Porous mirror for optical detection of an analyte in a fluid

Abstract

A porous mirror (1) for detecting an analyte (96) in a fluid (99) by optical detection, the porous mirror comprising a translucent sheet (2) having a front side (3) and a back side (4) facing away from the front side (3) wherein the front side (3) is adapted to be in contact with the fluid (99); and a reflective layer (5) located at the front side (3) of the translucent sheet (2), the reflective layer (5) being adapted to reflect light reaching the reflective layer out of the back side (4) of the translucent sheet (2) wherein the translucent sheet (2) comprises a hole (6) wherein the hole (6) is a dead end hole (6), and dead end holes (6) extending through the reflective layer (5) from respective openings (7) at the front side (3) into the translucent sheet (2) wherein the openings (7) of the holes (6) are dimensioned in cross-section so as to prevent larger particles or fragments from entering the holes (6) if the fluid contains any larger particles or fragments, and wherein the dead end holes (6) extend through the reflective layer (5) from the respective openings (7) at the front side (3) into the translucent sheet (2). Meanwhile, the analyte (96) in the fluid (99) is allowed to enter the hole (6) through diffusion.

Description

[0001] This application is a divisional application based on a patent application with application date of November 17, 2016, application number 201680064651.4 (PCT / EP2016 / 077943), and invention name: "Porous reflector for optical detection of analytes in fluids".

[0002] The present invention relates in one aspect to a porous reflector for optically detecting an analyte in a fluid. In another aspect, the present invention relates to a system for analyzing fractions of a fluid, the system comprising a porous reflector for optically detecting an analyte in the fluid. According to another aspect of the present invention, a system for analyzing whole blood comprises optically detecting bilirubin. According to yet another aspect of the present invention, a method for optically detecting an analyte in a fluid is provided.

[0003] According to a broader aspect, the present invention relates to a porous reflector for optically detecting substances in a fraction of a fluid. In another aspect, the present invention relates to a porous reflector for optically detecting substances in a plasma fraction of a whole blood sample. According to another aspect of the present invention, a system for analyzing a fluid includes a porous reflector for optically detecting substances in a fraction of a fluid. According to yet another aspect of the present invention, a method for optically detecting substances in a plasma fraction of whole blood is provided. Furthermore, the method for analyzing a fluid includes optically detecting substances in a fraction of a fluid. Background Art

[0004] There are many different ways to detect molecular subpopulations in a fluid. Variations range from membranes to chemical methods and biological processes. Many known processes use external forces for the filtration portion, which can have a detrimental effect on the filtrate. Other methods utilize a multi-step process with an initial filtration step followed by a detection step. This can complicate the process.

[0005] The one-step approach for filtering and detecting molecular subpopulations is applicable to a diverse range of technological fields, ranging from the food industry to wastewater treatment, pharmaceuticals and medical devices.

[0006] In the field of medical devices, most traditional filtration and detection methods involve large volumes of fluid that need to be filtered. Detection of drugs in, for example, whole blood is generally not feasible, and since plasma is required to analyze the drug of interest, a large volume of whole blood is drawn from the patient. If such analysis is required, the patient's vein is usually punctured regularly, so the patient is at risk of anemia.

[0007] In the field of food industry such as dairy industry, most traditional filtration and detection methods include filter paper, sieves, etc. for visual inspection of residues, spectroscopic analysis or bacteria counting. The disadvantage of this method is that it requires large volumes and is time-consuming.

[0008] In the field of environmental technology such as wastewater analysis and treatment, most traditional filtration and detection methods involve filter paper, sieves, etc. for spectral analysis of residues and bacterial counts. The disadvantages of this method are the same as those in the food industry: large volume and time-consuming.

[0009] One use of porous mirrors is related to the detection of analytes in patient samples. The analyte may be a laboratory test parameter used in blood analysis (e.g., spectrophotometry) that can be detected by light. Other methods for measuring the components present in a fraction of a fluid containing particles or other debris involve first separating the fraction from cellular components by microfiltration techniques, such as in a microfluidic device, and then analyzing the fraction in a dedicated measurement in the microfluidic device. For example, Archibong et al., a scientific paper published in Sensing and Bio-Sensing Research 3 (2015), p.1–6, discloses a micro-measurement chamber for optical analysis of a plasma fraction that has been separated from a whole blood sample. In this type of device, a micro-microfluidic chamber is attached to an interface of an optical fiber. The bottom of the microfluidic chamber consists of a porous membrane that allows fluids and chemical compounds to flow within the device while filtering out undesirable particles. The interior of the microfluidic chamber receiving the filtrate can be optically probed through a single optical fiber in a normal incidence reflection geometry.

[0010] However, such filtration-based methods have several disadvantages when used to analyze, for example, whole blood samples. Filtration devices inherently rely on a fluid flow of at least the filtrate from the sample feed through the pores of the filter to the filtrate analysis / measurement chamber. In a through-flow geometry, the retentate (here, red blood cells) gradually clogs the filter pores. In a cross-flow geometry, the retentate is guided along the surface of the filter membrane, thereby alleviating but not eliminating the clogging problem, especially if the system is intended for repeated use (more than 10-100 samples). The cross-flow geometry also causes friction and shear interactions between the retentate and the surface of the filter device. The disclosed device is most suitable as a disposable and not suitable for continuous reuse, because the complete washing out of the sample after the measurement may be difficult or at least very time-consuming and unreliable, and there is also a risk of cross-contamination between subsequent samples. In this particular type of device, the additional challenge of obtaining quantitative results by optical detection can be brought about because the filter membrane undergoes pressure-induced deformation, resulting in a change in the optical path used to detect the filtrate.

[0011] Therefore, there is a need for improved devices and methods for detecting analytes in fluids with a rapid and reliable response.More generally, there is a need for improved devices and methods for detecting substances in fractions of whole blood samples with a rapid and reliable response.

[0012] It is an object of the present invention to provide improved detection that overcomes at least some of the disadvantages of known sensors, systems and methods for detecting substances in the plasma fraction of a whole blood sample, in particular for detecting analytes in fluids.

[0013] The advantages of the present invention are that the filtration is performed by diffusion which does not require external energy and the diffusion is also rapid, so that the measurement of the liquid diffused into the pores of the porous mirror can be performed shortly after the fluid has been introduced into the porous mirror. The porous mirror is simple, has few parts, and no parts need to be moved or changed position during filtration and measurement. The size of the porous mirror is small and the volume required for measurement is very small compared to ordinary filtering devices. The porous mirror can be included in other applications or devices where filtration and subsequent measurement of fluids are required. Summary of the invention

[0014] A first aspect of the present invention relates to a porous reflector for optically detecting an analyte in a fluid, the porous reflector comprising a translucent plate having a front side and a back side facing away from the front side, wherein the front side is suitable for contacting the fluid; a reflective layer at the front side of the translucent plate, the reflective layer being suitable for reflecting light reaching the reflective layer out of the translucent plate; wherein the translucent plate is provided with dead-end holes extending from corresponding openings at the front side into the translucent plate, wherein the cross-sectional dimensions of the openings of the holes are set so as to prevent particles or debris (e.g., red blood cells when the fluid is whole blood) from entering the holes while allowing analytes in the fluid to enter the holes.

[0015] The term "fluid" refers to a whole blood sample, a plasma fraction of whole blood, spinal fluid, urine, pleural fluid, ascites, wastewater, a pre-prepared fluid for injection of any kind, a fluid having components detectable by spectroscopy, or a gas such as air, a gas containing carbon dioxide, a gas containing carbon monoxide.

[0016] The term "sample" refers to the portion of a fluid that is used or desired in an analysis performed using the porous mirror of the present invention.

[0017] The term "whole blood" refers to blood composed of plasma and cellular components. Plasma accounts for about 50% - 60% of the volume, and the cellular components account for about 40% - 50% of the volume. These cellular components are red blood cells (erythrocytes), white blood cells (leukocytes) and thrombocytes (platelets). Preferably, the term "whole blood" refers to the whole blood of a human subject, but it may also refer to the whole blood of an animal. Red blood cells account for about 90% - 99% of the total number of all blood cells. In the undeformed state, they are shaped like biconcave discs with a diameter of about 7µm and a thickness of about 2µm. Red blood cells are highly elastic, which allows them to pass through very narrow capillaries, thereby reducing the diameter of the red blood cells to as low as about 1.5µm. A core component of red blood cells is hemoglobin, which binds oxygen to be transported to tissues, then releases oxygen and binds carbon dioxide to be transported to the lungs as waste. Hemoglobin causes red blood cells, and therefore blood as a whole, to be red. White blood cells account for less than about 1% of the total number of all blood cells. White blood cells have a diameter of about 6 to about 20 µm. White blood cells are involved in the body's immune system, such as fighting off bacterial or viral invasions. Thrombocytes are the smallest blood cells, with a length of about 2 to about 4 µm and a thickness of about 0.9 to about 1.3 µm. Thrombocytes are cell fragments that contain enzymes and other substances that are important for blood clotting. Specifically, thrombocytes form temporary platelet plugs, which help seal breaks in blood vessels.

[0018] The term "plasma" refers to the liquid portion of blood and lymph, which accounts for about half of the volume of blood (e.g., about 50% ‒ 60% by volume). Plasma is cell-free. Plasma contains all coagulation factors, especially fibrinogen, and is about 90% - 95% water by volume. Plasma components include electrolytes, lipid metabolites, markers such as infectious diseases or tumors, enzymes, substrates, proteins, and additional molecular components.

[0019] The term "wastewater" refers to water that has been used for washing, flushing or in manufacturing processes and therefore contains waste products and / or particles and is therefore unsuitable for drinking and food preparation.

[0020] The term "translucent" refers to the property of a material that allows light to pass through it. The term "transparent" refers to the property of a material that allows light to pass through the material without scattering. The term "transparent" is therefore considered a subset of the term "translucent".

[0021] The porous reflector comprises a translucent sheet and a reflective layer applied to the front side of the translucent sheet. The translucent sheet contains small dead-end holes extending from the front side through the reflective layer into the translucent sheet. The porous reflector requires a light source and a detector arranged to optically detect the contents of the holes and generate a corresponding signal output representative of the analyte content in the fluid.

[0022] Each aperture has an opening at the front side of the translucent sheet material, through which the aperture can be communicated with the fluid space. Therefore, these holes penetrate the reflective layer to allow fluid communication between these holes and the fluid space. These holes extend from the corresponding openings at the front side into the translucent sheet material in the direction toward the back side. These holes are "dead-end", meaning that these holes terminate in the translucent sheet material. These holes do not continue through the translucent sheet material to reach the back side or to any common reservoir or receiver inside the sheet material. These holes are only in fluid communication with the fluid space at the front side of the translucent sheet material. It should be noted that in some embodiments, the dead-end holes can be cross-shaped, and therefore at least some of the holes can be connected to each other, thereby forming an X-shaped, Y-shaped, V-shaped or similar interconnected shape. This configuration is equivalently regarded as a dead end because these holes are only filled from the front side, and even if they cross each other, no significant net mass transport through these holes will occur under operation. By appropriately setting the size of the opening of the hole at the front side, debris in the fluid or red blood cells of the whole blood sample on the front side of the porous reflector can be prevented from entering the hole, while allowing relevant components in the plasma fraction of the whole blood sample or in the fluid to enter the hole, wherein the relevant components are substances to be measured / detected using the sensor that exist in the plasma fraction of the whole blood sample. Specifically, bilirubin and carbon dioxide are relevant components.

[0023] In operation, the front side of the translucent plate is in contact with a whole blood sample or fluid. The pores in the translucent plate are connected to the whole blood sample or fluid through the openings in the front side. The size of the pore opening is set to selectively extract a subsample of the plasma phase of the whole blood sample or extract a subsample of the fluid containing the analyte. Red blood cells cannot enter the pores through the openings on the front side of the translucent plate. Any substance larger than the pore size cannot enter the pores, thereby excluding any debris contained in the fluid, for example. As described, these holes are dead-end and are only connected to the front side of the translucent plate, that is, the subsample is extracted for optical detection in the hole, and after measurement, the subsample is discharged again through the same opening in the front side of the translucent plate. The subsample volume corresponds to the total internal volume of the hole. No filtration and net mass transport of any filtrate through the pore-containing layer occurs - neither into any common filtrate receiver nor to any filtrate outlet. Only the subsample contained in the hole will then be optically detected. The reflective layer optically separates the optical detection area in the translucent plate from the fluid space containing the whole blood sample or fluid. By optically separating the detection area from the fluid volume, any contribution to the detection signal from intact red blood cells of the whole blood sample or debris in the fluid is effectively suppressed. The measurement is therefore specific to the amount of analyte in the fluid.

[0024] This smaller subsample having a representative content of the relevant component can be transferred to the well in any suitable manner. These smaller dead-end wells allow very efficient and rapid extraction of subsamples for optical detection from whole blood samples or fluids through the openings in the front side by means of capillary forces and / or diffusion.

[0025] In a typical mode of operation, the front side surface of the translucent sheet is first contacted with a flushing fluid, and then the front side is contacted with a whole blood sample or fluid to be analyzed. Thus, the wells are "filled" with a pre-filled liquid compatible with the whole blood sample or fluid, in particular a liquid compatible with the plasma phase (if the fluid is whole blood), such as an aqueous solution commonly used for flushing, calibration and / or quality control purposes in blood analyzers. A typical flushing liquid used for washing out in, for example, a whole blood analyzer system can be used as such a liquid. The flushing liquid is a liquid containing K in a concentration corresponding to human plasma. + 、Na + , Cl - , Ca 2+ , O 2 , pH, CO 2 and HCO 3 - Aqueous solution. Non-limiting examples of suitable solutions commonly used for flushing, calibration and / or quality control purposes are further given below. When the whole blood sample or fluid is then brought into contact with the front surface filled with the plasma-compatible liquid / fluid-compatible liquid, a representative subsample of the components in the plasma phase of the whole blood sample or the fluid is extracted and transferred to the pre-filled wells in a very effective and gentle manner by means of diffusion of the relevant components. Specifically, any concentration gradient of the content of the analyte between the fluid in the well and the reference liquid drives the diffusive transfer, thereby producing a subsample in the well with an analyte concentration representative of the analyte concentration in the fluid.

[0026] In another mode of operation, it is also conceivable to bring the front side of the dry sensor into direct contact with a whole blood sample or fluid. It is further preferred that in this mode of operation, the inner surface of the hole is hydrophilic, so that a sub-sample is extracted from the whole blood sample or fluid into the hole at the front side of the translucent plate by means of capillary forces. When the porous reflector is operated in this mode, it can be calibrated via batch calibration, because porous reflectors made of the same batch of porous membrane materials tend to have equal sensitivity (when using porous reflectors made of different sheets of porous membrane materials from the same batch of porous membrane materials forming the translucent plate to measure the same fluid, they have equal light absorption). Alternatively, the hole of the translucent plate may contain a calibration dye with absorption characteristics different from the analyte. The calibration dye can be used to normalize / calibrate the optical detection signal while being spectrally distinguishable from the substance (e.g., bilirubin) in the plasma sample to be detected / measured. Since the calibration dye will not be present in the actual fluid, the calibrant dye will diffuse out of the sensor during measurement, while the analyte diffuses into the hole of the sensor. By optically probing the holes before and after fluid acquisition, a quantitative measure of the substance to be detected (eg, bilirubin) can be formed by comparison of a calibration reference with the fluid substance signal.

[0027] The contents of the wells can be conveniently optically detected from the back side of the translucent sheet, or more generally, from the side of the reflective layer facing the translucent sheet, wherein the reflective layer optically separates the optical detection area containing the wells from the fluid contacting the front side of the translucent sheet. The reflective layer is adapted to reflect light reaching the reflective layer out from the interior of the translucent sheet, thereby preventing the detection light from reaching and interacting with the fluid at the front side of the reflector. Thus, only a sub-sample within the well is optically detected selectively.

[0028] The porous reflector can be connected to a light source and a light detector, which are constructed for optical detection of the translucent sheet, the light source should be suitable for illuminating at least the hole, and the detector should be arranged to receive light emitted from the hole in response to illumination by the light source, and the detector should also be suitable for generating a signal representing the detected light.

[0029] The incident light is directed / guided to the optical detection region to ensure that the light traverses the holes and interacts with the subsample therein. Preferably, the detection light is sent into the detection region at an oblique incidence relative to the surface normal on the plane of the reflective layer to ensure that the light traverses the holes filled with the fluid to be detected, thereby ensuring that the optical interaction path length is maximized.

[0030] The light that emerges from the well in response to the illumination has interacted with the subsample in the well, thereby conveying information about the subsample. The emergent light and / or a signal representative of the emergent light may then be analyzed for this information to generate a value representative of the amount of analyte in the whole blood sample or in the fluid. The analysis may include spectral analysis of the emergent / detected light, and / or signal / data processing, for example, to compare the obtained signal with a signal obtained on a calibration / reference sample, to perform noise filtering, to apply corrections, and to remove artifacts.

[0031] In a particularly advantageous embodiment, the coloration of the plasma caused by bilirubin is optically detected, for example by using spectrally resolved absorbance measurements, or by measuring the spectrally integrated absorbance within a predetermined bandwidth in a spectral range indicative of the presence of bilirubin in the liquid subsample, such as in the spectral range of wavelengths 380nm - 750nm, such as in the spectral range of wavelengths 400nm - 520nm, or at about 455nm.

[0032] Further according to one embodiment of the porous mirror of the present invention, the cross-sectional size of the opening of the hole is about 1µm or less, about 800nm ​​or less, preferably about 500nm or less, or even about 400nm or less, and / or the length of the hole along the axial direction of the hole is less than 100µm, less than 50µm, preferably less than 30µm, or about 25µm.

[0033] By using pores having openings with a maximum cross-sectional dimension of about 1 µm or less, or preferably in the submicron range such as about 800 nm or less, such as about 500 nm or less, or even about 400 nm or less, in the plane of the front side of the translucent sheet, any cellular components including red blood cells, white blood cells and thrombocytes (platelets) are prevented from entering the pores.

[0034] It is also surprising that pores having openings with a cross-sectional size of about 500 nm or less have improved sensitivity compared to larger pores, such as pores having openings with a cross-sectional size of about 800 nm or more but having the same total pore volume / volumetric porosity.

[0035] Most preferably, the pore has a minimum opening and has a corresponding minimum pore volume, thereby allowing efficient extraction of a sufficiently large subsample that can still be detected with an acceptable signal-to-noise ratio. Advantageously, the pore has an opening of about 30 nm or more, or 50 nm or more, or 100 nm or more, or about 200 nm or more.

[0036] Suitable holes can be made, for example, of transparent polymer films with so-called track-etched holes, similar to those available from the company IT4IP (IT4IP sa / avenue Jean-Etienne Lenoir 1 / 1348 Louvain-la-Neuve / Belgium), modified in that the holes are closed at one end. The through-holes in the film can be closed, for example, by laminating a backing sheet to the back side of the porous film, or by decelerating the ions so that the ion bombardment tracks and thus the holes etched along these tracks terminate in the transparent polymer film to form dead-end holes. The film is usually supported by a rigid transparent element in order to provide sufficient mechanical strength for the translucent sheet.

[0037] The transparent sheet should preferably be made of a material that does not absorb light, while it should be possible to produce dead-end holes in the material, for example by etching tracks into the material. Suitable materials for this are polyethylene terephthalate (PET or PETE) or PET analogs (polyethylene terephthalate polyester (PETP or PET-P)) or polycarbonate (PC). The transparent sheet may include a hydrophilic coating, for example of polyethylene glycol (PEG), to enhance diffusion into the holes. The hydrophilic coating may be selected depending on the use of the porous reflector. In some use cases, the porous reflector will never dry out once in use, so it only needs to be hydrophilic at start-up. For other uses of the porous reflector, it is necessary to have a coating that permanently keeps the porous reflector hydrophilic so as to allow the porous reflector to dry out and still be usable later when the porous reflector is re-wetted for further use.

[0038] According to one embodiment of the porous mirror of the present invention, a given volume of the porous translucent sheet has a porosity between 50% and 5% by volume, between 30% and 10% by volume, or about 15% by volume.

[0039] These holes form the porosity in the translucent sheet (or in a given area of ​​the translucent sheet) and have a corresponding front surface area where the openings of the holes are distributed. The porosity can be characterized according to the volume of the voids formed by the holes in the translucent sheet (i.e., the pore volume), where the pore volume refers to the volume of the translucent sheet penetrated by the holes. This volume is defined here as the volume between the front area where the holes are distributed and the same parallel area shifted into the translucent sheet to the maximum penetration depth of the holes in the translucent sheet (as can be seen in the axial direction perpendicular to the front side of the translucent sheet).

[0040] In addition, the porosity can be characterized in terms of the integrated pore volume, which is equal to the subsample volume available for optical detection. The pore volume can be conveniently expressed as an equivalent pore volume depth DELTA, which is the pore volume related to the corresponding front side area where the pore openings are distributed. Therefore, the porosity of the translucent sheet can be converted to an equivalent pore volume depth DELTA as follows. The pores with openings within a given front side area A have a total pore volume V. The equivalent pore volume depth is then calculated as the total pore volume divided by the given front side area: DELTA=V / A.

[0041] Advantageously according to some embodiments, the equivalent pore volume depth DELTA is less than 20µm, or less than 15µm, or less than 10µm, or in the range of 3µm to 5µm, wherein the equivalent pore volume depth DELTA is defined as the total pore volume V divided by the front side area A of the openings in which the pores are distributed. Thus, a smaller subsample of the relevant component with a representative concentration is obtained. A smaller subsample volume is required to facilitate rapid subsample exchange, thereby reducing the response time of the porous mirror and the cycle time of the measurement performed using the porous mirror. A smaller subsample volume is also required in order to avoid the depletion effect of the boundary layer of the plasma fraction in the whole blood sample close to the front side of the translucent plate. Otherwise, such depletion effects may occur in a smaller number of static samples, where, for example, if the equivalent pore volume depth exceeds a critical value, the red blood cells may hinder the effective diffusion exchange of the relevant component from the volume of the whole blood sample toward the boundary layer at the front side of the translucent plate.

[0042] Preferably, the equivalent pore volume depth DELTA is at least 1 μm, or at least 2 μm, or in the range of 3 μm to 5 μm, wherein the equivalent pore volume depth is as defined above. A larger subsample volume is needed to achieve a better signal-to-noise level, since a larger subsample volume contributes to the optical detection information about the relevant components in the plasma.

[0043] Further according to some embodiments, for an equivalent pore volume depth DELTA in the range of 1µm to 20µm, preferably in the range of 2µm to 10µm or about 4µm-5µm, an effective compromise is found between reducing response time, reducing cycle time and / or avoiding depletion effects in less static whole blood samples or fluids on the one hand and the required or desired signal-to-noise ratio on the other hand.

[0044] Advantageously according to one embodiment, the translucent sheet is supported by a translucent backing attached to the back side of the translucent sheet. Thus, an enhanced mechanical stability is achieved.

[0045] Further in accordance with one embodiment of the porous reflector of the present invention, the transparent backing attached to the back side of the translucent sheet has a certain thickness so that 60° prisms (i.e., there are no 90° corners on the outside of the translucent sheet, but rather these corners are "cut off" to obtain a 60° surface) are positioned on the outside of the transparent backing so that the light from the light source and reaching the detector has an increased angle of incidence for the light reaching the aperture area. Having a 60° prism also increases the chance of light propagating into the interior of the translucent sheet because the light reflects at the surface of the backing so that there will be multiple reflections before the outgoing light reaches the detector.

[0046] According to one embodiment of the porous reflector of the present invention, the inner wall surface of the hole is hydrophilic, for example coated with a hydrophilic coating. Thus, effective capillary driven filling of liquid in the dry hole is achieved. In addition, the hydrophilic coating prevents certain hydrophobic substances such as hydrophobic dyes, hemoglobin and other proteins from being deposited in the hole, which would otherwise lead to gradual fouling of the sensor, making it difficult to wash out with aqueous solutions.

[0047] According to one embodiment of the porous reflector of the present invention, the light source is configured to provide an obliquely incident illumination beam from the back side of the translucent sheet, wherein the illumination angle is defined as the angle of the incident light beam relative to the surface normal of the reference plane defined by the front side of the translucent sheet. 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 detection area for detection by the detector. In addition, the detection light is prevented from penetrating into the fluid through the hole opening, which is due to the reduction of the apparent cross-section of the hole opening and the increase of scattered propagation light into the detection area instead of passing through the hole opening into the fluid space on the other side of the reflective layer.

[0048] The light source can in principle be any light source that transmits light in the region where the analyte in the hole absorbs light so that the system works, but preferably the light source should have a flat spectral characteristic (i.e., the spectrum does not contain peak amplitudes) because the flat characteristic will respond better. If the light source has a non-flat spectrum (i.e., the light source has peak amplitudes), then slight changes in the peaks can be misinterpreted as changes in absorption. Light emitting diodes are generally preferred due to their characteristics in terms of size, weight, efficiency, etc. Further according to one embodiment of the sensor of the present invention, the detector is configured to collect light emitted obliquely from the back side of the translucent plate, wherein the detection angle is defined as the angle of the surface normal of the propagation of the emitted light toward the detector relative to the reference plane defined by the front side of the translucent plate. The detector is configured to collect light emitted in response to illumination by the light source of the optical detection device. Detecting light emitted obliquely from the back side of the translucent plate reduces the contribution to the detected signal from light emitted from the whole blood sample and leaking back into the detection area through the reflective layer.

[0049] The detector may be a photodiode or a spectrometer capable of detecting absorption across the spectrum. Alternatively, an array or diodes may be used where each diode emits light of a different wavelength and the photodiodes are used as detectors. The diodes may be multiplexed to emit light in different intervals. The absorption is then derived by comparing the light emitted from the diode in that particular interval with the light detected by the photodiode.

[0050] According to an embodiment of the porous reflector of the present invention, the incident plane and the detection plane intersect at the surface normal to 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 about 90 degrees, wherein the incident plane is spanned by the direction of the illumination beam and the surface normal of the reference plane, and wherein the detection plane is spanned by the direction of propagation of the outgoing light toward the detector and the surface normal of the reference plane. Thereby, the contribution to the detected signal of glare partially reflected at the optical interface before passing through the detection area is reduced. Such glare of light that does not interact with the subsample in the detection area does not contain relevant information and is therefore detrimental to the signal-to-noise ratio.

[0051] The optical detection of light may be performed by any suitable optical detection device. Such an optical detection device may include directing only a beam of light to the back side of the translucent sheet and directing the input of the optical detector to the illuminated area. The optical device may include additional optical elements that improve the coupling of the detection light into the translucent sheet and improve the coupling of the light emitted from the translucent sheet into the detector input. Such optical elements may include one or more prisms and / or lens devices directly attached / glued to the back side of the translucent sheet. Preferably, the coupling optical device adapts to the "reflective" nature of optical detection, where the incident detection light and the detected exiting light remain on the same side of the reflective layer. Further improvements may be sought in enhancing the optical interaction of the detection light with the hole, for example by coupling the detection light into the translucent sheet at the first end, forcing the light in the detection area to propagate along the reflective surface and through the hole substantially in a direction parallel to the front side of the translucent sheet, and collecting the exiting light from the other end of the translucent sheet (which may be in a lateral or opposite direction to the first end).

[0052] As the light source ages, it may change characteristics, for example, emitting less light or drift may affect the peak amplitude. This can be compensated for using a feedback calibration process in which the detector measures the light received through the transparent sheet when the holes in the transparent sheet are expected to be relatively clean (i.e., the holes do not contain molecules that absorb light). If the amplitude of the received light is less than expected, the feedback loop to the light source may control the current or voltage to the light source to increase to compensate for the degradation of the light source. Alternatively, if the light source has changed characteristics, the calculation of the actual absorption may be adjusted at the time of measurement based on this change in the emitted light compared to the initial factory calibration.

[0053] According to one embodiment of the porous reflector of the present invention, the translucent plate is provided with additional reflective elements, which are arranged in the holes, in their mouths, adjacent to the openings at the front side of the translucent plate. The additional reflective elements are applied as a reflective coating on the inner wall of the holes, starting from the opening of each hole and extending into the hole. However, only the mouth close to the opening of the hole is covered. Providing the additional reflective elements around the opening of the hole improves the optical separation of the detection light from the fluid chamber, thereby preventing erroneous contributions to the detection signal by, for example, red blood cells in a whole blood sample in the fluid chamber. The reflective coating can be any suitable metal coating as discussed below. The additional reflective elements can be produced in the same step as the reflective layer covering the front side of the translucent plate.

[0054] According to one embodiment of the porous reflector of the present invention, the additional reflective element is arranged as a reflective coating covering only a portion of the circumference of the mouth of the hole near the opening, wherein the portion is about 70% or less, and preferably about 50% or less. By only partially covering the circumference of the hole, a small reflector with a concave reflective surface facing the inside of the hole is provided in each hole. This partial coverage can be produced, for example, by directional deposition of a metal layer, wherein the front side of the semi-transparent plate is inclined relative to the deposition direction. The opening of the hole in the plane of the front side of the semi-transparent plate acts as a shadow mask. The shadow mask only allows deposition on a portion of the circumferential inner wall of the hole in the mouth region of the shadow mask (i.e., close to the opening). Thereby an array of small concave reflector elements all oriented in the same direction can be produced.

[0055] When these small reflector elements are illuminated from the concave side, the resulting exit light is directed in a preferential direction. By placing the detector in this preferential direction, an improved signal-to-noise ratio is achieved compared to other directions and compared to embodiments without such additional small directional reflector elements.

[0056] According to some embodiments with small reflector elements (i.e. with additional reflective elements with directional properties), an increase in the intensity of the exiting light by a factor of about 3 was observed compared to embodiments with additional reflective elements without directional properties. In addition to this, it has been surprisingly observed that when using small reflector elements applied to the inner surface of the hole at its mouth, for example when detecting absorbance, a further enhancement of the relevant signal of about 50% or more occurs. This therefore results in a surprising overall improvement in the signal-to-noise ratio of at least about 4 to 5 times.

[0057] It should be noted that also when using additional reflective elements, a reflective layer at the front side of the translucent sheet is still required in order to ensure optical separation of the optical detection area in the translucent sheet containing the hole and the fluid chamber containing the whole blood sample. A reflective layer on the front side of the translucent sheet is also required, for example, in order to simultaneously illuminate and detect from the back side.

[0058] Typically, the small reflector element is symmetrical with respect to a central reflector plane. Advantageously, the incident plane (as determined by the incident light beam) and the detection plane (as determined by the detection direction) are also arranged symmetrically with respect to the central reflector plane. According to a simplified embodiment, the incident plane and the detection plane coincide and are parallel to the central reflector plane of the small reflector element.

[0059] Advantageously according to one embodiment, the reflective layer and / or the further reflective element is made of metal. Such metal coatings having sufficient reflectivity can be applied in a relatively cost-effective and well-controlled manner.

[0060] Advantageously according to one embodiment, the reflective layer is made of platinum, palladium or an alloy containing platinum or palladium as a main component. These materials show good reflectivity in the spectral range of the electromagnetic spectrum (deep violet to blue) relevant for the detection of free hemoglobin (e.g., by absorbance detection). In addition, these materials are biocompatible and do not, for example, produce artificial hemolysis. In addition, these materials are chemically stable and in the chemical environment of the whole blood sample.

[0061] Alternatively, according to some embodiments, the reflective layer may be made of silver or aluminum. Further advantageously, according to some embodiments, the surface of the reflective layer facing the fluid volume is encapsulated by an additional passivation layer, thereby increasing the lifetime of the device, particularly when silver or aluminum is used as the material for the reflective layer. A suitable passivation layer may be made of, for example, a thin layer of SiO2, which is preferably made transparent and must be thin enough so as not to block the opening of the pores. These materials may also provide good reflectivity in the relevant spectral range and are biocompatible and chemically stable in the environment.

[0062] Advantageously according to one embodiment, the thickness of the reflective layer is between 10 nm - 100 nm, depending on the metal used. Such a layer thickness allows the reflective layer to be applied by evaporation techniques without blocking the opening of the holes at the front side of the translucent sheet. At the same time, the layer thickness must be sufficient to provide sufficient attenuation of the light propagating to the fluid volume in order to ensure a correct optical separation between the detection area and the fluid volume containing the whole blood sample or fluid. Preferably, within the spectral range of the detection, i.e. within the spectral range from which the signal representing the relevant plasma component is formed, such as in the range of 380 nm to 750 nm, 400 to 520 nm, or at about 455 nm, the transmitted light is less than 5%, less than 1% or even less than 0.1%.

[0063] Advantageously according to one embodiment, the detector comprises a spectrophotometer and the optical detection means are configured for spectrophotometric analysis of light emerging from a detection region in the translucent sheet. This allows the spectral signature of one or more components of interest to be resolved in the light emerging from the subsample in the detection region.

[0064] According to a further particularly advantageous embodiment, the optical detection device is configured to measure absorbance. Thus, a surprisingly significant signal is obtained using relatively simple optical equipment. This allows the sensor to be easily integrated with more complex analytical equipment (such as a blood analyzer system).

[0065] Several optically active components may be present in the blood, such as bilirubin, carbon dioxide (CO 2 ), Patent Blue V, and Methylene Blue. The membrane makes it possible to detect bilirubin with a sensitivity high enough to report natural adult bilirubin concentrations. The dye Patent Blue V can be used in lymphangiography and sentinel lymph node biopsy to stain lymphatic vessels. The dye Patent Blue V can also be used as a stain in plaque indicators to show dental plaque on teeth. Methylene blue is used in the treatment of patients with high methemoglobin concentrations in the body and in the treatment of some urinary tract infections.

[0066] When analyzing the spectrum obtained from the porous mirror, it is obvious that the absorption spectrum from whole blood or plasma has a negative baseline. This negative baseline is caused by the porous mirror reflecting a higher proportion of the incident light towards the detector when measuring on whole blood or plasma compared to flushing. This effect can be seen at high wavelengths (600 to 700nm) that hemoglobin does not absorb. This effect is caused by the higher refractive index caused by the high protein content in plasma compared to flushing. The effect is about 5mAbs, compared to about 15mAbs for hemoglobin at the hemoglobin peak wavelength (416nm). In the case where the detector is determined by the reference of the light intensity from the light source, the protein (human serum albumin, HSA) content of the whole blood sample can be detected with a detection limit of about 1-5g / L.

[0067] The membrane configuration can be used to determine CO in whole blood samples or in fluids in the infrared region. 2 .CO 2 It has several absorption peaks in the IR region. 2 O has an overlapping absorption band. The same wavelength can be used for porous mirrors, where the fluid path length in the membrane is about 20 µm. 2 Determined systems can extend the fluid path length because CO 2 Such a high diffusion coefficient.

[0068] The improved porous mirror can consist of a 1.0 to 10 µm thick sheet of silicone rubber (preferably without holes) laminated to a transparent support material. The top of the silicone rubber (the side facing the fluid) has a porous structure attached to it, which allows CO 2 The advantage of using silicone rubber sheet is that silicone rubber has CO 2 The absorption window is open, and water is slightly soluble in silicone rubber. The solubility of carbon dioxide in silicone rubber is about 5 times that in water, which also ensures high sensitivity.

[0069] The porous reflector can be used as a readout device for color producing / color consuming assays. An advantage is that no plasma preparation is required prior to the assay.

[0070] The following types of assays can be used with porous mirrors:

[0071] •Sandwich assay, in which the receptor ligand is allowed to bind within a membrane channel.

[0072] • Assays where one part is bound to the well, such as the bromocresol green albumin assay, which uses bromocresol green to form a colored complex specifically with albumin. The intensity of the color measured at 620 nm is directly proportional to the albumin concentration in the fluid.

[0073] • Enzyme activity assays, such as, for example, the Aspartate Aminotransferase (AST) activity assay kit, in which the amino group is transferred from aspartate to α-ketoglutarate to generate glutamate, thereby generating a colorimetric (450 nm) product proportional to the AST enzyme activity present.

[0074] Porous mirrors can also be used in non-medical applications such as beer brewing, wastewater analysis, food testing, and dye production. In beer brewing, accurate color is required. Porous mirrors can be used to measure on a liquid and compare the reading with a liquid of the correct color to determine if the beer has the desired color. Wastewater can be analyzed for the presence of a component. In food testing (liquids such as milk, juice, and other slurries), porous mirrors can be used to analyze for the presence of a component or analyte. Other chemical reactors such as the dye industry can use porous mirrors to obtain the desired color, content, or other chemical characteristics of their liquids.

[0075] Advantageously according to some embodiments, the porous mirror or a blood analysis system comprising the porous mirror further comprises a processor configured to compare the signal generated by the detector with a predetermined calibration reference to form a quantitative measure of the analyte level in the fluid.

[0076] Further advantageously according to some embodiments, the calibration reference is obtained on a dye-based calibration solution, such as an aqueous solution comprising tartrazine dye.Preferably, the dye-based aqueous solution is prepared from a typical rinse solution to which a calibrant dye, such as tartrazine, is added.

[0077] According to another aspect of the present invention, a system for analyzing a fluid comprises (a) a fluid chamber having an inlet port and an outlet port for feeding and discharging the fluid; (b) a first detector adapted to provide a first signal representing the level of an analyte in the fluid; and (c) one or more additional detectors, each of the additional detectors adapted to provide a corresponding additional signal representing the analyte in the fluid; wherein the first detector and the additional detectors are operable to obtain the first signal and the one or more additional signals from the same fluid, wherein the first detector is constructed as a porous reflector for optically detecting the analyte according to any embodiment disclosed herein.

[0078] As discussed above, with this design, it is achieved that only by contacting the front side surface of the porous reflector with the fluid, a sub-sample of the relevant components of the plasma containing a representative amount can be filled in the hole from the front side, and the sub-sample thus extracted can be conveniently optically detected separately from the fluid. The relevant components can be substances present in the plasma phase of the whole blood sample and to be measured / detected using a sensor. The representative sub-sample of the plasma phase can be extracted from the whole blood sample and transferred to the hole by means of diffusion and / or capillary forces. As also discussed above, the hole is preferably pre-filled with a liquid compatible with the plasma phase, such as an aqueous solution commonly used for flushing, calibration and / or quality control purposes in a blood analyzer. Non-limiting examples of suitable solutions are further given below. Filling the hole with such a known liquid allows the sub-sample representing the relevant components in the plasma to be extracted into the hole by diffusion alone.

[0079] Advantageously according to one aspect of the present invention, a method for optically detecting an analyte such as bilirubin in a fluid is provided, as described in detail below. The method achieves at least the same advantages as discussed above for the corresponding embodiments of a porous mirror for detecting an analyte or a system comprising such a porous mirror.

[0080] According to some embodiments, a method for optically detecting an analyte in a fluid comprises the following steps: providing a porous reflector as disclosed above; contacting the porous reflector with a reference liquid so as to fill the reference liquid in the wells; contacting the front side of the porous reflector with the fluid; waiting for a diffusion time to allow the diffusion of the analyte in the fluid into the wells to reach stability; optically detecting the fluid in the wells; and establishing the analyte level of the fluid based on the result of the optical detection. Preferably, the reference liquid is an aqueous solution compatible with the fluid, in particular compatible with its fraction that can enter the wells, such as a liquid for flushing, calibration and / or quality control. In some embodiments, it is conceivable to omit the step of contacting the front side of the reflector with the reference liquid before introducing the fluid. However, including this step allows purely diffusive sub-sample extraction, which is very efficient and results in surprisingly fast detection responses and surprisingly short measurement cycle times. Most advantageously, the analyte is optically detected in the wells by a color change caused by the presence of a representative amount of the analyte in the extracted sub-sample.

[0081] Advantageously according to some embodiments, the optical probing comprises illuminating the translucent sheet from the back side with a probing light and performing a spectrophotometric analysis of light emitted from the back side of the translucent sheet as an optical response to the probing light.

[0082] Advantageously according to some embodiments, the optical detection is the measurement of absorbance.

[0083] Advantageously according to some embodiments the method further comprises the step of comparing the optical response to a predetermined calibration reference to form a quantitative measure of the level of analyte in the fluid.

[0084] Further advantageously according to some embodiments of the method, the calibration reference is obtained on a dye-based calibration solution such as an aqueous solution comprising tartrazine dye.Preferably, the dye-based aqueous solution is prepared from a typical rinse solution to which a calibrant dye such as tartrazine is added. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings, which show the following views:

[0086] Figure 1 schematically illustrates a porous mirror device in operating conditions according to one embodiment,

[0087] Figure 2 schematically illustrates a cross-sectional detail of a hole with an additional reflective element according to one embodiment;

[0088] Figure 3a / b schematically shows two cross-sectional side views of a detail of a hole with an additional reflective element according to another embodiment;

[0089] Figure 4 schematically shows a cross-sectional side view of a measurement unit;

[0090] Figure 5 Shows Figure 4 A top elevation view of a measuring unit;

[0091] Figure 6a / b schematically shows two cross-sectional side views of a measuring cell having a prismatic exterior with a transparent backing according to another embodiment;

[0092] Figure 7 Shows Figure 6a A top elevation view of a measuring unit;

[0093] Figure 8 A graph is shown showing an example of the response of bilirubin in plasma;

[0094] Fig. 9 shows a coordinate diagram showing CO 2 and H 2 IR spectrum of O (obtained from http: / / www.randombio.com / co2.html on November 8, 2016);

[0095] Fig.10 A graph is shown illustrating an example of using a dye (tartrazine) as a calibration and quality control reference for spectrophotometric measurements;

[0096] Fig.11 Shown is a graph showing an example of the response to different concentrations of a protein (HSA) in human whole blood. DETAILED DESCRIPTION

[0097] Figure 1 A cross-sectional view of a porous reflector 1 according to one embodiment is schematically shown. The porous reflector 1 comprises a translucent sheet 2 having a front side 3 and a back side 4. The front side 3 is provided with a reflective layer 5. The translucent sheet 2 further comprises dead-end holes 6 extending from an opening 7 at the front side 3 through the reflective layer 5 into the body of the translucent sheet 2, where the dead-end holes terminate. Figure 1, but the holes do not have to be perpendicular to the front side 3 or parallel to each other. In operation, the front side 3 of the porous reflector with the hole opening 7 is in contact with the fluid 99. The fluid may have a cell fraction or a specific fraction containing red blood cells or particles 98, and a plasma fraction / fluid fraction 97 containing the relevant component to be detected (here, the analyte 96). The cross-sectional size of the opening 7 of the hole 6 is set so as to prevent the red blood cells or particles 98 from entering the hole 6, while allowing the analyte 96 to enter the hole 6.

[0098] The wells 6 may be pre-filled with a flushing solution 8 that is compatible with the fluid 99, and in particular with the fluid fraction 97. When the fluid 99 contacts the front side 3 of the porous mirror 1 having the pre-filled wells 6, a diffusive transfer of the analyte 96 into the wells 6 occurs, thereby establishing a subsample 9 within the wells 6 having a concentration of the analyte 96 that is representative of the concentration of the analyte 96 in the fluid 99.

[0099] The flushing solution 8 used to pre-fill the well 6 can be any aqueous solution compatible with the fluid 99. Suitable flushing solutions include those flushing solutions commonly used for flushing, calibration and / or quality control purposes in blood parameter analyzers. Such solution compositions typically include organic buffers, inorganic salts, surfactants, preservatives, anticoagulants, enzymes, colorants, and sometimes metabolites. Optical detection is performed from the back side using an optical detection device having a light source 10 and a detector 20. The light source 10 illuminates the detection volume in the porous portion of the translucent sheet 2 from the side of the reflective layer 5 facing away from the fluid 99. The detection light 11 is an oblique incident beam that interacts with the subsample 9 in the well 6. The exiting light 21 is detected by the detector 20, which is also arranged to observe the detection area at an oblique angle. The detector 20 generates a signal representing the exiting light, in particular, containing information about the concentration of the analyte 96 due to the interaction with the subsample 9 in the well 6. Processing the generated signal allows the formation of the analyte level in the fluid. When calibration is used, the analyte level in the fluid can be quantitative. The optical detection technique used for all measurements in the following examples uses spectrally resolved absorbance measurements in the visible range of the electromagnetic spectrum (eg, wavelengths in the range between about 380 nm and 750 nm, between about 400 nm and 520 nm, or at about 455 nm).

[0100] The measurement cycle ends with flushing out the fluid using a flushing solution such as the flushing solution 8 used to prime the wells 6. The sensor device is thereby reinitialized and ready to receive the next fluid.

[0101] Figure 2 A detail of a porous reflector according to another embodiment is shown. A single hole 6 in a translucent sheet 2 is schematically shown. The hole 6 comprises an additional reflective element in the form of a reflective collar 51 produced by depositing a reflective material into the mouth at the opening 7 of the hole 6.

[0102] Figure 3a and Figure 3b Two cross-sectional views of a detail of a porous reflector according to a further embodiment are shown. Again, a single hole 6 in a semi-transparent sheet 2 is schematically shown. The hole 6 comprises an additional reflective element in the form of a small reflector element 52 produced by directed deposition of reflective material into the mouth at the opening 7 of the hole 6, wherein the reflector covers only a portion of the circumference of the opening / mouth, as shown in FIG. Figure 3a and Figure 3b As indicated in the two views of FIG. 2 , the small reflector element 52 is concave when viewed from the inside of the hole. The small reflector elements are produced by directional evaporation of a suitable reflective material (preferably metal) on the inclined porous translucent plate 2, so that all the reflector elements 52 are formed at the same time and point to the same direction. Thus, when the detection light 11 is incident from the concave side of the small reflector element 52, a preferential direction of the outgoing light 21 is achieved. Therefore, the signal-to-noise ratio of the signal generated by the light emitted in the preferential direction is significantly improved.

[0103] All the examples given below have been measured using a sensor configuration with an additional small reflector element obtained by directional sputtering evaporation of Pd onto the front side of a translucent polymer sheet 2 with an evaporation direction at an inclination angle of 25 degrees relative to the surface normal on the front side 3 until a reflective layer 5 with a thickness of 30 nm is obtained on the front side 3 of the translucent sheet 2. The translucent sheet 2 is made of a translucent, preferably transparent polymer material and has track-etched dead-end holes 6 with a substantially circular cross section. These holes have an opening 7 with a diameter of 400 nm, a depth of 25 μm, and are distributed with a porosity of 15% by volume. Together, the pores distributed within a given front side surface area A have a total volume V and an equivalent pore volume depth DELTA = V / A. For the above specified fluids used for the measurements in the examples given below, the equivalent pore volume depth DELTA is about 4 μm.

[0104] Figure 4 and Figure 5 A measuring cell 100 is schematically shown, comprising a porous mirror 1, the front side 3 of which faces a fluid volume 101 within the measuring cell 100. The fluid volume is connected to fluid input and output ports (not shown) for feeding and draining fluid and performing filling, flushing and washing out steps. The back side of the porous mirror is mechanically stabilized by a transparent backing carrier 30, which also acts as a window for light to enter the detection area from the back side 4 of the porous mirror 1. Using the method described above in conjunction with Figure 1The device with the light source 10 and the detector 20 performs optical detection, wherein the detection beam and the detection direction are inclined at corresponding angles to the surface normal on the plane of the front side 3 of the porous reflector 1. In addition, Figure 5 As best shown, the planes of the incident probe light 11 and the detection 21 preferably intersect each other at an angle less than 180 degrees to avoid glare effects, and preferably intersect at an acute angle of about 90 degrees or less. In the measurements of the embodiments given below, the planes of the incident probe light 11 and the outgoing light 21 are arranged symmetrically with respect to a direction parallel to the symmetry plane of the small reflector element 52.

[0105] Figure 6a , Figure 6b and Figure 7 A transparent backing carrier 31 is schematically shown in direct contact with the back side 4 of the translucent plate 2 of the porous reflector 1. When the incident probe light 11 enters the back side carrier 4 of the translucent plate 2 having a surface at the 60° prism 32, the refractive index offset between the air and the polymer does not affect the incident probe light 11, and the light enters the hole 6 (not shown) of the translucent plate 2 without changing the angle of the light, and the outgoing light 21 reaches the detector 20. Figure 6b It is shown that the incident detection light 11 can be reflected several times in the transparent backing carrier 31 before the outgoing light 21 reaches the detector 20. In addition, as shown in FIG. Figure 7 As best shown, the planes of the incident detection light 11 and the outgoing light 21 preferably intersect each other at an angle less than 180 degrees to avoid glare effects, and preferably intersect at an acute angle of about 90 degrees or less, and the prism 32 does not affect the incident detection light 11 or the outgoing light 21.

[0106] Example

[0107] See below Figures 8 to 11 , data from test run measurements are given as examples illustrating different aspects of the performance of a porous mirror according to an embodiment of the invention.

[0108] The porous mirror used for the experiments in these examples was made of a transparent PETP film with a total thickness of 49 µm and provided with linear holes etched by single-sided tracks. The holes had a hole depth of 25 µm and a hole diameter of 0.4 µm and were treated with hydrophilic PVP. The hole surface density was 1.2E8 / cm 2. The hole is therefore dead-ended, with an opening at one side of the PETP membrane and essentially terminating halfway into the PETP membrane acting as a translucent sheet. The porous side of the membrane (translucent sheet) was sputter coated with palladium at an angle of 25 degrees and an approximate layer thickness of 30nm. This gives a metal coating on the porous front side of the membrane (translucent sheet) and a small coating on one side inside the hole, forming a small concave mirror in the mouth of the hole adjacent to its opening toward the front side. The sputtered porous PETP membrane was laminated to a custom cuvette using double-sided tape so that the concave side of the small mirror in the hole pointed halfway between the light guide from the light source and the light guide from the spectrometer input. A drop of approximately 10μL of silicone rubber was pipetted onto the membrane and a cover glass was then fixed to the back side of the membrane as a mechanical backing for the sensor membrane (translucent sheet). The porous mirror was mounted in a test bench for automated handling of liquids, time intervals and data sampling. Data acquisition lasted approximately 3s and was delayed until 14s after fluid acquisition.

[0109] The test bench is equipped with two light emitting diodes (violet and "white" LEDs) as light sources and a miniature spectrometer as detector. The standard slit in the miniature spectrometer has been replaced with a 125µm slit in order to increase light and sensitivity. Since the measurement is a reflection measurement, both the light source and the detector are arranged on the back side (the side without holes) of the porous membrane. The porous metal coated side of the membrane is positioned on the inside of the measurement chamber, so the mirror and the holes are directly exposed to the fluid in the chamber. The light from the two light emitting diodes is guided by a common fiber optic light guide, which has a lens at the end in order to collimate the light to a small spot (approximately 2 mm x 2 mm) of the porous mirror membrane. Referring to Cartesian coordinates, the plane of the membrane (the front side of the translucent sheet) can be defined as the ZX plane of the coordinate system. The light enters the outer surface of the membrane (the back side of the translucent sheet) at an angle of 45° relative 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 60° polar angle relative to the Y axis and is rotated at an azimuth angle relative to the YZ plane that is 90° relative 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 relative to the Y axis provide increased sensitivity for detection of hemoglobin because the collected light has traveled a greater length of the subsample in the well.

[0110] Fluids were prepared by spiking whole blood samples with bilirubin. Plasma-based interference solutions were prepared by spiking plasma with the interferants to the specified values. Plasma was generated by centrifugation at 1500G for 15 minutes. As a reference, the absorption spectra of centrifuged plasma from all whole blood samples tested were also measured on a Perkin Elmer Lambda 19 UV-Vis spectrometer.

[0111] spectrum Figure 8Spectrally resolved absorbance data for two fluids are shown: one is plasma containing bilirubin, the other is plasma alone. At a wavelength of about 455 nm, a clear peak is observed, where the absorbance maxima of the different fluids clearly scale linearly with their bilirubin content.

[0112] spectrum Fig. 9 The carbon dioxide (CO 2 ) and water (H 2 Compared with water, the 2 The non-overlapping peaks of 0.04 and 0.08 indicate that the porous mirror of the present invention can be used in CO-containing fluids even when water is present in the fluid. 2 Determine CO in the fluid 2 content.

[0113] spectrum Fig.10 An example of a series of spectrally resolved absorbance data obtained on a dye-based calibration solution and on a rinse solution for comparison is shown. Spectra are obtained in successive cycles immediately following each other. The dye-based calibration solution is a rinse solution to which 0.5g of tartrazine is added per 1L of rinse solution. The sequence of the measured solutions is as follows: first the rinse solution, then the dye-based calibration solution, then the rinse solution again, again the same dye-based solution, and the sequence of three consecutive measurements is performed on the rinse solution. All spectra are drawn on the same scale and on top of each other. This experiment again shows the excellent stability and reproducibility of the results obtained. However, more importantly, the data show the surprising clear separation of two dye-based solution spectra that overlap on top of each other and all five rinse solution spectra that also overlap on top of each other. It should be noted that these optical data are all detected in the detection volume of the porous reflector. This indicates that a very effective and complete diffusion exchange of extracting and washing out subsamples in the hole is also used when using a dye-based spectrophotometric calibration solution (such as the rinse solution stained with tartrazine mentioned above).

[0114] spectrum Fig.11Spectrally resolved absorbance data of negative baselines caused by higher refractive index produced by high protein content in plasma compared with flushing are shown. Compared with flushing, the porous reflector reflects a higher proportion of incident light toward the detector when measuring on whole blood or plasma. This effect can be seen at high wavelengths (600 to 700nm) that hemoglobin in whole blood does not absorb. The effect is about 5mAbs, while hemoglobin has about 10-15mAbs at the hemoglobin peak wavelength (416nm). The content of protein (HSA) in whole blood samples can be detected with a detection limit of about 1-5g / L. Two different HSA concentrations (20% and 8%) are measured, and higher concentrations are also measured in the presence of free hemoglobin (i.e., hemoglobin other than red blood cells) in the fluid. The presence of hemoglobin in the fluid only affects the part of the spectrum below 600nm. Above 600nm, HSA content is the main influencing factor of the spectrum, and the larger the negative baseline value, the higher the protein content in the whole blood sample.

[0115] Although the devices and methods of the present invention have been specifically discussed in conjunction with the detection of bilirubin, according to a broader aspect, the devices and methods discussed herein are equally applicable to the detection of other optically active substances in the plasma fraction of a whole blood sample or in a fluid, wherein the term "optically active" refers to substances that can be directly detected by spectroscopic optical detection techniques. Such substances may include, but are not limited to, metabolites, medicinal substances, drugs, or vitamins.

Claims

1. A porous reflector (1) for detecting an analyte (96) in a fluid (99) by optical detection, comprising: a translucent sheet (2) having a front side (3) and a back side (4) facing away from the front side (3), wherein the front side (3) is suitable for contact with a fluid (99), and wherein the translucent sheet (2) comprises a hole (6), wherein the hole (6) is a dead-end hole (6) extending from a corresponding opening (7) at the front side (3) through the reflective layer (5) into the translucent sheet (2), wherein the cross-sectional dimensions of the opening (7) of the pore (6) are set so as to prevent any larger particles or debris from entering the pore (6) if the fluid (99) contains any larger particles or debris, while allowing the analyte in the fluid (99) to enter the pore (6) via diffusion, The porous reflector (1) further comprises a light source (10), wherein the light source (10) is adapted to illuminate at least the hole (6) in the translucent sheet (2), wherein the porous mirror (1) further comprises a light detector (20), wherein the detector (20) is arranged to receive light (21) emitted from the holes (6) in response to illumination (11) by the light source (10), and wherein the detector (20) is adapted to generate a signal representative of the detected light, wherein the incident plane and the detection plane intersect at the surface normal to enclose an azimuth angle greater than 0 degrees and less than 160 degrees, wherein the incident plane is spanned by the direction of the illumination light beam and the surface normal of a reference plane defined by the front side of the translucent plate, and wherein the detection plane is spanned by the direction of the outgoing light propagating toward the detector and the surface normal of the reference plane.

2. The porous reflector (1) according to claim 1, wherein the incident plane and the detection plane intersect at the surface normal to enclose an azimuth angle greater than 0 degrees and less than 130 degrees.

3. The porous reflector (1) according to claim 1, wherein the incident plane and the detection plane intersect at the surface normal to enclose an azimuth angle greater than 0 degrees and less than 90 degrees.

4. A porous reflector (1) according to claim 1, wherein the cross-sectional size of the opening (7) of the hole (6) is 1µm or less or 800nm ​​or less, and / or wherein the length of the hole (6) in the axial direction along the hole (6) is less than 100µm or less than 50µm.

5. A porous reflector (1) according to claim 1, wherein the cross-sectional size of the opening (7) of the hole (6) is 500nm or less or even 400nm or less, and / or wherein the length of the hole (6) in the axial direction along the hole (6) is less than 100µm or less than 50µm.

6. A porous reflector (1) according to claim 1, wherein the cross-sectional size of the opening (7) of the hole (6) is 1µm or less or 800nm ​​or less, and / or wherein the length of the hole (6) in the axial direction along the hole (6) is less than 30µm, or 25µm.

7. A porous reflector (1) according to claim 1, wherein the cross-sectional size of the opening (7) of the hole (6) is 500nm or less or even 400nm or less, and / or wherein the length of the hole (6) in the axial direction along the hole (6) is less than 30µm, or 25µm.

8. A porous mirror (1) according to any one of the preceding claims, wherein the opening of the pores is 30 nm or more, or 50 nm or more, or 100 nm or more, or 200 nm or more.

9. The porous reflector (1) according to claim 1, wherein the porosity of a given volume of the translucent sheet (2) including holes (6) is between 50% and 5% by volume, between 30% and 10% by volume, or is 15% by volume.

10. A porous reflector (1) according to claim 1, wherein the equivalent pore volume depth (DELTA) is less than 20µm, alternatively less than 10µm, wherein the equivalent pore volume depth (DELTA) is defined as the total volume (V) of the pores divided by the front side area (A), and the openings (7) of the pores (6) are distributed on the front side area (A).

11. A porous reflector (1) according to claim 10, wherein the equivalent pore volume depth (DELTA) is 5 µm or less, wherein the equivalent pore volume depth (DELTA) is defined as the total volume (V) of the pores divided by the front side area (A), and the openings (7) of the pores (6) are distributed on the front side area (A).

12. A porous reflector (1) according to claim 1, wherein the equivalent pore volume depth (DELTA) is at least 1µm, or at least 2µm, or in the range of 3µm to 5µm, wherein the equivalent pore volume depth (DELTA) is defined as the total volume (V) of the pores divided by the front side area (A), and the openings (7) of the pores (6) are distributed on the front side area (A).

13. A porous reflector (1) according to claim 1, wherein the equivalent pore volume depth (DELTA) is in the range of 1µm to 20µm, wherein the equivalent pore volume depth (DELTA) is defined as the total volume (V) of the pores divided by the front side area (A), and the openings (7) of the pores (6) are distributed on the front side area (A).

14. A porous reflector (1) according to claim 13, wherein the equivalent pore volume depth (DELTA) is in the range of 2µm to 10µm or 4µm-5µm, wherein the equivalent pore volume depth (DELTA) is defined as the total volume (V) of the pores divided by the front side area (A), and the openings (7) of the pores (6) are distributed on the front side area (A).

15. The porous reflector (1) according to claim 1, wherein the inner wall surface of the hole (6) is coated with a hydrophilic coating.

16. The porous reflector (1) according to claim 1, wherein the translucent plate (2) is provided with an additional reflective element (51, 52), which is arranged in the hole (6), in its mouth, adjacent to the opening (7) located at the front side (3) of the translucent plate (2).

17. A porous reflector (1) according to claim 16, wherein the additional reflective element (51, 52) is arranged as a reflective coating covering only a portion of the circumference of the mouth of the hole (6) near the opening (7), wherein the portion is 70% or less.

18. A porous reflector (1) according to claim 17, wherein the additional reflective element (51, 52) is arranged as a reflective coating covering only a portion of the circumference of the mouth of the hole (6) near the opening (7), wherein the portion is 50% or less.

19. The porous reflector (1) according to claim 1, wherein the transparent backing carrier 31 of the translucent plate (2) is provided with a 60° inclined surface (32) to minimize the effect of the refractive index shift between the external air and the transparent backing carrier (31).

20. The porous mirror (1) according to claim 1, wherein the holes (6) are track-etched in the translucent sheet (2) and the reflective layer (5).

21. The porous mirror (1) according to claim 1, wherein the holes (6) are flushed by diffusion.

22. The porous reflector (1) according to claim 1, wherein the translucent sheet (2) is made of a transparent polymer.

23. A method for optically detecting an analyte (96) in a fluid (99), comprising: - providing a porous mirror (1) according to any one of the preceding claims, - bringing the porous mirror (1) into contact with a reference liquid so as to fill the holes (6) with the reference liquid, - bringing the front side of the porous mirror (1) into contact with a fluid (99), - waiting for a diffusion time to allow the diffusion of the analyte (96) in the fluid (99) into the pore (6) to stabilize, - optical detection of the fluid in the hole (6) from the side of the reflective layer, and - establishing an analyte level of said fluid based on the results of said optical detection.

24. The method for optically detecting an analyte (96) in a fluid (99) according to claim 23, wherein the analyte is bilirubin and the fluid is whole blood.

25. A system for analyzing a fluid (99), comprising: a fluid chamber (100) having an inlet port and an outlet port for feeding and discharging the fluid (99), - a first detector (20) adapted to provide a first signal representative of the level of an analyte (96) in the fluid (99), and - one or more further detectors, each further detector being adapted to provide a respective further signal representative of said analyte (96) of said fluid (99), wherein the first detector and the further detector are operable to obtain the first signal and the one or more further signals from the fluid (99), The first detector is configured as a porous mirror (1) for optically detecting the analyte (96) according to any one of claims 1-22.

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