toward the boundary layer near the front of the light-transmitting element.

[0133] Preferably, the equivalent pore volume depth DELTA is at least 1 μm, alternatively at least 2 μm, or in the range of 3 μm to 5 μm, where the equivalent pore volume depth is defined as above. Larger subsample volumes are desirable to achieve better signal-to-noise levels, as larger subsample volumes contribute to optically probed information regarding relevant components in the plasma.

[0134] Furthermore, according to some embodiments, a useful compromise between reducing response time, reducing cycle time, and / or avoiding diminishing effects in small static whole blood samples or liquids, and on the other hand, the required or desired signal-to-noise ratio, can be found for equivalent pore volume depths DELTA in the range of 1 μm to 20 μm, preferably in the range of 2 μm to 10 μm, or about 4 μm to 5 μm.

[0135] Advantageously, according to one embodiment, the translucent element is supported by a translucent backing material attached to the rear side of the translucent element. This achieves enhanced mechanical stability. According to one embodiment, a light-transmitting element is presented in which the transparent backing slide of the light-transmitting element is provided with surfaces angled to 45° to 75°, such as a surface angled to 60° with respect to the front surface, in order to minimize the effect of the shift in refractive index between the outside air and the transparent backing slide (for example, there is no 90° corner on the outside of the light-transmitting element, and the corner is “cut off” to obtain a surface of 45° to 75°, for example, 60° instead).

[0136] Furthermore, according to one embodiment of the light-transmitting element according to the present invention, the transparent backing material attached to the back of the light-transmitting element has a thickness such that a 60° prism (i.e., there is no 90° angle on the outside of the light-transmitting element, and the angle is "cut off" to instead obtain a 60° surface) is positioned on the outside of the transparent backing material, so that light from the light source and to the detector has an increased angle of incidence for the light reaching the perforation area. For example, a possible advantage of having a 60° prism is also that it increases the chance of light traveling inside the light-transmitting element because light is reflected off the surface of the backing material, and therefore there are multiple reflections before the emitted light reaches the detector.

[0137] Furthermore, according to one embodiment of the translucent element according to the present invention, the inner wall surface of the pore is hydrophilic and, for example, covered with a hydrophilic coating. This achieves efficient capillary drive filling of the dry pore with liquid. Moreover, the hydrophilic coating prevents certain hydrophobic substances, such as hydrophobic dyes, hemoglobin, and other proteins, from accumulating inside the pore, otherwise resulting in gradual fouling of the sensor that would otherwise be difficult to wash away with aqueous solutions. Thus, an improved device for detecting analytes in liquids with high-speed and highly reliable response can be enabled.

[0138] According to one embodiment, a light-transmitting element is presented in which the inner wall surface of the pore is covered with a hydrophilic coating. Furthermore, according to one embodiment of the apparatus according to the present invention, the light source is configured to provide an obliquely incident illumination beam from the rear of the translucent element, and the illumination angle is defined as the angle of the incident beam with respect to the surface normal of a reference plane defined by the front of the translucent element. Thus, an increased optical interaction length is achieved, thereby enhancing the interaction of the incident light with the contents of the hole before the incident light leaves the probe region for detection by the detector. Moreover, penetration of probe light into the liquid through the hole opening is prevented due to the reduced obvious cross-section of the hole opening, as well as increased scattering that spreads the light into the probe region rather than into the liquid space on the opposite side of the reflective layer through the hole opening.

[0139] The light source can, in principle, be any light source that transmits light in a region where the analyte in the pore absorbs light for the system to function, but preferably the light source should have a flat spectral characteristic, i.e., the spectrum does not contain peak amplitude, because a flat characteristic results in a better response. If the light source has a non-flat spectrum, i.e., if the light source has peak amplitude, a slight change in the peak may be misinterpreted as a change in absorption. Light-emitting diodes are often preferred due to their properties in terms of size, weight, efficiency, etc. Furthermore, according to one embodiment of the sensor according to the present invention, the detector is configured to collect light that is obliquely emitted from the rear side of a translucent element, and the detection angle is defined as the angle of propagation of the emitted light toward the detector with respect to the plane normal of a reference plane defined by the front side of the translucent element. The detector is configured to collect light that is emitted in response to illumination by a light source in an optical probe configuration. Detecting light emitted obliquely from the back of the translucent element reduces the contribution to the detection signal from light emitted from the whole blood sample that leaks into the probe area through the front surface and one or more layers (if any).

[0140] The detector may be a photodiode or spectrometer capable of detecting absorbance across the entire spectrum. Alternatively, an array or diodes may be used, each emitting light at a different wavelength, and the photodiodes are used as detectors. The diodes may be multiplexed to emit light at different intervals. Absorbance is then found by comparing the light emitted from the diodes at that particular interval with the light detected by the photodiodes.

[0141] Furthermore, according to one embodiment of the apparatus according to the present invention, the incident surface and the detection surface intersect by surface normals and enclose an azimuth angle of at least 0 degrees and less than 180 degrees, preferably less than 160 degrees, preferably less than 130 degrees, or preferably less than about 90 degrees, the incident surface is stretched by the direction of the illumination beam and the surface normal to the reference plane, and the detection surface is stretched by the direction of the outgoing light propagation toward the detector and the surface normal to the reference plane. Thus, the contribution of glare from partial reflection at the optical interface before passing through the probe region to the detection signal is reduced. Such glare of light that did not interact with the subsample within the probe region does not contain relevant information and is therefore detrimental to the signal-to-noise ratio.

[0142] The optical probe light can be implemented by any suitable optical probe configuration. Such an optical probe configuration may involve directing the light beam only to the rear side of the translucent element and directing the input of the photodetector to the illumination area. The optical configuration may include further optical elements that improve the coupling of the probe light into the translucent element and the coupling of the light emanating from the translucent element into the detector input. Such optical elements may include one or more prism and / or lens configurations that are directly attached / bonded to the rear side of the translucent element. Preferably, the coupling optical system corresponds to the “reflectivity” of the optical probe, and the incoming probe light and detected outgoing light are maintained on the same side of the front surface of the translucent element. Further improvements may be found, for example, in enhancing the optical interaction of the probe light with the hole by coupling the probe light into the transparent element at the first end, forcing the light within the probe region to propagate essentially along the front surface of the transparent element in a direction parallel to the front of the transparent element and traverse the hole, thereby collecting the emitted light from another end of the transparent element that may traverse the first end or the opposite end.

[0143] As light sources age, their characteristics may change, for example, by emitting less light, or drift may affect the peak amplitude. This can be compensated for by using a feedback calibration process in which the detector measures light received through a transparent element, such as a transparent element, in situations where it is expected that the pores in the transparent element are clean, i.e., do not contain molecules in the pores that absorb light. If the amplitude of the received light is smaller than expected, the feedback loop to the light source can be controlled to increase the current or voltage to the light source to compensate for the degradation of the light source. Alternatively, if the light source changes its characteristics, the calculation of the actual absorbance at the time of measurement can be adjusted for such changes in emitted light compared to the original factory calibration.

[0144] Advantageously, according to one embodiment, the detector includes a spectrophotometer, and the optical probe device is configured for spectrophotometric analysis of light emitted from a probe region within a translucent element. This allows for the decomposition of the spectral characteristics of one or more relevant components in the light emitted from a subsample within the probe region.

[0145] Furthermore, according to a particularly advantageous embodiment, the optical probe device is configured to measure absorbance. Thus, a remarkably significant signal is obtained in a relatively simple optical setup. This allows for easy integration of the sensor with more complex analytical setups, such as blood analyzer systems.

[0146] Several optically active components can be found in blood, for example, bilirubin, carbon dioxide (CO2), Patent Blue V, and methylene blue. Translucent elements allow for the detection of bilirubin with sufficient sensitivity to report normal adult bilirubin concentrations. The dye Patent Blue V can be used in lymphangiography and sentinel lymph node biopsy to color lymphatic vessels. It can also be used in plaque-staining tablets as a staining agent to show dental plaque. Methylene blue is used in the treatment of high methemoglobin levels in patients and in the treatment of some urinary tract infections.

[0147] When analyzing the resulting spectra from translucent elements, it was found that absorbance spectra from whole blood or plasma have a negative baseline. This negative baseline is caused by the translucent element reflecting a higher proportion of incoming light to the detector compared to rinse when measuring whole blood or plasma. This effect can be observed at high wavelengths (600-700 nm) where hemoglobin does not absorb light. This effect stems from a higher refractive index due to the higher protein content in plasma compared to rinse. This effect is approximately 5 mAbs compared to hemoglobin, which has approximately 15 mAbs at the hemoglobin peak wavelength (416 nm) (absorbance Abbs is a unit of light, where 1 Abbs represents a 10% attenuation of the original light intensity, and mAbs refers to milliabs). Detectors that utilize the determination of a baseline of light intensity from the light source can detect the protein (human serum albumin, HSA) content of whole blood samples with a detection limit of approximately 1-5 g / L.

[0148] However, the amplitude (and sign of the amplitude) of the baseline (shift) depends on the geometry of the setup (a positive baseline is observed when the angle between the incoming and outgoing light is small (less than approximately 40°), and the opposite when the angle is large).

[0149] Translucent elements can be used as reading devices for color-developing / color-consumable assays. An advantage is that it eliminates the need to produce plasma before the assay. The following types of assays can be used with light-transmitting elements: • A sandwich assay in which the receptor ligand is bound inside the membrane channel. • Assays in which some components bind within the pore, such as the bromocresol green albumin assay, which uses bromocresol green to form a colored complex, particularly using albumin. The intensity of the color, measured at 620 nm, is directly proportional to the albumin concentration in the liquid. • The transfer of an amino group from aspartate to α-ketoglutarate results in the production of glutamate, and the resulting colorimetric analysis (450nm) product is proportional to the present AST enzyme activity, for example, as an aspartate aminotransferase (AST) activity assay kit.

[0150] Translucent elements can also be used in non-medical applications such as beer brewing, wastewater analysis, food testing, and dye production. In beer brewing, precise color is desired. Translucent elements can be used to determine whether beer has the desired color by measuring a liquid and comparing it to a reading from a liquid of the correct color. Wastewater can be analyzed for the presence or absence of certain components. In food testing of liquids such as milk, juice, and other slurries, translucent elements can be used to analyze the presence or absence of certain components or analytes. Other chemical reactors, such as the dye industry, may use translucent elements to obtain desired color, content, or other chemical properties of their liquids.

[0151] Advantageously, according to some embodiments, a translucent element, or a blood analysis system comprising a translucent element, further comprises a processor configured to compare the signal generated by the detector with a predetermined calibration standard in order to develop a quantitative measure of the analyte level in the liquid.

[0152] More advantageously, according to some embodiments, the calibration standard is obtained for a dye-based calibration solution, such as an aqueous solution containing a tartrazine dye. Preferably, the dye-based aqueous solution is prepared from a typical rinse solution to which a calibration dye such as tartrazine is added.

[0153] According to some embodiments, a method for optically detecting an analyte in a liquid includes the steps of: providing a translucent element as disclosed above; contacting the translucent element with a reference solution to fill a pore with a reference solution; contacting the front side of the translucent element with the liquid; waiting for a diffusion time to allow the analyte in the liquid to diffuse into the pore and stabilize; optically probe the liquid inside the pore; and establishing the analyte level of the liquid based on the results of the optical probe. Preferably, the reference solution is an aqueous solution that has affinity for the liquid and, in particular, for fractions of the liquid that may enter the pore, such as liquids for rinsing, calibration, and / or quality control. Most advantageously, the analyte is optically detected in the pore by a color change resulting from the presence of the analyte in a representative amount in the extracted subsample.

[0154] Advantageously, according to some embodiments, the optical probe includes illuminating a translucent element from behind with probe light and performing spectrophotometric analysis of the light emitted from behind the translucent element as an optical response to the probe light.

[0155] Advantageously, according to some embodiments, the optical probe measures absorbance. Advantageously, according to some embodiments, the method further includes a step of comparing the photoresponse to a predetermined calibration standard in order to develop a quantitative measure of the analyte level in a liquid.

[0156] More advantageously, according to some embodiments of the present method, the calibration standard is obtained for a dye-based calibration solution, such as an aqueous solution containing a tartrazine dye. Preferably, the dye-based aqueous solution is prepared from a typical rinse solution to which a calibration dye such as tartrazine is added.

[0157] According to one embodiment, the method is such that the analyte is Cellless hemoglobin, Bilirubin, and / or, This is the total protein content. A method is presented.

[0158] In one embodiment, a method is presented in which the liquid is either a whole blood sample or the plasma phase of a whole blood sample. According to one embodiment, the method is as follows: A step of bringing a translucent element into contact with a reference solution, for example, to fill the pores with the reference solution by diffusion, and / or A method is presented which further includes the step of waiting for a diffusion time to allow the analyte in the liquid to diffuse into the pores and stabilize.

[0159] In the context of point-of-care measurement systems or devices (also referred to in the art as 'bedside' systems or devices) and similar laboratory environments, blood gas analysis is often performed by users, such as nurses, who may not be trained in the use of blood gas analyzers.

[0160] According to another aspect of the present invention, the use of an apparatus according to a second aspect of the present invention is presented for point-of-care (POC) determination of a difference scale, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes in a liquid such as whole blood, including in a whole blood sample.

[0161] POC measurement is also referred to as 'bedside' measurement in the art. In this context, the term 'point-of-care measurement' should be understood to mean a measurement performed in close proximity to the patient, i.e., a measurement not performed in a laboratory. Therefore, according to this embodiment, a user of a device such as a blood gas analyzer performs the measurement of a whole blood sample in a handheld blood sample container in close proximity to the patient from whom the blood sample is taken, for example, in a hospital room or ward housing the patient's bed, or in a nearby room in the same department. In such use, the user's level of expertise often varies from novice to expert, and therefore, the ability of a blood gas analyzer to automatically output commands that match the skill level of each individual user based on sensor input is particularly beneficial in such environments.

[0162] According to the third aspect, a computer program such as a computer program product, where the program is executed by a computer, Receiving one or more signals representing received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light. Determining one or more time response values ​​based on one or more signals, and Determining a difference measure, such as an absolute or relative difference, in the concentrations of two or more analytes based on one or more time response values. A computer program is presented that includes instructions for the computer to perform the following action.

[0163] In an alternative third aspect, a computer program is presented, such as a computer program product, which includes instructions causing an apparatus according to the second aspect (or an apparatus according to the second aspect in which a data processing device is further operably connected to one or more light sources and / or detectors) to perform a step of the method of the first aspect.

[0164] In a further embodiment, a computer-readable medium is presented on which a computer program of the third embodiment and / or an alternative computer program of the third embodiment are stored.

[0165] Each of the first, second, and third aspects of the present invention can be combined with any of the other aspects. These and other aspects of the present invention will be revealed and clarified with reference to the embodiments described below.

[0166] The apparatus, method, and computer program according to the present invention will be described in more detail thereafter with reference to the accompanying figures. The figures illustrate one way of implementing the invention and should not be construed as limiting to other possible embodiments that fall within the scope of the accompanying set of claims.

[0167] Preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings. [Brief explanation of the drawing]

[0168] [Figure 1] This diagram schematically shows a porous unit according to one embodiment under operating conditions. [Figure 2] This diagram schematically shows a porous unit that comes into direct contact with a liquid. [Figure 3] This diagram schematically shows a porous unit containing a low refractive index layer. [Figure 4] This diagram schematically shows a cross-sectional side view of the measurement cell. [Figure 5] Figure 4 is a top elevation view of the measurement cell. [Figure 6] Figure 6a is a schematic cross-sectional side view of a measuring cell having a prism-like structure on the outside of a transparent backing material according to a further embodiment. Figure 6b is a schematic cross-sectional side view of a measuring cell having a prism-like structure on the outside of a transparent backing material according to a further embodiment. [Figure 7] Figure 6a is a top elevation view of the measurement cell. [Figure 8] This graph shows an example of the reaction of bilirubin in plasma. [Figure 9] This graph shows the IR spectra of CO2 and H2O (obtained from http: / / www.randombio.com / co2.html on November 8, 2016). [Figure 10] This graph shows an example of using a dye (taltrazine) as a calibration and quality control standard for spectrophotometric measurements. [Figure 11] This graph shows examples of responses to different concentrations of human whole blood protein (HSA). [Figure 12] This graph shows the time-resolved signals for the porous unit. [Figure 13] This figure shows multiple time-resolved signals at wavelengths WL1 and WL4 for four different concentrations of cfHb. [Figure 14] This figure shows characteristic tau times derived from the data in Figure 13. [Figure 15] This figure shows tau_ratio and tau_WL1 as functions of cfHb. [Figure 16] This figure shows tau_ratio and tau_WL1 as functions of cfHb. [Modes for carrying out the invention]

[0169] References to WL1 throughout this application may be understood to refer to a first specific wavelength, such as 415 nm (advantageously, it may be used due to its identity or approximation to the hemoglobin peak wavelength at 415 nm or 416 nm).

[0170] References to WL4 throughout this application may be understood to refer to a second specific wavelength, such as 450 nm (advantageously, due to its remoteness to the hemoglobin peak wavelength at 415 nm or 416 nm, which may make 450 nm suitable for use as a reference wavelength).

[0171] Figure 1 schematically shows a cross-sectional view of the porous unit 1. The porous unit 1 comprises a translucent element 2 with a front side 3 and a rear side 4. The front side 3 is provided with one or more layers 5 that allow internal reflection (in an alternative embodiment, one or more layers are absent, in yet another alternative embodiment, one or more translucent layers are present, and in yet another alternative embodiment, one or more light-absorbing layers are present). The translucent element 2 further comprises non-through holes 6 that extend from an opening 7 in the front side 3 through one or more layers 5 into the bulk of the translucent element 2, where the non-through holes 6 terminate. As shown in the schematic diagram of Figure 1, the holes do not need to be perpendicular to the front side 3 or parallel to each other. Under operation, the front side 3 of the porous unit having the hole opening 7 is in contact with the liquid 99. The liquid may have a cell fraction or specific fraction containing red blood cells or particles 98, and a plasma fraction / liquid fraction 97 having the relevant component to be detected, in this case the analyte 96. The cross-sectional dimensions of the opening 7 of the pore 6 are determined to allow the analyte 96 to enter the pore 6 while preventing red blood cells or particles 98 from entering the pore 6.

[0172] The pores 6 can be pre-filled with liquid 99 and a rinse solution 8 that is particularly compatible with the liquid fraction 97. When liquid 99 comes into contact with the front side 3 of the porous unit 1 having the pre-filled pores 6, diffusion of the analyte 96 into the pores 6 occurs, thereby establishing a subsample 9 inside the pores 6 having a concentration of analyte 96 that represents the concentration of analyte 96 in liquid 99.

[0173] The rinse solution 8 used to pre-fill the pore 6 can be any aqueous solution with affinity for the liquid 99. Suitable rinse solutions include those commonly used in blood parameter analyzers for rinsing, calibration, and / or quality control purposes. Such solution compositions typically include organic buffers, inorganic salts, surfactants, preservatives, anticoagulants, enzymes, colorants, and sometimes metabolites. Photodetection is performed from the rear using an optical probe configuration with a light source 10 and a detector 20. The light source 10 illuminates the probe volume in the porous portion of the translucent element 2 from the side of one or more layers 5 facing away from the liquid 99. The probe light 11 is an obliquely incident beam that interacts with the subsample 9 in the pore 6. The emitted light 21 is detected by the detector 20, which is similarly positioned to view the probe region at an oblique angle. The detector 20 generates a signal representing the emitted light, which, due to its interaction with the subsample 9 in the pore 6, contains information in particular about the concentration of the analyte 96. By processing the generated signal, it is possible to determine the level of the analyte in the liquid. With calibration, the level of the analyte in the liquid can be quantitative. The optical probe techniques used for all measurements in the following examples utilize spectrally resolved absorbance measurements within the visible range of the electromagnetic spectrum, for example, in the range of approximately 380 nm to 750 nm, approximately 400 nm to 520 nm, or 455 nm or approximately 455 nm.

[0174] The measurement cycle is concluded by rinsing the liquid with a rinse solution, such as rinse solution 8, which is used to pre-fill the holes 6. The sensor device is then initialized again and ready to receive the next liquid.

[0175] Figure 2 shows a porous unit similar to that in Figure 1, except that the porous unit in Figure 2 does not contain one or more layers, i.e., the translucent element is in direct contact with the liquid (one or more layers are not present in front of the translucent layer).

[0176] Figure 3 shows a porous unit that includes a low refractive index layer 5 (compared to the refractive index of the transparent element) at the interface between the light-transmitting element and the light layer 5, where the light reaches the interface from the light-transmitting element, enabling internal reflection such as total internal reflection.

[0177] Figures 4 and 5 schematically show the measurement cell 100, which contains a porous unit 1 with its front side 3 facing into a liquid volume 101 inside the measurement cell 100. The liquid volume communicates with liquid input and output ports (not shown) for supplying and discharging liquid, and for performing priming, rinsing, and washing steps. The rear side of the porous unit is mechanically stabilized by a transparent backing slide 30, which also acts as a window for optical access to the probe area from the rear side 4 of the porous unit 1. The optical probe is implemented using a configuration with a light source 10 and a detector 20 as described above in relation to Figure 1, and the directions of the probe beam and detection are inclined at angles to the surface normal on the surface of the front side 3 of the porous unit 1, respectively. Furthermore, as is best seen in Figure 5, the plane of the incident probe light 11 and the plane of the detection 21 intersect each other preferably at an angle of less than 180 degrees, and preferably at a sharp angle of about 90 degrees or less, to avoid glare effects. In the measurement of the example provided below, the plane of the incident probe light 11 and the plane of the exit light 21 are positioned symmetrically with respect to a direction parallel to the plane of symmetry of the small mirror element 52.

[0178] Figures 6a, 6b, and 7 schematically show a transparent backing slide 31 in direct contact with the back side 4 of the translucent element 2 of the porous unit 1. When the incident probe light 11 enters the backing slide 4 of the translucent element 2, which has a surface in the 60° prism 32, the shift in refractive index between air and polymer does not affect the incident probe light 11, and the light enters the hole 6 (invisible) of the translucent element 2 without a change in the angle of light, and the emitted light 21 reaches the detector 20. Figure 6b shows that the incident probe light 11 may be reflected several times within the transparent backing slide 31 before the emitted light 21 reaches the detector 20. Furthermore, as is best seen in Figure 7, the plane of the incident probe light 11 and the plane of the outgoing light 21 intersect each other preferably at an angle of less than 180 degrees, and preferably at a sharp angle of about 90 degrees or less, to avoid glare effects, and the prism 32 does not affect either the incident probe light 11 or the outgoing light 21.

[0179] In Figures 1, 4, 5, 6a, 6b, and 7, the hole is optically probed from the rear side 4 of the light-transmitting element 2, that is, the incident probe light 11 into the hole 6 travels in the direction from the rear side 4 to the front side 3, that is, it enters the light-transmitting element 2 via the rear side 4 in the direction from the rear side 4 to the front side 3, and the light 21 emitted from the hole 6 to the receiving unit such as the photodetector 20 is emitted in the direction from the front side to the rear side, that is, from the rear side away from the front side.

[0180] In Figures 1, 4, 5, 6a, 6b, and 7, incident and emitted light are depicted as propagating through air or empty space. However, in embodiments, the incident and emitted light may propagate within an optical assembly comprising a light guide core, the light guide core comprising an input branch, an output branch, and a coupling interface positioned to contact the rear 4 of the translucent element 2 opposite the front 3. For example, the input and output branches are located on a common waveguide positioned perpendicular to the front surface. [Examples]

[0181] Translucent element with reflective palladium layer - Stability measurement Referring below to Figures 8 to 11, the data from the test run measurements are provided as examples illustrating different aspects of the performance of the porous mirror, where the porous mirror corresponds to a porous unit including a reflective palladium layer in front of a translucent element (in this example, a slab), and the reflective palladium layer is adapted to reflect light reaching the reflective palladium layer from behind the translucent element, and the data in Figures 8 and 11 from the porous mirror are presented as useful examples for understanding the porous unit according to embodiments of the present invention.

[0182] The porous microscopes used for these example experiments were produced from a transparent PETP film with a total thickness of 49 μm, providing linear holes etched on one side. The holes were treated with hydrophilic PVP to have a pore depth of 25 μm and a pore diameter of 0.4 μm. The pore density was 1.2E8 / cm². 2 Therefore, the pores are non-penetrating, with openings on one side of the PETP film, and essentially terminate halfway into the PETP film, which functions as a translucent slab. The porous side of the film (translucent slab) is sputter-coated with palladium at a 0-degree angle and with an approximate layer thickness of 100 nm. This provides a metallic coating on the porous front side of the film (translucent slab) and a small coating on one side of the inside of the pores, thus forming a small concave mirror at the mouth portion of the pores adjacent to the opening of the pores facing forward. The sputtered porous PETP film is laminated to a custom cuvette using double-sided adhesive tape so that the concave surface of the small mirror in the pores points midway between the light guide from the light source and the light guide from the spectrometer input. Approximately 10 μL of silicone rubber droplets are pipetted onto the film, and then a coverslip is fixed to the rear side of the film as mechanical support for the sensor film (translucent slab). The porous microscope is mounted on a test stand for automated handling of liquid, time intervals, and data sampling. Data acquisition takes approximately 3 seconds and is delayed up to 14 seconds after liquid acquisition.

[0183] The test stand is equipped with two light-emitting diodes (purple and 'white' LEDs) as light sources and a miniature spectrometer as a detector. The standard slit in the miniature spectrometer has been replaced with a 125 μm slit to increase light and sensitivity. Since the measurement is a reflectance measurement, both the light source and detector are placed on the back side (non-porous side) of the porous film. The porous metal-coated side of the film is positioned inside the measurement chamber, and therefore the mirror and pores are directly exposed to the liquid within the chamber. Light from the two photodiodes is guided through a common conductor optical fiber with lenses at its ends to focus the light onto a small spot (approximately 2 mm × 2 mm) on the porous mirror film. Referring to Cartesian coordinates, the surface of the film (the front side of the translucent slab) can be defined as the ZX plane of the coordinate system. Light enters the outer surface of the film (the back side of the translucent slab) at a 45° angle to the Y axis, i.e., the surface normal to the ZX plane (and in the YZ plane of the coordinate system). The detector is positioned at a polar angle of 60° with respect to the Y-axis and rotated relative to the YZ plane by an azimuth of 90° with respect to the plane of incidence of the light source (e.g., in the YX plane). The relatively high angles of the light incidence and detection directions with respect to the Y-axis result in improved detection sensitivity for hemoglobin, as the collected light travels through a larger length of the subsample within the pore.

[0184] The liquid is prepared by mixing bilirubin with the whole blood sample. The plasma-based interference solution is prepared by mixing the interference substance with the plasma to a specific level. The plasma is produced by centrifugation at 1500G for 15 minutes. As a reference, the absorbance spectra of the centrifugated plasma from all whole blood samples to be tested are also measured on a Perkin Elmer Lambda19 UV-Vis spectrometer.

[0185] Spectroscopic Figure 8 shows spectrally resolved absorbance data for two liquids: one containing bilirubin-containing plasma and the other containing only plasma. A prominent peak is observed at a wavelength of approximately 455 nm, and the maximum absorbance values ​​for the different liquids clearly expand linearly according to their bilirubin content.

[0186] Spectroscopic Figure 9 shows the spectrally decomposed infrared data for carbon dioxide (CO2) and water (H2O). The non-overlapping peaks of CO2 compared to water indicate that the CO2 content can be determined using the porous microscope of the present invention in a CO2-containing fluid, even if water is present in the fluid.

[0187] Spectral Figure 10 shows an example using a series of spectroscopically resolved absorbance data obtained for a dye-based calibration solution, and for comparison, data obtained for a rinse solution. The spectra were obtained in a continuous cycle immediately following each other. The dye-based calibration solution is a rinse solution with the addition of 0.5 g taltrazine per 1 L of rinse. The series of measurement solutions are as follows: firstly, the rinse solution, then the dye-based calibration solution, then the rinse solution again, and again the same dye-based solution, with all three consecutive measurements performed on the rinse solution. All spectra are plotted on the same scale and on each other. The experiment again demonstrates very good stability and reproducibility of the obtained results. More importantly, the data show a remarkably clear separation of the two dye-based solution spectra that match on each other, and similarly, all five rinse solution spectra that match on each other. Note that all optical data are probed in the probe volume of a porous microscope. This demonstrates highly efficient and complete diffusion exchange for the extraction and rinsing of subsamples within the pores, even when using dye-based spectrophotometric calibration solutions, such as the tartradine-stained rinse solution mentioned above.

[0188] Spectroscopic Figure 11 shows spectrally resolved absorbance data with a negative baseline, caused by a higher refractive index due to the higher protein content in plasma compared to rinse. The porous microscope reflects a higher proportion of incoming light to the detector when measuring whole blood or plasma compared to rinse. This effect is observed at higher wavelengths (600-700 nm) where hemoglobin in whole blood does not absorb light. This effect is approximately 5 mAbs compared to hemoglobin, which has approximately 10-15 mAbs at the hemoglobin peak wavelength (416 nm). It is possible to detect the protein (HSA) content of whole blood samples with a detection limit of approximately 1-5 g / L. Two different HSA concentrations (20% and 8%) were measured, with the higher concentration also being measured by free hemoglobin in the liquid (i.e., hemoglobin outside red blood cells). The presence of hemoglobin in the liquid only affects the portion of the spectrum below 600 nm. Above 600 nm, HSA content is the primary influence on the spectrum, and a more negative baseline indicates a higher protein content in the whole blood sample.

[0189] While the devices and methods of the present invention are discussed in particular in relation to the detection of bilirubin in a wide range of embodiments, the devices and methods disclosed herein are equally applicable to the detection of other optically active substances in plasma fractions of whole blood samples or in liquids, where the term “optically active” refers to substances that can be directly detected by spectroscopic optical probe techniques. Such substances may include, but are not limited to, metabolites, pharmaceutical substances, drugs, or vitamins.

[0190] Layerless translucent element - multiple time-resolved (transient) signals Figure 12 shows the optically detected (absorbance) signals for a setup similar to the one used to acquire the data presented in Figures 8–11, and in particular, for a setup where the porous unit is in direct contact with the liquid (i.e., no layers). All scales are linear. The horizontal axis represents time in seconds. The vertical axis represents the optical (absorbance) signal. All curves show time evolution (diffusion), and the subgraphs, from left to right, represent liquids with hematocrit (Hct) levels of 0, 45, 55, and 65%, respectively. In each subgraph, four curves (or a set of markers that effectively depict four curves) are shown, each corresponding to a different wavelength (however, the same four wavelengths (WL1, WL2, WL3, and WL4) are used in each subgraph).

[0191] The porous unit and configuration may be similar to, or otherwise similar to, the porous mirror and configuration described with respect to Figures 8 to 11, and modifications to the configuration in particular include the absence of one or more layers on the front side of the porous unit (and in particular, the absence of reflective, metallic, or palladium layers), such that the porous unit is in direct contact with the liquid.

[0192] Time response values ​​that enable the identification of Hap-cfHb and cfHb. Haptoglobin (Hap) binds to cell-free hemoglobin (cfHb) and transports it to the liver, where it is broken down and iron (Fe) can be reused. Plasma contains an average of approximately 160 mg / dL of Hap, which can bind approximately 100 mg / dL of cfHb. During in vivo hemolysis, Hap is rapidly depleted, so determining haptoglobin-bound cell-free hemoglobin (Hap-cfHb) can potentially be used to determine whether hemolysis occurred in vivo or in vitro. This example demonstrates the possibility of quantifying Hap-cfHb using time-response values, as obtained by embodiments of the present invention. conclusion Haptoglobin determination is particularly relevant in samples with a hemolysis interval exceeding a short interval (100-165 mg / dL cfHb). In severely hemolyzed samples (cfHb > 330 mg / dL), possible corrections (for values ​​affected by interference due to cfHb, etc.) are not very accurate. Therefore, the presence of Hap may only need to be identified within the hemolysis interval of 100-330 mg / dL. The presence of haptoglobin in a hemolyzed sample can be identified at key hemolysis intervals by an increase in the ratio of the characteristic ('tau'-) time tau_WL1 at the first wavelength to the characteristic ('tau'-) time tau_WL4 at the second wavelength, where 'tau' is determined as a feature in the first-order time-invariant system, as previously described. The tau_WL1 / tau_WL4 tau increase is brought about by Hap binding to cfHb and, therefore, by increasing the mean MW and diffusive tau. The WL4 signal is used as an internal tau reference within the sample. The results are groundbreaking because they demonstrate the ability of the apparatus having a translucent element according to the present invention to distinguish between two compounds having the same optical properties (at least for the above apparatus) but different molecular weights (MW). This is not possible with conventional spectrophotometers. Hap can be particularly well detected in samples with a cfHb level of 165 mg / dl or around that level. data When cfHb binds to Hap, the MW of the complex increases. This instrument allows for the determination of the time constant for signal construction and the determination of whether Hap is present in the sample. To obtain a value insensitive to interference, the tau (understandable as the characteristic time) of the cfHb signal at WL1 (understandable as the first wavelength) can be compared to the tau of the plasma signal (WL4 (understandable as the second wavelength)). Average plasma protein content is colorless, but the higher refractive index (RI) of plasma causes a signal at all wavelengths (WLs), and no Hb absorbance is present at WL4. Determining the tau_ratio (tau_ratio=100*((tau_WL1-tau_WL4) / tau_WL4) therefore elucidates whether the sample contains Hap.

[0193] Figure 13 shows multiple time-resolved normalized signals at wavelengths WL1 and WL4 at four different concentrations of cfHb. The figure shows that at concentrations of 0 and 330 mg / dL, the characteristic tau times are approximately similar, but at 165 mg / dL, WL1 is slower than WL4, and the opposite is true at 1000 mg / dL. As previously mentioned, mean serum Hap can bind approximately 100 mg cfHb, and therefore the tau ratio is expected to be less affected at ccfHb = 1000 mg / dL (ccfHb is an abbreviation for the concentration c of cell-free cf hemoglobin Hb). Similarly, at ccfHb 0 mg / dL, cfHb is absent, and tau_WL1 is determined by the same protein that constitutes the WL4 signal, and therefore the tau_ratio is expected to be close to zero.

[0194] Figure 14 shows characteristic tau times, as derived from the data in Figure 13. Figure 15 shows tau_ratio(tau_WL1 / tau_WL4) and tau_WL1 as a function of ccfHb. The samples are either hemolyzed (HB) samples or blood samples (WB-HSA) in which plasma is replaced with 8% HSA to obtain a Hap-free sample. All samples contain a 45% hematocrit (Hct.) value.

[0195] As can be observed in Figure 15, the HB sample shows a higher tau ratio at ccfHb = 165 mg / dL compared to 0 and 1000 mg / dL. Figure 16 shows tau_ratio(tau_WL1 / tau_WL4) and tau_WL1 as a function of ccfHb. The sample is either hemolyzed plasma (PL) or 8% HSA (PL-HSA) to obtain a Hap-free sample. Both sample types are red blood cell-free.

[0196] As can be observed in Figure 16, the PL sample shows a higher tau ratio at ccfHb = 165 mg / dL compared to 0 and 1000 mg / dL. Figures 15-16 show that the tau ratio is quite insensitive to Hct.

[0197] Furthermore, Figures 14-16 show that a difference scale, such as an absolute or relative difference in concentrations between two or more predetermined analytes in a liquid, can be provided based on one or more time response values. For example, it is possible to estimate or determine the difference scale in binary (for at least a specific relevant concentration of cfHb) based on the tau ratio tau_WL1 / tau_WL4, or, for example, tau_WL1 alone (the ratio between the Hap-cfHb concentration and the concentration of Hap-unbound cfHb, concentration Hap-cfHb / concentration cfHb This exceeds a certain threshold, which results in a tau_WL1 / tau_WL4 ratio that, for example, (significantly and measurably) exceeds the threshold. For example, the value of tau_WL1 / tau_WL4 versus the concentration Hap-cfHb / concentration cfHb By quantitative calibration of the ratio value, the concentration can be obtained from the measured value of the ratio tau_WL1 / tau_WL4. Hap-cfHb / concentration cfHb It is possible to obtain quantitative relative values ​​of the ratio. Furthermore, for example, the absolute concentration of the sum of Hap-cfHb and Hap-unbound cfHb (concentration Hap-cfHb +concentration cfHbBy measuring this, it is possible to determine the respective concentrations and absolute differences (scale) of Hap-cfHb and Hap-unbound cfHp.

[0198] While the present invention has been described in relation to specific embodiments, it should be construed as not being limited in any way to the examples presented. The scope of the invention is specified by the appended set of claims. In the context of the claims, the terms “equipped with” or “equipped with” do not exclude other possible elements or steps. Nor should references such as “one (a)” or “one (an)” be construed as excluding plurals. The use of reference numerals in the claims for elements shown in the figures should also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims may possibly be advantageously combined, and references to these features in different claims do not exclude whether or not the combination of features is possible or not. This specification includes the following disclosures of inventions. [Item 1] A method for determining a difference scale, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes (96) in a liquid (99), The steps to be provided, i. A light-transmitting element comprising a pore (6), wherein the pore (6) is a non-through pore (6) extending into the light-transmitting element from each opening (7) within the light-transmitting element, and the cross-sectional dimensions of the pore (6) to the opening (7) are determined to allow the two or more predetermined analytes in the liquid (99) to enter the pore (6) by diffusion, while preventing larger particles or fragments from entering the pore (6), ii. One or more light sources (10), and iii. The step of providing a photodetector (20), The steps include bringing the pores of the light-transmitting element into contact with the liquid (99), The steps include illuminating at least the holes (6) in the light-transmitting element (2) using one or more light sources, The steps include receiving light (21) emitted from the hole (6) in response to the illumination (11) at each of the multiple time points, A step of generating one or more signals based on the received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light; A step of determining one or more time response values ​​based on the one or more signals, A method comprising the step of determining a difference measure, such as an absolute or relative difference, in the concentrations between two or more predetermined analytes (96) in a liquid (99), based on one or more time response values. [Item 2] The method according to Item 1, wherein the two or more default analytes are cell-free hemoglobin that does not bind to haptoglobin, and haptoglobin-bound hemoglobin such as hemoglobin-haptoglobin complexes. [Item 3] A step of determining the presence of the hemoglobin-haptoglobin complex based on the difference scale, and / or The method according to item 2, further comprising the step of determining the absence of the hemoglobin-haptoglobin complex based on the difference scale. [Item 4] The method according to any one of Items 1 to 3, further comprising the steps of bringing the apparatus (1) into contact with the reference solution so as to fill the holes (6) with the reference solution, and / or waiting for a diffusion time to allow the two or more predetermined analytes (96) in the liquid (99) into the holes (6) to reach a stable state. [Item 5] The above method is The further step includes providing a data processing device equipped with a processor, wherein the data processing device includes the following steps: A step of controlling one or more light sources to illuminate at least the holes (6) in the light-transmitting element (2), A step of controlling the detector to receive light (21) emitted from the hole (6) in response to the illumination (11) at each of a plurality of time points, A step of receiving one or more signals based on the received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light. A step of determining one or more time response values ​​based on one or more signals, and The system is configured to perform one or more of the steps of determining a difference scale, such as an absolute or relative difference, in the concentrations between two or more predetermined analytes (96) in a liquid (99), based on the one or more time response values, For example, the method according to any one of items 1 to 4, further comprising the step, wherein the two or more default analytes are cell-free hemoglobin that does not bind to haptoglobin and haptoglobin-bound hemoglobin such as a hemoglobin-haptoglobin complex. [Item 6] The above method is, The method according to any one of items 1 to 5, further comprising the step of acquiring a plurality of signals for different wavelength intervals, for example, each signal in the plurality of signals being acquired for a wavelength interval unique to it with respect to the wavelength intervals for the remaining signals in the plurality of signals. [Item 7] The method according to any one of Items 1 to 6, wherein the step of determining the difference scale indicating the difference in concentration between the two or more predetermined analytes (96) is based on a plurality of time response values ​​obtained for different wavelength intervals, for example, one response value serving as a reference for another response value. [Item 8] The step of determining the difference scale that shows the difference in concentration between the two or more predetermined analytes is based on a plurality of time response values, and more specifically, The time response value obtained for the first wavelength interval, such as 415 nm, The method according to any one of items 1 to 7, based on the ratio of the time response value obtained for a second wavelength interval, such as 450 nm. [Item 9] The first wavelength interval is 415 nm, The method according to item 8, wherein the second wavelength interval is 450 nm. [Item 10] The one or more time response values ​​are, Based on one or more differences in the signal values ​​within each of the one or more signals, the signal values ​​are obtained at different time points, for example, each of the one or more time response values ​​is the result of subtracting the signal value at the first time point from the signal value at the second time point. and / or, The method according to any one of items 1 to 9, wherein one or more characteristic time intervals, for example, each of the one or more characteristic time intervals represents the duration of change or a specific amount of change in a parameter. [Item 11] The method according to any one of Items 1 to 10, further comprising the step of determining, based on the one or more signals, the concentration of cell-free hemoglobin not bound to haptoglobin in the liquid, and / or the concentration of haptoglobin-bound hemoglobin, such as a hemoglobin-haptoglobin complex analyte. [Item 12] The method according to Item 11, wherein the determined concentration of the analyte in the liquid is adjusted based on one or more time response values. [Item 13] Apparatus for determining a difference scale, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes (96) in a liquid (99), A light-transmitting element comprising a pore (6), wherein the pore (6) is a non-through pore (6) extending into the light-transmitting element from each opening (7) within the light-transmitting element, and the cross-sectional dimensions of the pore (6) to the opening (7) are determined to allow the two or more predetermined analytes in the liquid (99) to enter the pore (6) by diffusion, while preventing larger particles or fragments from entering the pore (6), One or more light sources (10) adapted to illuminate at least the holes (6) in the light-transmitting element (2), and Each of the multiple time points includes a photodetector adapted to receive light (21) emitted from the hole (6) in response to illumination (11) by one or more light sources, The photodetector is further adapted to generate one or more signals based on the received light, each of which is temporally decomposed and represents at least a portion of the received light. The aforementioned device is The data processing device further comprises a processor, and the data processing device is Based on the one or more signals, one or more time response values ​​are determined. An apparatus configured to determine a difference scale, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes (96) in a liquid (99), based on one or more time response values. [Item 14] The apparatus according to Item 13, wherein the two or more specified analytes are cell-free hemoglobin that does not bind to haptoglobin, and haptoglobin-bound hemoglobin such as a hemoglobin-haptoglobin complex. [Item 15] The one or more light sources and / or the photodetector are operably coupled to a data processing device comprising a processor, and the data processing device comprising the processor The acquisition of multiple signals over different wavelength intervals, for example, each signal in the multiple signals being acquired over a wavelength interval unique to that of the remaining signals in the multiple signals. Determining a plurality of time response values ​​by determining a time response value for each of the signals in the plurality of signals, for example, determining a plurality of time response values ​​where each time response value is determined based on signals obtained for different wavelength signals, such as being obtained for a specific wavelength interval with respect to the wavelength intervals of the remaining signals in the plurality of signals, and The apparatus according to item 13 or 14, further configured to perform, based on the plurality of time response values, a difference scale indicating a difference, such as an absolute or relative difference, in the concentrations between two or more predetermined analytes (96) in a liquid (99). [Item 16] The data processing device is Determining a tuned time response value, wherein the tuned time response value is determined based on at least two time response values ​​among a plurality of time response values, for example, one time response value serving as a reference for another response value, and the two time response values ​​are obtained for different, e.g., unique, signals obtained for wavelength intervals, and The apparatus according to item 15, further configured to perform, based on the adjusted time response value, determining the difference scale, which indicates a difference such as an absolute or relative difference in the concentrations between two or more predetermined analytes (96) in a liquid (99). [Item 17] The one or more light sources and / or the photodetector are operably coupled to a data processing device comprising a processor, and the data processing device comprising the processor The first signal is obtained at a first wavelength interval, such as a first wavelength interval centered at 415 nm. The method involves obtaining a second signal at a second wavelength interval, wherein the second wavelength interval is different from, for example, unique to, the first wavelength interval, and for example, the second wavelength interval is substantially centered at 450 nm. The purpose is to determine the ratio, A first time response value obtained for a first wavelength interval, To determine the ratio with the second time response value obtained for the second wavelength interval, and The apparatus according to any one of items 13 to 16, further arranged to perform, based on the ratio, determining the difference scale which indicates a difference such as an absolute or relative difference in the concentrations between two or more predetermined analytes (96) in a liquid (99). [Item 18] The light-transmitting element has a transmittance coefficient that, in some cases partially or entirely, diffuses light passing through the material, ignoring any interfacial effects, at least 50% for a length of 100 micrometers of material. For example, the proportion of light that does not pass through the length of the material is at least in the range of 380 nm to 750 nm, such as 400 to 520 nm, for example, in the range of 400 to 460 nm, for example, in the range of 415 to 420 nm, for example, 415 nm or about 415 nm, or 416 nm or about 416 nm. Apparatus according to any one of items 13 to 17, comprising, for example, mainly comprising, for example, comprising at least 50 w / w%, for example, consisting of, for one wavelength of m, 450 nm or about 450 nm, or 455 nm or about 455 nm, having an attenuation coefficient such that it is 50% or less of 100 micrometers, for example, 40% or less of 100 micrometers, for example, 20% or less of 100 micrometers, for example, 10% or less of 100 micrometers, for example, 5% or less of 100 micrometers. [Item 19] The apparatus according to any one of items 13 to 18, further comprising an optical assembly having a light guide core, the light guide core having an input branch, an output branch, and a coupling interface positioned to contact the rear side (4) of the translucent element opposite to the front side, for example, the input branch and the output branch being positioned in a common waveguide positioned perpendicular to the front surface. [Item 20] The apparatus according to any one of items 13 to 19, further comprising a housing through which a channel defining an axial direction is passed, the channel comprising a sample space, and the porous unit with a front side is positioned to define a sensor surface for contact with the liquid, such as when the liquid is in the sample space, the sensor surface facing the sample space, and for example the holes being configured with respect to the analyte in the liquid for diffusive liquid communication with the sample space. [Item 21] The apparatus according to any one of items 13 to 20, wherein the apparatus is positioned to optically probe the liquid disposed inside the hole (6) from the side of the front (3) facing the rear (4). [Item 22] The apparatus according to any one of items 13 to 21, wherein each of the one or more light sources and the detector is located on the front side, opposite to the rear side, such as outside the light-transmitting element on the same side as the rear side. [Item 23] The one or more light sources (10) are adapted to illuminate at least the holes (6) in the light-transmitting element (2) from the side of the front side (3) facing the rear side (4), The apparatus according to any one of items 13 to 22, wherein the detector (20) is arranged to receive light (21) emitted from the hole (6), such as emitted in response to illumination (11) from one or more light sources (10), and the photodetector (20) is adapted to generate a signal representing the received light, for example, mainly emitted, emitted from the hole in a direction away from the front side (3) in a direction opposite to the rear side (4). [Item 24] The one or more light sources (10) are adapted to illuminate at least the holes (6) in the light-transmitting element (2) from, for example, the front side (3) opposite to the rear side (4), and the light from the one or more light sources reaching the holes is fluidly connected to the holes and does not need to cross the volume outside the light-transmitting element, such as the front side opposite to the rear side. The photodetector (20) is arranged to receive light (21) emitted from the hole (6), such as being emitted in response to illumination (11) by one or more light sources, such as the one or more light sources (10), and the photodetector (20) is adapted to generate a signal representing the received light, and the light emitted from the hole (6) and reaching the photodetector (20) is fluidly connected to the hole and does not need to cross the volume outside the light-transmitting element, such as on the front side, opposite to the rear side, as described in any one of items 13 to 23. [Item 25] The apparatus described in any one of items 13 to 24, configured for measuring absorbance, such as the absorbance of the liquid in the hole. [Item 26] A computer program such as a computer program product, where the program is executed by a computer, To cause the apparatus described in any one of items 13 to 25, such as an apparatus in a second embodiment in which a data processing device is further operably connected to one or more light sources and / or detectors, to perform the steps of the method described in any one of items 1 to 12, and / or Receiving one or more signals representing received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light. Determining one or more time response values ​​based on the aforementioned one or more signals, and A computer program comprising instructions to cause the computer to determine a difference measure, such as an absolute or relative difference, in the concentrations of two or more predetermined analytes, based on one or more time response values.

Claims

1. A method for determining a difference scale indicating the difference in concentration between two or more predetermined analytes (96) in a liquid (99), The steps to be provided, i. A light-transmitting element comprising a pore (6), wherein the pore (6) is a non-through pore (6) extending into the light-transmitting element from each opening (7) within the light-transmitting element, and the cross-sectional dimensions of the pore (6) of the opening (7) are determined to allow the two or more predetermined analytes in the liquid (99) to enter the pore (6) by diffusion, while preventing larger particles or fragments from entering the pore (6), ii. One or more light sources (10), and iii. The step of providing a photodetector (20), The steps include bringing the pores of the light-transmitting element into contact with the liquid (99), The steps include illuminating at least the holes (6) in the light-transmitting element (2) using one or more light sources (10), The steps include receiving light (21) emitted from the hole (6) in response to the illumination (11) at each of the multiple time points, A step of generating one or more signals based on the received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light. A step of determining one or more time response values ​​based on the one or more signals, The step of determining a difference scale indicating the difference in concentration between two or more predetermined analytes (96) in a liquid (99) based on one or more time response values, The step of determining the difference scale that shows the difference in concentration between the two or more predetermined analytes (96) is based on a plurality of time response values, and more specifically, The time response obtained for the first wavelength interval, A method based on the ratio of the time response value obtained for a second wavelength interval.

2. The method according to claim 1, wherein the two or more predetermined analytes are haptoglobin-free cell-free hemoglobin and haptoglobin-bound hemoglobin.

3. A step of determining the presence of a hemoglobin-haptoglobin complex based on the difference scale, and / or The method according to claim 2, further comprising the step of determining the absence of a hemoglobin-haptoglobin complex based on the difference scale.

4. The method according to any one of claims 1 to 3, further comprising the steps of bringing the translucent element into contact with the reference solution so as to fill the pores (6) with the reference solution, and / or waiting for a diffusion time to allow the two or more predetermined analytes (96) in the liquid (99) to reach a stable diffusion state into the pores (6).

5. The aforementioned method, The further step includes providing a data processing device equipped with a processor, wherein the data processing device includes the following steps: A step of controlling one or more light sources (10) to illuminate at least the holes (6) in the light-transmitting element (2), A step of controlling the photodetector (20) to receive light (21) emitted from the hole (6) in response to the illumination (11) at each of a plurality of time points, A step of receiving one or more signals based on the received light, wherein each of the one or more signals is temporally decomposed and represents at least a portion of the received light. A step of determining one or more time response values ​​based on one or more signals, and The method according to any one of claims 1 to 4, further comprising the step of performing one or more of the steps of determining the difference scale indicating a difference in the concentration between two or more predetermined analytes (96) in a liquid (99) based on one or more time response values.

6. The aforementioned method, The method according to any one of claims 1 to 5, further comprising the step of acquiring a plurality of signals for different wavelength intervals.

7. The method according to any one of claims 1 to 6, wherein the step of determining the difference scale indicating the difference in concentration between two or more predetermined analytes (96) is based on a plurality of time response values ​​obtained for different wavelength intervals, where one response value serves as a reference for another response value.

8. The first wavelength interval is 415 nm. The method according to any one of claims 1 to 7, wherein the second wavelength interval is 450 nm.

9. The method according to any one of claims 1 to 8, further comprising the step of determining the concentration of haptoglobin-unbound cell-free hemoglobin and / or haptoglobin-bound hemoglobin in the liquid based on one or more signals.

10. The method according to claim 9, wherein the determined concentration of the analyte in the liquid is adjusted based on one or more time response values.

11. An apparatus for determining a difference scale that indicates the difference in concentration between two or more predetermined analytes (96) in a liquid (99), A light-transmitting element comprising a pore (6), wherein the pore (6) is a non-through pore (6) extending into the light-transmitting element from each opening (7) within the light-transmitting element, and the cross-sectional dimensions of the pore (6) of the opening (7) are determined to allow the two or more predetermined analytes in the liquid (99) to enter the pore (6) by diffusion, while preventing larger particles or fragments from entering the pore (6), One or more light sources (10) adapted to illuminate at least the holes (6) within the light-transmitting element (2), and Each of the multiple time points includes a photodetector (20) adapted to receive light (21) emitted from the hole (6) in response to illumination (11) by one or more light sources (10), The photodetector (20) is further adapted to generate one or more signals based on the received light, each of which is temporally decomposed and represents at least a portion of the received light. The aforementioned device is The data processing device further comprises a processor, and the data processing device is Based on the one or more signals, one or more time response values ​​are determined, Based on the one or more time response values, the system is configured to determine a difference scale indicating the difference in concentration between two or more predetermined analytes (96) in the liquid (99), The one or more light sources (10) and / or the photodetector (20) are operably coupled to the data processing device which includes a processor, and the data processing device which includes a processor Acquiring multiple signals at different wavelength intervals, Determining a time response value for each of the signals in the plurality of signals, thereby determining a plurality of time response values, and The apparatus is further configured to determine the difference scale, which indicates the difference in concentration between two or more predetermined analytes (96) in a liquid (99), based on the plurality of time response values.

12. The apparatus according to claim 11, wherein the two or more predetermined analytes are haptoglobin-free cell-free hemoglobin and haptoglobin-bound hemoglobin.

13. The aforementioned data processing device is Determining a regulated time response value, wherein the regulated time response value is determined based on at least two time response values ​​among a plurality of time response values, one time response value serving as a reference for another response value, and the two time response values ​​being obtained for signals obtained for different wavelength intervals, and The apparatus according to claim 11 or 12, further configured to determine the difference scale indicating the difference in concentration between two or more predetermined analytes (96) in a liquid (99) based on the adjusted time response value.

14. The one or more light sources (10) and / or the photodetector (20) are operably coupled to the data processing device which includes a processor, and the data processing device which includes a processor To acquire a first signal at a first wavelength interval centered around 415 nm, The method involves acquiring a second signal at a second wavelength interval, wherein the second wavelength interval differs from the first wavelength interval, and the second wavelength interval is centered at 450 nm. The purpose is to determine the ratio, A first time response value obtained for a first wavelength interval, To determine the ratio with the second time response value obtained for the second wavelength interval, and The apparatus according to any one of claims 11 to 13, further arranged to determine the difference scale indicating the difference in concentrations between two or more predetermined analytes (96) in a liquid (99) based on the ratio.

15. The apparatus according to any one of claims 11 to 14, wherein the light-transmitting element comprises a material having an attenuation coefficient such that, at least one wavelength in the range of 380 nm to 750 nm, the transmittance coefficient when light passing through the material passes through a length of 100 micrometers in the material is at least 50%, in an amount of at least 50 w / w% and 100 w / w% or less.

16. The apparatus according to claim 15, wherein the at least one wavelength is in the range of 415 to 420 nm.

17. The apparatus according to claim 15, wherein the at least one wavelength is 415 nm, 416 nm, 450 nm, or 455 nm.

18. The apparatus according to any one of claims 11 to 17, further comprising an optical assembly having a light guide core, the light guide core having an input branch, an output branch, and a coupling interface positioned to contact the front side (3) of the light-transmitting element and the rear side (4) of the light-transmitting element opposite to it.

19. The apparatus according to claim 18, further comprising a housing through which a channel defining an axial direction is passed, the channel comprising a sample space, the front side (3) of a translucent element being positioned to define a sensor surface for contact with the liquid, the sensor surface facing the sample space, and the hole being configured with respect to the analyte in the liquid for diffusive liquid communication with the sample space.

20. The apparatus according to claim 18 or 19, wherein the apparatus has an arrangement that optically probes the liquid disposed inside the hole (6) from the side of the front side (3) that faces the rear side (4).

21. The apparatus according to any one of claims 18 to 20, wherein each of the one or more light sources (10) and the photodetector (20) is placed on the outside of the light-transmitting element behind the light-transmitting element.

22. The one or more light sources (10) are configured to illuminate at least the holes (6) in the light-transmitting element (2) from the side of the front side (3) facing the rear side (4), The photodetector (20) is arranged to receive light (21) emitted from the hole (6) in response to illumination (11) from one or more light sources (10), and the photodetector (20) is positioned so that light (21) is emitted from the hole in the direction opposite to the rear side (4) from the front side (3) The apparatus according to any one of claims 18 to 21, which is adapted to generate a signal representing the received light emitted in a moving direction.

23. The apparatus according to any one of claims 12 to 22, wherein the apparatus is configured to measure the absorbance of the liquid in the hole.

24. A computer program, wherein when the computer program is executed by a computer, A computer program comprising an instruction causing the computer to perform a step of the method according to any one of claims 1 to 11.

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