Device for detecting dynamic oxygen carrying and oxygen release of membranous hemoglobin solution, absorbance detection and calculation method, equipment, medium and program product

Abstract

The invention provides a device for detecting dynamic oxygen carrying and oxygen release of a membranous hemoglobin solution, an absorbance detection and calculation method, equipment, a medium and a program product, and relates to the technical field of biological detection. A detection bin is arranged in the device, and the device further comprises a sample assembly unit located in the detection bin and used for containing a to-be-detected sample; the sample assembly unit comprises a pore plate bracket, a polytetrafluoroethylene film and cover glass which are sequentially arranged from bottom to top; the polytetrafluoroethylene film tightly covers the upper end of the pore plate bracket, and a to-be-detected sample is added to the upper end of the polytetrafluoroethylene film; and an optical detection unit. The device supports synchronous detection of multi-sample absorbance of a film-shaped hemoglobin solution, carries a polytetrafluoroethylene film, realizes gas exchange based on the characteristics of chemical inertness, ultra-thinness, porosity and the like of the polytetrafluoroethylene film, and simulates a real scene of gas interaction between a hemoglobin spray and the surrounding environment on the surface of a wound surface.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically, to a device for detecting dynamic oxygen carrying and oxygen release of a membranous hemoglobin solution, and a method, equipment, medium and program product for detecting and calculating absorbance. Background Art

[0002] Chronic wounds are injuries to the skin and subcutaneous tissue caused by various reasons that fail to heal within the normal healing period (usually 3-4 weeks) and show no clear healing tendency. The healing process of chronic wounds is influenced by multiple complex factors, among which hypoxia is a key factor. Hypoxia negatively impacts the function and activity of repair cells in chronic wounds, significantly slowing the rate and quality of wound healing. Hemoglobin has the ability to carry and release oxygen deep into wound tissue through wound exudate. This spray has entered clinical use in some European and American countries to improve chronic wound hypoxia and promote wound healing. However, this spray lacks a specific mechanism of action and supporting data in practical applications, and its dynamic oxygen-carrying and oxygen-releasing processes in chronic wounds remain unclear, significantly limiting its further optimization and wider application.

[0003] At present, the dynamic oxygen carrying / release in hemoglobin solution is usually detected by absorbance method, such as enzyme labeling method and spectrophotometer method. These two detection methods use enzyme labeling plate and cuvette as sample pool respectively, so that the thickness of the sample to be tested is usually several millimeters to tens of millimeters, which cannot fully exchange gas with the surrounding air. When the hemoglobin spray is used, the hemoglobin solution is sprayed out through the nozzle and evenly covers the wound surface, forming only a film-like hemoglobin with a thickness of about several microns to tens of microns. During the contact with the wound, it combines oxygen from the air with higher oxygen partial pressure and releases oxygen on the wound surface with lower oxygen partial pressure. By repeating the oxygen carrying / release process, the wound hypoxia state is improved. Therefore, the existing conventional methods cannot truly simulate the dynamic oxygen carrying / release process of hemoglobin spray when it is used to treat chronic wounds. In addition, although there are literature reports that a device constructed based on the diffusion chamber principle can be used to simulate the dynamic oxygen carrying / release process of hemoglobin spray, the device construction process used in this method is very cumbersome and complicated, and the required detection components need to be customized. In actual application, there will be operational difficulties and technical barriers, and only one sample can be tested at a time, and the detection efficiency is low. Summary of the Invention

[0004] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention constructs a simple device that supports absorbance detection and gas exchange in membranous hemoglobin solutions, as well as a method for detecting and calculating absorbance using this device. This method and device are primarily used for studying hemoglobin function in the treatment of chronic wounds. The device possesses two key functions: First, it supports simultaneous absorbance detection of multiple samples of membranous hemoglobin solutions, reversing the previous inefficient model of individual detection to acquire absorbance data for multiple samples simultaneously, improving detection efficiency. Second, it incorporates a polytetrafluoroethylene (PTFE) film. Leveraging its chemically inert, ultrathin, and porous properties, it enables gas exchange, simulating the actual interaction between hemoglobin spray on the wound surface and the surrounding environment. This simple device allows researchers to accurately simulate the state of hemoglobin spray on the wound surface, enabling scientific and effective evaluation of the dynamic oxygen transport and release of membrane hemoglobin solutions, providing stronger technical support for the treatment of chronic wounds.

[0005] In a first aspect, the present application discloses a device for detecting dynamic oxygen carrying and oxygen release of a membranous hemoglobin solution, wherein a detection chamber is provided in the device, and the device further comprises:

[0006] The sample assembly unit is located in the detection chamber and is used to accommodate the sample to be tested. The sample assembly unit includes a well plate holder, a polytetrafluoroethylene film, and a cover glass arranged in order from bottom to top. The polytetrafluoroethylene film tightly covers the upper end of the well plate holder, and the sample to be tested is added to the upper end of the polytetrafluoroethylene film.

[0007] The optical detection unit is used to collect and convert the optical signal of the sample to be tested and calculate the absorbance of the sample to be tested in different bands.

[0008] The device supports simultaneous absorbance detection of multiple samples of membranous hemoglobin solution. It is equipped with a polytetrafluoroethylene film and, based on the chemical inertness, ultra-thinness, and porosity of the polytetrafluoroethylene film, achieves gas exchange, simulating the real scene of gas interaction between the hemoglobin spray on the wound surface and the surrounding environment.

[0009] In some embodiments, the thickness of the polytetrafluoroethylene film includes: 0.75-2 μm; preferably 1 μm;

[0010] Optionally, the orifice plate support is a bottomless orifice plate; the number of holes in the bottomless orifice plate is greater than 1;

[0011] Optionally, the sample to be tested is a hemoglobin solution;

[0012] Optionally, the source of the hemoglobin solution to be tested includes any one of the following: whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and chemically modified or physically assembled hemoglobin.

[0013] In some embodiments, the optical detection unit includes a light source, a photodetector, and an optical absorption detection device. The light source generates light of different wavelengths. The photodetector faces the detection chamber and reflects the light, which then enters the detection chamber and passes through the sample to be tested in the detection chamber and is absorbed by the optical absorption detection device.

[0014] Optionally, a line connecting the reflection point on the photodetector and the center of the optical absorption detection device is perpendicular to a horizontal reference line;

[0015] Optionally, there is an angle between the direction in which the light enters the photoelectric detector and the direction in which the light enters the detection chamber.

[0016] In some embodiments, a plate carrier is provided in the detection chamber, the sample assembly unit is placed on the plate carrier, and the plate carrier is placed in the detection chamber;

[0017] Optionally, the device further includes:

[0018] Temperature control unit, used to simulate the temperature environment of chronic scenes;

[0019] Gas control unit, used to simulate the moist environment of chronic wounds;

[0020] Optionally, the temperature control unit includes at least two heating plates and at least two temperature sensors, and the number of the heating plates and the temperature sensors is the same; the two heating plates are respectively located at the upper and lower ends of the plate carrier, and there is a distance between the heating plates and the plate carrier; the temperature sensors are respectively arranged at one end of the temperature sensor close to the plate carrier;

[0021] Optionally, the gas control unit includes a nitrogen group and an air group, the nitrogen group and the air group respectively include a gas humidifying bottle, a connecting pipeline and a gas flow regulating valve, the two ends of the connecting pipeline are connected to the gas humidifying bottle and the detection chamber, and the gas flow regulating valves are respectively provided in the connecting pipeline;

[0022] Optionally, the connection points between the two connecting pipes and the detection chamber are respectively located on the detection chamber wall corresponding to the space where the heating plate is away from the plate carrier.

[0023] The second aspect of the present application discloses a method for detecting and calculating absorbance, the method comprising:

[0024] 101, adding the sample to be tested to the corresponding position of the well plate holder covered with a polytetrafluoroethylene gas permeable film, covering it with a cover glass, and obtaining an assembled sample assembly unit;

[0025] 102. Place the assembled sample assembly unit on the plate carrier of the device described in the first aspect of the present application and operate it to calculate the absorbance at different wavelengths; the wavelengths include: 560nm, 570nm, 576nm, 630nm and 700nm.

[0026] In some embodiments, the operation mode in 102 includes: adjusting the gas control unit, introducing nitrogen and air in sequence, and switching conditions: nitrogen is introduced when the deoxyhemoglobin content is lower than 10%, and air is introduced when the deoxyhemoglobin content is higher than 90%, and ventilation is circulated multiple times.

[0027] In some embodiments, the method further comprises: calculating the percentage of hemoglobin in different states in the hemoglobin solution based on the absorbance;

[0028] Optionally, the different states of hemoglobin include any one or more of the following: carboxyhemoglobin COHb, oxyhemoglobin OxyHb, methemoglobin MetHb, deoxyhemoglobin DeoxyHb;

[0029] Optionally, the source of the hemoglobin solution to be tested includes any one of the following: whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and chemically modified or physically assembled hemoglobin;

[0030] Optionally, the hemoglobin concentration to be measured is less than or equal to 10 g / dl.

[0031] A third aspect of the present application discloses a computer device, comprising: a memory and a processor; the memory is used to store a computer program; and the processor executes the computer program to implement the steps of the above method.

[0032] In a fourth aspect, the present application discloses a computer-readable storage medium having a computer program stored thereon, which implements the steps of the above-mentioned method when the computer program is executed by a processor.

[0033] In a fifth aspect, the present application discloses a computer program product, comprising a computer program, which implements the steps of the above method when executed by a processor.

[0034] This application specifically improves the microplate reader, using a membrane to simulate this extremely thin test. Conventional 96-well plates cannot achieve this thickness. Furthermore, the two devices they built were complex and only capable of single-detection testing. We used a microplate reader with some clever tricks to simulate gas and temperature control. The improved device and method have the following advantages:

[0035] (1) Cost-effectiveness: The device has a simple structure, and its main components are common laboratory consumables, which are low-cost and easy to prepare and replace. This greatly reduces the cost of equipment procurement and maintenance, and improves the cost-effectiveness of testing.

[0036] (2) Ease of operation: The operation process is simple and intuitive, and does not require complex debugging and operation training by professional technicians. Ordinary laboratory personnel can quickly get started, reducing operational errors and improving detection efficiency and repeatability.

[0037] (3) Detection Accuracy: This invention combines an optimized calculation formula with a specific thin-film sample processing method, while fully considering the effects of temperature and humidity on hemoglobin. This multi-factor comprehensive optimization design effectively reduces the interference of external factors on the test results, can accurately measure the characteristic parameters of the hemoglobin solution, and provides more accurate data for studying the function of hemoglobin in chronic wound environments.

[0038] (4) Wide concentration adaptability: The thin film type test can flexibly detect hemoglobin solutions with different concentration ranges by adjusting parameters such as the volume of the hemoglobin solution added, the dilution ratio, or the film area thickness. The highest detectable hemoglobin concentration is 10g / dl. This feature enables the device and method to meet different experimental needs and clinical sample detection requirements, broadening its application range.

[0039] (5) Strong compatibility with sample types: Thin-film detection is applicable to hemoglobin solutions from a variety of sources, including whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and hemoglobin after chemical modification or physical assembly. Simply by treating the solution to meet the requirements of thin-film diffusion, the test can be performed, providing a platform for different sample testing in biopharmaceutical R&D and clinical diagnosis.

[0040] Compared with existing complex devices, the present invention greatly improves detection efficiency on the basis of considering temperature and humidity control, and can simultaneously detect multiple samples; the detection accuracy is not limited by membrane permeability, and can accurately measure different concentrations and multiple sample types, with obvious advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0042] Figure 1 is a schematic diagram of a device provided by the first aspect of an embodiment of the present invention;

[0043] Figure 2is a schematic flow chart of the method provided by the second aspect of an embodiment of the present invention;

[0044] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention;

[0045] Figure 4 is a schematic diagram of the architecture of an exemplary computing device provided by an embodiment of the present invention;

[0046] Figure 5 is a schematic diagram of a storage medium provided by an embodiment of the present invention;

[0047] Figure 6 Schematic diagram of the test results corresponding to the two thicknesses of 20 μm and 1 μm of the polytetrafluoroethylene film provided in an embodiment of the present invention;

[0048] Figure 7 Schematic diagram of the deoxyhemoglobin content test results provided by an embodiment of the present invention;

[0049] In the figure, 11, sample to be tested; 12, orifice plate holder; 13, polytetrafluoroethylene film; 14, cover glass; 21, light source; 22, photodetector; 23, optical absorption detection device; 31, temperature sensor; 32, heating plate; 41, gas humidification bottle; 42, gas flow regulating valve; 5, detection chamber; 6, plate carrier; 7, signal processing system. DETAILED DESCRIPTION

[0050] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0051] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or may be executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish between different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., and do not represent the order of precedence, nor do they limit "first" and "second" to be different types.

[0052] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0053] Figure 1 Schematic diagram of a device for dynamic oxygen carrying and oxygen release detection of a membranous hemoglobin solution provided by an embodiment of the present invention. Specifically, a detection chamber is provided in the device, and the device further includes:

[0054] The sample assembly unit is located in the detection chamber and is used to accommodate the sample to be tested. The sample assembly unit includes a well plate holder, a polytetrafluoroethylene film, and a cover glass arranged in order from bottom to top. The polytetrafluoroethylene film tightly covers the upper end of the well plate holder, and the sample to be tested is added to the upper end of the polytetrafluoroethylene film.

[0055] The optical detection unit is used to collect and convert the optical signal of the sample to be tested and calculate the absorbance of the sample to be tested in different bands.

[0056] The device supports simultaneous absorbance detection of multiple samples of membranous hemoglobin solution. It is equipped with a polytetrafluoroethylene film and, based on the chemical inertness, ultra-thinness, and porosity of the polytetrafluoroethylene film, achieves gas exchange, simulating the real scene of gas interaction between the hemoglobin spray on the wound surface and the surrounding environment.

[0057] In some embodiments, the thickness of the polytetrafluoroethylene film includes: 0.75-2 μm; preferably 1 μm;

[0058] Optionally, the orifice plate support is a bottomless orifice plate; the number of holes in the bottomless orifice plate is greater than 1;

[0059] Optionally, the sample to be tested is a hemoglobin solution; optionally, the source of the hemoglobin solution to be tested includes any one of the following: whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and chemically modified or physically assembled hemoglobin.

[0060] In some embodiments, the optical detection unit includes a light source, a photodetector, and an optical absorption detection device. The light source generates light of different wavelengths. The photodetector faces the detection chamber and reflects the light, which then enters the detection chamber and passes through the sample to be tested in the detection chamber and is absorbed by the optical absorption detection device.

[0061] Optionally, a line connecting the reflection point on the photoelectric detector and the center of the optical absorption detection device is perpendicular to a horizontal reference line; and an angle is formed between a direction in which the light enters the photoelectric detector and a direction in which the light enters the detection chamber.

[0062] In some embodiments, a plate carrier is provided in the detection chamber, the sample assembly unit is placed on the plate carrier, and the plate carrier is placed in the detection chamber;

[0063] Optionally, the device further comprises: a temperature control unit for simulating the temperature environment of a chronic wound; a gas control unit for simulating the moist environment of a chronic wound;

[0064] Optionally, the temperature control unit includes at least two heating plates and at least two temperature sensors, and the number of the heating plates and the temperature sensors is the same; the two heating plates are respectively located at the upper and lower ends of the plate carrier, and there is a distance between the heating plates and the plate carrier; the temperature sensors are respectively arranged at one end of the temperature sensor close to the plate carrier; the temperature in the detection chamber is controlled to be accurately adjusted and stably maintained at a temperature close to human body temperature, thereby simulating the physiological temperature environment of the human body where the chronic wound is located;

[0065] Optionally, the gas control unit includes a nitrogen group and an air group, and the nitrogen group and the air group respectively include a gas humidification bottle, a connecting pipe and a gas flow regulating valve. The two ends of the connecting pipe are connected to the gas humidification bottle and the detection chamber, and the gas flow regulating valves are respectively arranged in the connecting pipe; the gas humidification bottle humidifies the gas before passing it into the detection chamber to simulate the actual moist environment around the chronic wound; there are two gas inlet holes in total, through which nitrogen and air are respectively introduced. The purpose of introducing nitrogen is to make oxygenated hemoglobin release oxygen, and the purpose of introducing air is to make deoxygenated hemoglobin carry oxygen again, simulating the oxygen carrying and oxygen release process of hemoglobin in a low oxygen partial pressure environment. Optionally, the connection points of the two connecting pipes and the detection chamber are respectively located on the detection chamber wall corresponding to the space where the heating plate is away from the plate carrier.

[0066] The enzyme-linked immunosorbent assay reader (ELISA) provided in this embodiment, also known as an enzyme-linked immunosorbent assay (ELISA), is an instrument used in laboratories to detect and analyze enzyme-linked immunosorbent assay (ELISA) results. The main components of the ELISA reader are as follows:

[0067] Light source: Provides light of the required wavelength, typically using a tungsten lamp, halogen lamp, or light-emitting diode (LED).

[0068] Filters: Select specific wavelengths of light. Microplate readers are usually equipped with multi-wavelength filters to allow samples to be detected at different wavelengths.

[0069] Cuvette or microplate placement area: used to place microplates containing samples to be tested.

[0070] Detector: Typically a photomultiplier tube (PMT) or other photodetector that measures the intensity of light passing through the microplate.

[0071] Optical system: This system includes a series of lenses and mirrors that guide light through the microplate and to the detector.

[0072] Data processor: It is used to process the light signal collected by the detector and convert it into understandable numerical values, such as absorbance or fluorescence intensity.

[0073] Control Panel: Users enter parameters, start tests, and view results through the control panel.

[0074] Software: used to operate the microplate reader, collect data, process data and output reports.

[0075] Mechanical structure: includes chassis, doors, covers, etc., used to protect internal components and provide necessary working space.

[0076] Communication interface: such as USB, RS-232, etc., used to communicate with external devices such as computers and transmit data.

[0077] Internal calibration system: Some microplate readers have an automatic calibration function to ensure the accuracy and reproducibility of the test.

[0078] Figure 2 1 is a flow chart of a method for detecting and calculating absorbance provided by an embodiment of the present invention. Specifically, the method comprises the following steps: 101, adding a sample to be tested to a corresponding position of a well plate holder covered with a polytetrafluoroethylene gas permeable film, covering it with a cover glass, and obtaining an assembled sample assembly unit;

[0079] 102. Place the assembled sample assembly unit on the plate carrier of the device described in the first aspect of the present application and operate it to calculate the absorbance at different wavelengths; the wavelengths include: 560nm, 570nm, 576nm, 630nm and 700nm.

[0080] In some embodiments, the operation mode in 102 includes: adjusting the gas control unit, introducing nitrogen and air in sequence, and switching conditions: nitrogen is introduced when the deoxyhemoglobin content is lower than 10%, and air is introduced when the deoxyhemoglobin content is higher than 90%, and ventilation is circulated multiple times.

[0081] In some embodiments, the hemoglobin spray is tested for its oxygen carrying / release capacity in the air, simulating the oxygen carrying / release capacity of the spray on the skin surface.

[0082] In some embodiments, the method further comprises: calculating the percentage of hemoglobin in different states in the hemoglobin solution based on the absorbance;

[0083] Optionally, the different states of hemoglobin include any one or more of the following: carboxyhemoglobin COHb, oxyhemoglobin OxyHb, methemoglobin MetHb, deoxyhemoglobin DeoxyHb;

[0084] Optionally, the source of the hemoglobin solution to be tested includes any one of the following: whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and chemically modified or physically assembled hemoglobin.

[0085] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention, such as Figure 3 As shown, the device 2000 may include: one or more processors 2010, and one or more memories 2020; wherein the memories store computer-readable codes, and when the computer-readable codes are run by the one or more processors, they may execute the method described above.

[0086] The processor in this embodiment can be an integrated circuit chip with signal processing capabilities. The above-mentioned processor can be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, a discrete gate or transistor logic device, or a discrete hardware component. It can implement or execute the various methods, operations, and logic block diagrams disclosed in the embodiments of the present disclosure. The general-purpose processor can be a microprocessor or any conventional processor, etc., and can be an X86 architecture or an ARM architecture.

[0087] In general, various example embodiments of the present disclosure may be implemented in hardware or dedicated circuitry, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the disclosed embodiments are illustrated or described as block diagrams, flow charts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented, as non-limiting examples, in hardware, software, firmware, dedicated circuitry or logic, general-purpose hardware or a controller or other computing device, or some combination thereof.

[0088] For example, the method or apparatus according to the embodiment of the present disclosure may also be implemented by Figure 4 The architecture of the computing device 3000 shown in FIG. Figure 4As shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, an input / output component 3060, a hard disk 3070, etc. The storage device in the computing device 3000, such as the ROM 3030 or the hard disk 3070, may store various data or files used for processing and / or communication of the method provided in the present disclosure, as well as program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 The architecture shown is only exemplary and can be omitted according to actual needs when implementing different devices. Figure 4 One or more components of a computing device are shown.

[0089] The embodiment of the present invention further provides a computer-readable storage medium, such as Figure 5 As shown, it is a schematic diagram of a storage medium 4000 provided in an embodiment of the present invention, and computer-readable instructions 4010 are stored on the computer storage medium 4020. When the computer-readable instructions 4010 are executed by the processor, the method according to the embodiment of the present disclosure described with reference to the above figures can be executed. The computer-readable storage medium in the embodiment of the present disclosure can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. The non-volatile memory can be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM) or a flash memory. The volatile memory can be a random access memory (RAM), which is used as an external cache. By way of example and not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDRSDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory. It should be noted that the memory of the methods described herein is intended to include, but is not limited to, these and any other suitable types of memory.

[0090] The embodiments of the present disclosure further provide a computer program product or system, including a computer program, which implements the steps of the above method when executed by a processor.

[0091] In some embodiments, this embodiment also discloses a system for detecting and calculating absorbance, such as Figure 2 As shown, the system includes:

[0092] A test sample adding module is used or configured to add the test sample to a corresponding position of the well plate holder covered with a polytetrafluoroethylene breathable film and cover it with a cover glass to obtain an assembled sample assembly unit;

[0093] An absorbance calculation module is used or configured to place the assembled sample component unit on the plate carrier of the device described in the first aspect of the present application and operate it to calculate the absorbance at different wavelengths; the wavelengths include: 560nm, 570nm, 576nm, 630nm and 700nm.

[0094] In some embodiments, the system further comprises: a hemoglobin percentage calculation module, configured to calculate the percentage of hemoglobin in different states in the hemoglobin solution based on the absorbance. Specific embodiments

[0095] (1) Device structure: The device schematic is as attached Figure 1 The detection device of the present invention consists of a sample assembly unit, an optical detection unit, a temperature control unit, and a gas control unit. By controlling both temperature and humidity, the impact of environmental factors on the properties of the hemoglobin solution is effectively reduced, ensuring that the test results more accurately and reliably reflect the true function of hemoglobin in chronic wound environments.

[0096] The sample assembly unit consists of a bottomless 96-well plate, a 1μm thick polytetrafluoroethylene breathable film, and a cover glass: the bottomless 96-well plate serves as the basic support structure, providing a stable carrying platform for the sample. In addition, the row and column markings on the 96-well plate allow the position of each well to be clearly and accurately located, making it convenient for detection; the polytetrafluoroethylene breathable film tightly covers the well plate, and its good air permeability ensures that gases can be freely exchanged during the detection process. At the same time, it has chemical stability and will not react with the hemoglobin solution. During the experiment, polytetrafluoroethylene breathable films with a thickness of 20μm and 1μm were compared. At a wavelength of 426nm, the transmittance of the 1μm thick polytetrafluoroethylene film was 55%, while the transmittance of the 20μm thick polytetrafluoroethylene film was only 3.53% (as shown in the attached figure). Figure 6 As shown in the figure, low membrane transmittance can lead to poor contrast in sample detection and affect measurement accuracy. Therefore, a thickness of 1 micron is preferred (multiple thicknesses are possible, but 1 micron works best). The cover glass is used to cover the hemoglobin solution to allow the solution to diffuse, and the thickness can be controlled at around 40-50 μm, which is consistent with the thickness used for hemoglobin sprays reported in the literature.

[0097] The optical detection unit consists of a light source, a photodetector, an optical absorption detection device and a signal processing system. It is responsible for collecting and converting the optical signals of the sample in the sample assembly unit and calculating the absorbance of the sample in a specific band.

[0098] The temperature control unit consists of the microplate reader's built-in heating plate and temperature sensor, which can accurately adjust the temperature in the detection chamber and stably maintain it at a temperature close to human body temperature (37°C), thereby simulating the physiological temperature environment of the human body where chronic wounds are located.

[0099] The gas control unit consists of a gas cylinder, a gas humidifier, and the microplate reader's built-in gas flow control valve. The humidifier humidifies the gas before it enters the detection chamber, simulating the actual moist environment surrounding chronic wounds. There are two gas inlet ports, one for nitrogen and one for air. Nitrogen is introduced to cause oxyhemoglobin to release oxygen, while air is introduced to cause deoxyhemoglobin to re-carry oxygen, simulating the oxygen-carrying and oxygen-releasing process of hemoglobin under low oxygen partial pressure.

[0100] (2) Detection method: First, approximately 50 μl of hemoglobin solution was added dropwise to the corresponding position of a bottomless 96-well plate covered with a polytetrafluoroethylene gas-permeable film. The plate was quickly covered with a coverslip to allow the solution to spread evenly. The absorbance of the hemoglobin solution at specific wavelengths was measured using a microplate reader: 560 nm, 560 nm, 570 nm, 576 nm, 630 nm, and 700 nm.

[0101] (3) Calculation method: Based on the Lambert-Beer law, the percentage of hemoglobin in different states in the hemoglobin solution is calculated using the different wavelength method. The Lambert-Beer law is the basic law of spectrophotometry, which describes the relationship between the intensity of a substance's absorption of light of a certain wavelength and the concentration of the absorbing substance and the thickness of the liquid layer; A=K1b, k1 is the proportionality coefficient, and b is the liquid layer thickness.

[0102] When the hemoglobin states in the solution are primarily carboxyhemoglobin (COHb), oxyhemoglobin (OxyHb), and methemoglobin (MetHb), calculations were performed using three-wavelength (570 nm, 576 nm, 630 nm), four-wavelength (560 nm, 570 nm, 576 nm, 630 nm), three-wavelength (570 nm, 576 nm, 630 nm, 700 nm) corrected at 700 nm, and four-wavelength (560 nm, 570 nm, 576 nm, 630 nm, 700 nm) corrected at 700 nm. Comparison with blood gas analysis revealed that the wavelength formula with an absolute error within 2.0% and good repeatability was preferred. Ultimately, the three-wavelength formula corrected at 700 nm was confirmed to be optimal, with the smallest error compared to blood gas analysis.

[0103] The calculation formula of the selected three-wavelength method calibrated at 700nm is as follows:

[0104] C COHb =1.5062*(OD 560 -OD 700 )-0.4421*(OD 630 -OD 700 )-1.1188*(OD 576 -OD 700 );

[0105] C OxyHb =1.368*(OD 576 -OD 700 )-1.0156*(OD 560 -OD 700 )-0.5311*(OD 630 -OD 700 );

[0106] C MetHb =2.7517*(OD 630 -OD 700 )+0.0141*(OD 576 -OD 700 )-0.0525*(OD 570 -OD 700 );

[0107] F COHb =[C COHb / (C COHb +C oxyHb +C MetHb )]*100%;

[0108] F OxyHb =[C OxyHb / (CCOHb +C oxyHb +C MetHb )]*100%;

[0109] F MetHb =[C MetHb / (C COHb +C oxyHb +C MetHb )]*100%.

[0110] b. When the hemoglobin states in the solution are primarily oxyhemoglobin, deoxyhemoglobin (DeoxyHb), and methemoglobin, a three-wavelength formula (560nm, 576nm, 630nm, 700nm) calibrated at 700nm is used for calculation. Compared with blood gas analysis, this formula shows an absolute error of less than 2.0% and good repeatability.

[0111] The calculation formula of the selected three-wavelength method calibrated at 700nm is as follows:

[0112] C DeoxyHb =1.373*(OD 560 -OD 700 )-0.747*(OD 576 -OD 700 )-0.737*(OD 630 -OD 700 );

[0113] C OxyHb =1.013*(OD 576 -OD 700 )-0.3269*(OD 630 -OD 700 )-0.7353*(OD 560 -OD 700 );

[0114] C MetHb =2.985*(OD 630 -OD 700 )+0.194*(OD 576 -OD 700 )-0.4023*(OD 560 -OD 700 );

[0115] F DeoxyHb =[C DeoxyHb / (C DeoxyHb +C oxyHb +C MetHb )]*100%;

[0116] F OxyHb =[COxyHb / (C DeoxyHb +C oxyHb +C MetHb )]*100%;

[0117] F MetHb =[C MetHb / (C DeoxyHb +C oxyHb +C MetHb )]*100%.

[0118] The specific operation process is as follows:

[0119] 1. Device Assembly: Select a standard bottomless 96-well plate. Flatly cover the plate with a polytetrafluoroethylene (PTFE) film cut to the appropriate size. Prepare a clean, scratch-free glass slide for subsequent covering with the hemoglobin solution.

[0120] 2. Sample Preparation: Use a pipette to draw up 50 μl of hemoglobin solution and slowly add it dropwise to the corresponding well of the film-covered plate, avoiding splashing and bubbles. Cover with a glass slide and allow the solution to spread evenly over the desired area and thickness.

[0121] 3. Sample Testing: Place the assembled sample in a microplate reader and adjust the gas flow path connected to the microplate reader to introduce nitrogen and then air. Switch the gas flow conditions so that nitrogen is introduced when the deoxyhemoglobin content is less than 10%, and air is introduced when the deoxyhemoglobin content is greater than 90%. Repeat the ventilation cycle multiple times. Measure the sample's absorbance at 560nm, 576nm, 630nm, and 700nm. Substitute the absorbance values into the test formula to calculate the percentages of OxyHb, DeoxyHb, and MetHb in the hemoglobin solution.

[0122] 4. Experimental results: as attached Figure 7As shown in Figure 2 . The sample used in this experiment was an OxyHb solution with an initial DeoxyHb content of 2.02%. In the first cycle, nitrogen was introduced into the system for 35 minutes, during which the DeoxyHb content gradually increased to 90.66%. Subsequently, air was introduced for 15 minutes, at which point a rapid decrease in the DeoxyHb content from 90.66% to 4.41% was clearly observed. Of particular note, the MetHb content in the composite system remained extremely low throughout the entire cycle, remaining below 5%. Continuous monitoring of the changes in DeoxyHb content during both nitrogen and air purging revealed that the second, third, fourth, and fifth cycles exhibited remarkably similar trends compared to the first cycle. This result strongly demonstrates the reproducibility of hemoglobin's oxygen-carrying and oxygen-release processes, fully demonstrating hemoglobin's ability to stably and reliably carry and release oxygen. This provides important experimental evidence and theoretical support for in-depth research into hemoglobin's functional properties and related physiological mechanisms.

[0123] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible implementation architectures, functions and operations of the systems, methods and computer program products according to various embodiments of the present disclosure. In this regard, each box in the flowchart or block diagram can represent a module, program segment, or a part of code, and the module, program segment, or a part of code contains one or more executable instructions for implementing the specified logical functions. It should also be noted that in some alternative implementations, the functions marked in the boxes can also occur in an order different from that marked in the accompanying drawings. For example, two boxes represented in succession can actually be executed substantially in parallel, and they can sometimes be executed in the opposite order, depending on the functions involved. It should also be noted that each box in the block diagram and / or flowchart, and the combination of boxes in the block diagram and / or flowchart, can be implemented using a dedicated hardware-based system that performs the specified functions or operations, or can be implemented using a combination of dedicated hardware and computer instructions.

[0124] In general, the various example embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Certain aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that may be executed by a controller, microprocessor, or other computing device. When various aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flow charts, or using certain other graphical representations, it will be understood that the boxes, devices, systems, techniques, or methods described herein may be implemented as non-limiting examples in hardware, software, firmware, dedicated circuits or logic, general-purpose hardware or controllers or other computing devices, or some combination thereof. Those skilled in the art will clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices, and units described above may refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0125] In the several embodiments provided in this application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are merely schematic. For example, the division of the units is merely a logical function division. In actual implementation, there may be other division methods, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0126] The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of these units may be selected to achieve the purpose of this embodiment according to actual needs.

[0127] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. Those skilled in the art will appreciate that various modifications and combinations may be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A device for dynamic oxygen carrying and oxygen release detection of membranous hemoglobin solution, wherein a detection chamber is provided in the device, characterized in that: The device further comprises: The sample assembly unit is located in the detection chamber and is used to accommodate the sample to be tested. The sample assembly unit includes a well plate holder, a polytetrafluoroethylene film, and a cover glass arranged in order from bottom to top. The polytetrafluoroethylene film tightly covers the upper end of the well plate holder, and the sample to be tested is added to the upper end of the polytetrafluoroethylene film. The optical detection unit is used to collect and convert the optical signal of the sample to be tested and calculate the absorbance of the sample to be tested in different bands.

2. The device for dynamic oxygen carrying and oxygen release detection of membranous hemoglobin solution according to claim 1, characterized in that: The thickness of the polytetrafluoroethylene film includes: 0.75-2 μm; preferably 1 μm; Optionally, the orifice plate support is a bottomless orifice plate; the number of holes in the bottomless orifice plate is greater than 1; Optionally, the sample to be tested is a hemoglobin solution; Optionally, the source of the hemoglobin solution to be tested includes any one of the following: whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and chemically modified or physically assembled hemoglobin.

3. The device for dynamic oxygen carrying and oxygen release detection of membranous hemoglobin solution according to claim 1, characterized in that: The optical detection unit includes a light source, a photodetector, and an optical absorption detection device. The light source generates light of different wavelengths. The photodetector faces the detection chamber and reflects the light, which then enters the detection chamber and is absorbed by the optical absorption detection device after passing through the sample to be tested in the detection chamber. Optionally, a line connecting the reflection point on the photodetector and the center of the optical absorption detection device is perpendicular to a horizontal reference line; Optionally, there is an angle between the direction in which the light enters the photoelectric detector and the direction in which the light enters the detection chamber.

4. The device for dynamic oxygen carrying and oxygen release detection of membranous hemoglobin solution according to claim 1, characterized in that: A plate carrier is provided in the detection chamber, the sample assembly unit is placed on the plate carrier, and the plate carrier is placed in the detection chamber; Optionally, the device further includes: Temperature control unit, used to simulate the temperature environment of chronic scenes; Gas control unit, used to simulate the moist environment of chronic wounds; Optionally, the temperature control unit includes at least two heating plates and at least two temperature sensors, and the number of the heating plates and the temperature sensors is the same; the two heating plates are respectively located at the upper and lower ends of the plate carrier, and there is a distance between the heating plates and the plate carrier; the temperature sensors are respectively arranged at one end of the temperature sensor close to the plate carrier; Optionally, the gas control unit includes a nitrogen group and an air group, the nitrogen group and the air group respectively include a gas humidifying bottle, a connecting pipeline and a gas flow regulating valve, the two ends of the connecting pipeline are connected to the gas humidifying bottle and the detection chamber, and the gas flow regulating valves are respectively provided in the connecting pipeline; Optionally, the connection points between the two connecting pipes and the detection chamber are respectively located on the detection chamber wall corresponding to the space where the heating plate is away from the plate carrier.

5. A method for detecting and calculating absorbance, characterized in that: The method comprises: 101, adding the sample to be tested to the corresponding position of the well plate holder covered with a polytetrafluoroethylene gas permeable film, covering it with a cover glass, and obtaining an assembled sample assembly unit; 102. Place the assembled sample assembly unit on the plate carrier of the apparatus of claim 4 and operate it to calculate the absorbance at different wavelengths; the wavelengths include: 560nm, 570nm, 576nm, 630nm and 700nm.

6. The method for detecting and calculating absorbance according to claim 5, wherein: The operation mode in 102 includes: adjusting the gas control unit, introducing nitrogen and air in sequence, and switching conditions: nitrogen is introduced when the deoxyhemoglobin content is lower than 10%, and air is introduced when the deoxyhemoglobin content is higher than 90%, and ventilation is circulated multiple times.

7. The method for detecting and calculating absorbance according to claim 5, wherein: The method further comprises: calculating the percentage of hemoglobin in different states in the hemoglobin solution based on the absorbance; Optionally, the different states of hemoglobin include any one or more of the following: carboxyhemoglobin COHb, oxyhemoglobin OxyHb, methemoglobin MetHb, deoxyhemoglobin DeoxyHb; Optionally, the source of the hemoglobin solution to be tested includes any one of the following: whole blood lysate, recombinant hemoglobin products, hemoglobin extracted from red blood cells, and chemically modified or physically assembled hemoglobin; Optionally, the hemoglobin concentration to be measured is less than or equal to 10 g / dl.

8. A computer device, characterized in that: The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 5 to 7.

9. A computer-readable storage medium, characterized in that A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 5 to 7 are implemented.

10. A computer program product comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the method according to any one of claims 5 to 7 are implemented.

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