Device for detecting phenylketonuria and control method thereof

Abstract

The application discloses a device for detecting phenylketonuria and a control method thereof. The device comprises a main body, a microfluidic detection chip, a driving assembly, a light-emitting module and a detection module. The main body is provided with a detection bin, and the microfluidic detection chip is detachably installed in the detection bin. The microfluidic detection chip is provided with a sample quantification cavity, a reagent quantification cavity, a mixing cavity and a detection cavity. The sample quantification cavity is communicated with the mixing cavity, the reagent quantification cavity is communicated with the mixing cavity, and the mixing cavity is communicated with the detection cavity. The detection cavity is provided with a freeze-dried reagent ball capable of reacting with phenylalanine in a sample through an enzymatic reaction. The driving assembly is arranged on the main body and is used for driving the microfluidic detection chip to rotate in the detection bin. The light-emitting module is arranged in the detection bin and is used for emitting light of a specific wavelength to the detection cavity. The detection module is arranged on the main body and is used for detecting and analyzing the intensity of the light passing through the detection cavity. The application solves the problem of low accuracy of the test result of phenylketonuria in the related art.

Description

Technical Field

[0001] This application relates to the field of bioinformatics detection technology, and more specifically, to a detection device for phenylketonuria and its control method. Background Technology

[0002] In related technologies, methods for detecting phenylketonuria (PKU) include fluorescence analysis, which is based on the principle that phenylalanine reacts with reagents such as ninhydrin to produce a fluorescent substance. An ELISA reader or a dedicated fluorescence detector is used to measure the fluorescence intensity of this substance, and the phenylalanine concentration is calculated using a standard curve. The detection procedure involves collecting finger-prick blood, adding it to a reagent strip, inserting it into the detection device, where a heating module controls the reaction temperature, a light source excites fluorescence, a photodetector receives the fluorescence signal, and a processor calculates the L-phenylalanine concentration in the blood based on the signal intensity and displays the result.

[0003] However, the detection methods in related technologies suffer from significant fluctuations in fluorescence intensity due to the difficulty in uniformly mixing reagents and samples, ultimately resulting in low accuracy of test results. Summary of the Invention

[0004] The main objective of this application is to provide a detection device and control method for phenylketonuria (PKU) to solve the problem of low accuracy of PKU test results in related technologies.

[0005] According to one aspect of this application, a device for detecting phenylketonuria is provided, comprising: The main body, on which a detection chamber is provided; A microfluidic detection chip is detachably installed in the detection chamber. The microfluidic detection chip is provided with a sample quantification chamber, a reagent quantification chamber, a mixing chamber, and a detection chamber. The sample quantification chamber is connected to the mixing chamber, the reagent quantification chamber is connected to the mixing chamber, and the mixing chamber is connected to the detection chamber. The distances from the sample quantification chamber, the mixing chamber, and the detection chamber to the center of the microfluidic detection chip gradually increase along the direction from the center of the microfluidic detection chip to the outer edge of the microfluidic detection chip. The distances from the reagent quantification chamber, the mixing chamber, and the detection chamber to the center of the microfluidic detection chip gradually increase. A lyophilized reagent ball that undergoes an enzymatic reaction with phenylalanine in the sample is disposed in the detection chamber. A driving component is disposed on the main body and is used to drive the microfluidic detection chip to rotate within the detection chamber; A light-emitting module is disposed in the detection chamber and is used to emit light of a specific wavelength into the detection chamber; A detection module is disposed in the main body and is used to detect and analyze the intensity of light passing through the detection cavity.

[0006] In some embodiments, the lyophilized reagent spheres include at least: An enzyme catalytic component, wherein the enzyme catalytic component is used to catalyze an enzymatic reaction of phenylalanine in a sample; The coenzyme component is used as a hydrogen acceptor to cooperate with the enzyme catalytic component and participate in the phenylalanine enzymatic reaction.

[0007] In some embodiments, the enzyme-catalyzing component includes phenylalanine dehydrogenase; Wherein, the specific activity of the phenylalanine dehydrogenase is greater than or equal to 6 U / mg; and / or, The content A of phenylalanine dehydrogenase in the lyophilized reagent ball satisfies the following relationship: 0.1U≤A≤0.5U.

[0008] In some embodiments, the coenzyme component includes oxidized nicotinamide adenine dinucleotide; The content B of the oxidized nicotinamide adenine dinucleotide in the lyophilized reagent ball satisfies the following relationship: 10 nmol ≤ B ≤ 50 nmol.

[0009] In some embodiments, the lyophilized reagent ball further includes a protective layer located on the periphery of the enzyme catalytic component and the coenzyme component, and the protective layer is formed by lyophilization of a protective agent.

[0010] In some embodiments, the protective agent includes at least trehalose, sucrose, bovine serum albumin, mannitol, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, and ethylenediaminetetraacetic acid; The mass-volume percentage concentration of trehalose is 2%-8%. The mass-volume percentage concentration of sucrose is 1%-3%; The mass-volume percentage concentration of bovine serum albumin is 0.1%-1%. Mannitol has a mass-volume percentage concentration of 1%-3%; The concentration of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer in the protective agent is 20 mM-50 mM; The concentration of ethylenediaminetetraacetic acid in the protective agent is 0.1 mM-1 mM.

[0011] In some embodiments, the diameter R of the lyophilized reagent spheres satisfies the relationship: 1.3 mm ≤ R ≤ 1.7 mm; and / or, The water content of the lyophilized reagent balls is less than or equal to 3%.

[0012] In some embodiments, the phenylketonuria detection device further includes a heating component disposed in the detection chamber, the heating component being used at least to heat the liquid in the detection chamber to a predetermined temperature.

[0013] On the other hand, this application also provides a control method for a phenylketonuria (PKU) detection device, the control method being used to control the PKU detection device, the control method comprising: The sample and reagent are injected into the sample quantification chamber and the reagent quantification chamber, respectively. The microfluidic detection chip is rotated so that the sample enters the mixing chamber in a first predetermined volume under the action of centrifugal force, and the reagent enters the mixing chamber in a second predetermined volume under the action of centrifugal force, and mixes with the sample to form a mixed liquid. Rotate the microfluidic detection chip to allow the mixed liquid in the mixing chamber to enter the detection chamber, thereby dissolving and mixing the lyophilized reagent ball into the mixed liquid; The mixed liquid is heated to a predetermined temperature and left to stand for a predetermined time. Then, the light-emitting module emits light of a predetermined wavelength to irradiate the mixed liquid, and the intensity of the light passing through the detection cavity is detected and analyzed by the detection module.

[0014] In some embodiments, the step after introducing the mixed liquid in the mixing chamber into the detection chamber further includes: After rotating the microfluidic detection chip forward at a predetermined speed for a predetermined time, the microfluidic detection chip is then rotated backward at a predetermined speed for a predetermined time, and this forward and reverse rotation is repeated multiple times.

[0015] In this application, the microfluidic detection chip is provided with a sample quantification chamber, a reagent quantification chamber, a mixing chamber, and a detection chamber. The distances from the sample quantification chamber, the mixing chamber, and the detection chamber to the center of the microfluidic detection chip gradually increase along the direction from the center of the microfluidic detection chip to its outer edge, and the distances from these distances to the center of the microfluidic detection chip also gradually increase. After the sample and reagent are injected into the sample quantification chamber and the reagent quantification chamber respectively, rotating the microfluidic detection chip causes the sample to enter the mixing chamber in a first predetermined volume under centrifugal force, and the reagent to enter the mixing chamber in a second predetermined volume under centrifugal force, where they are thoroughly mixed. Then, rotating the microfluidic detection chip again causes the mixed liquid in the mixing chamber to enter the detection chamber. Since the detection chamber contains lyophilized reagent beads that undergo an enzymatic reaction with phenylalanine in the sample, when the lyophilized reagent beads dissolve and mix with the mixed liquid, reduced nicotinamide adenine dinucleotide is generated in the mixed liquid. At this time, light of a specific wavelength emitted by the light-emitting module shines on the mixed liquid. Since the reduced nicotinamide adenine dinucleotide absorbs some of the light, the intensity of the light passing through the detection cavity will decrease. Therefore, the intensity of the light passing through the detection cavity can be used to analyze whether the person providing the sample has phenylketonuria.

[0016] Unlike related technologies, this solution utilizes a microfluidic detection chip for sample detection. This chip, with its quantitative function, allows samples and reagents to enter the mixing chamber in specific volumes, preventing excessive dilution due to large differences in their relative proportions. Furthermore, the samples and reagents are thoroughly mixed under centrifugal force. On the other hand, because the detection chamber contains lyophilized reagent beads that undergo an enzymatic reaction with phenylalanine in the sample, there is no need to inject additional enzymes into the microfluidic detection chip. When the mixed liquid enters the detection chamber, centrifugal force dissolves the lyophilized reagent beads and mixes them uniformly with the dissolved reagents. This prevents significant fluctuations in light intensity passing through the mixed liquid due to uneven mixing of the sample, reagents, and specific enzymes during detection. It's important to understand that this solution tests the intensity of light passing through the mixed liquid, while existing technologies test the intensity of fluorescence generated by a light source. Fluorescence intensity is affected by the light source, while the intensity of light passing through the mixed liquid is only related to the light source itself and is not affected by other light sources. Attached Figure Description

[0017] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, are illustrative and descriptive, serving to explain this application and do not constitute an undue limitation thereof. In the drawings: Figure 1 This is a simplified diagram of the phenylketonuria detection device disclosed in the embodiments of this application; Figure 2 This is a schematic diagram of the microfluidic detection chip disclosed in an embodiment of this application; Figure 3 This is a schematic diagram of the detection process of the microfluidic detection chip disclosed in the embodiments of this application; Figure 4 This is a graph showing the relationship between different concentrations of phenylalanine in the lyophilized reagent balls and the detection absorbance in the embodiments of this application; Figure 5 for Figure 4 Magnified schematic diagram of the 0-200μM region; Figure 6 Different NAD values ​​in the lyophilized reagent balls in the embodiments of this application + Graph showing the relationship between content and absorbance.

[0018] The above figures include the following reference numerals: 10. Main body; 20. Microfluidic detection chip; 30. Driving component; 40. Light emission module; 50. Detection module; 60. Heating component; 70. Lyophilized reagent ball; 201. Sample quantification chamber; 202. Reagent quantification chamber; 203. Mixing chamber; 204. Detection chamber. Detailed Implementation

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps described in these embodiments do not limit the scope of this application. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail. Any specific values ​​in all examples shown and discussed herein should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0022] To address the problems existing in the relevant technologies, see [link to relevant documentation]. Figures 1 to 3 As shown in the figure, this application provides a detection device for phenylketonuria. The detection device includes a main body 10, a microfluidic detection chip 20, a driving component 30, a light-emitting module 40, and a detection module 50. The main body 10 is provided with a detection chamber, and a microfluidic detection chip 20 is detachably installed in the detection chamber. The microfluidic detection chip 20 is provided with a sample quantification chamber 201, a reagent quantification chamber 202, a mixing chamber 203, and a detection chamber 204. The sample quantification chamber 201 is connected to the mixing chamber 203, the reagent quantification chamber 202 is connected to the mixing chamber 203, and the mixing chamber 203 is connected to the detection chamber 204. Along the direction from the center of the microfluidic detection chip 20 to its outer edge, the distances from the sample quantification chamber 201, the mixing chamber 203, and the detection chamber 204 to the center of the microfluidic detection chip 20 gradually increase, as do the distances from the reagent quantification chamber 202, the mixing chamber 203, and the detection chamber 204 to the center of the microfluidic detection chip 20. A lyophilized reagent ball 70 that undergoes an enzymatic reaction with phenylalanine in the sample is disposed in the detection chamber 204. A driving assembly 30 is disposed in the main body 10 and is used to drive the microfluidic detection chip 20 to rotate within the detection chamber. The light-emitting module 40 is disposed in the detection chamber and is used to emit light of a specific wavelength into the detection cavity 204. The detection module 50 is disposed in the main body 10 and is used to detect and analyze the intensity of light passing through the detection cavity 204.

[0023] In this application, the microfluidic detection chip 20 is provided with a sample quantification chamber 201, a reagent quantification chamber 202, a mixing chamber 203, and a detection chamber 204. Along the direction from the center of the microfluidic detection chip 20 to its outer edge, the distances from the sample quantification chamber 201, the mixing chamber 203, and the detection chamber 204 to the center of the microfluidic detection chip 20 gradually increase, as do the distances from the reagent quantification chamber 202, the mixing chamber 203, and the detection chamber 204 to the center of the microfluidic detection chip 20. When a sample and a reagent are injected into the sample quantification chamber 201 and the reagent quantification chamber 202 respectively, and the microfluidic detection chip 20 is rotated, the sample enters the mixing chamber 203 in a first predetermined volume under the action of centrifugal force, and the reagent enters the mixing chamber 203 in a second predetermined volume under the action of centrifugal force, and mixes thoroughly with the sample. Next, the microfluidic detection chip 20 is rotated again, and the mixed liquid in the mixing chamber 203 enters the detection chamber 204. Since the detection chamber 204 contains lyophilized reagent balls 70 that undergo an enzymatic reaction with phenylalanine in the sample, when the lyophilized reagent balls 70 dissolve and mix with the mixed liquid, reduced nicotinamide adenine dinucleotide is generated in the mixed liquid. At this time, light of a specific wavelength emitted by the light-emitting module 40 irradiates the mixed liquid. Because reduced nicotinamide adenine dinucleotide absorbs some of the light, the intensity of the light passing through the detection chamber 204 decreases. Therefore, the intensity of the light passing through the detection chamber 204 can be used to analyze whether the person providing the sample has phenylketonuria. In this embodiment, the driving component 30 can be a drive motor, the light-emitting module 40 can be an LED, and the detection module 50 can be a photodetector.

[0024] Unlike related technologies, this solution uses a microfluidic detection chip 20 to detect samples. The microfluidic detection chip has a quantitative function, allowing the sample and reagent to enter the mixing chamber 203 in specific volumes. This avoids excessive dilution of the sample due to large differences in the relative proportions of the sample or reagent, and ensures thorough mixing of the sample and reagent under centrifugal force. Furthermore, since the detection chamber 204 contains lyophilized reagent beads 70 that undergo an enzymatic reaction with phenylalanine in the sample, there is no need to inject additional enzymes into the microfluidic detection chip 20 to react with phenylalanine. When the mixed liquid enters the detection chamber 204, centrifugal force dissolves the lyophilized reagent beads 70 and mixes them uniformly with the dissolved reagent. This prevents large fluctuations in the intensity of light passing through the mixed liquid during detection due to uneven mixing of the sample, reagent, and specific enzyme. Meanwhile, it is understood that this solution tests the intensity of light passing through the mixed liquid, while existing technologies test the intensity of fluorescence generated by a light source. The intensity of fluorescence is affected by the light source, while testing the intensity of light passing through the mixed liquid is only related to the light source and is not affected by other light sources. On the other hand, the quantification and mixing of samples and reagents, as well as the dissolution of the lyophilized reagent balls 70, are all completed within the microfluidic detection chip 20. Furthermore, the microfluidic detection chip 20, the driving component 30, the light-emitting module 40, and the detection module 50 are all integrated into one unit, resulting in a simple structure and easy detection operation.

[0025] In some embodiments, the lyophilized reagent sphere 70 includes at least an enzyme catalytic component and a coenzyme component. The enzyme catalytic component is used to catalyze the enzymatic reaction of phenylalanine in the sample, and the coenzyme component is used as a hydrogen acceptor to cooperate with the enzyme catalytic component and participate in the phenylalanine enzymatic reaction.

[0026] Specifically, the enzyme-catalyzed component reacts with L-phenylalanine in the sample to generate reduced nicotinamide adenine dinucleotide (NADH). The coenzyme component acts as a hydrogen acceptor, receiving hydrogen removed from phenylalanine and ultimately reducing it back to NADH. Since NADH absorbs light of a specific wavelength, its concentration can be calculated by measuring the light intensity passing through the mixed liquid. Compared to fluorescence methods, which require excitation from a light source and can lead to interference between fluorescence and the light source, resulting in false positives, the enzyme-catalyzed reaction offers higher specificity. It only requires measuring the absorbance of light before and after passing through the mixed liquid to determine the NADH concentration, eliminating interference between the light source and fluorescence and preventing false positives. Furthermore, it is known that although the enzymatic reaction between the sample and L-phenylalanine is a routine chemical reaction, due to the integrated setup of the detection device, the sample and reagent quantification, mixing, reconstitution of the lyophilized reagent ball 70, NADH detection, and the pre-setting of the lyophilized reagent ball 70 containing the enzyme catalytic component and coenzyme component in the microfluidic detection chip 20, the detection of phenylketonuria can be completed simply by injecting the sample, controlling the drive component 30, and irradiating the detection cavity 204 with the light-emitting module 40. This can significantly shorten the detection time for phenylketonuria.

[0027] In some embodiments, the enzyme catalytic component includes phenylalanine dehydrogenase. The specific activity of the phenylalanine dehydrogenase is greater than or equal to 6 U / mg. In this embodiment, specific activity refers to the oxidation of 1.0 μmol of L-phenylalanine per minute at pH 10.5 and 30°C. Using a high-purity phenylalanine dehydrogenase with a specific activity ≥6 U / mg ensures sufficient catalytic activity within a limited reagent volume, enabling the enzymatic reaction to be completed within 3 to 5 minutes, significantly shortening the detection time. Furthermore, the phenylalanine dehydrogenase in this embodiment is a lyophilized powder.

[0028] In some embodiments, the content A of phenylalanine dehydrogenase in the lyophilized reagent bulb 70 satisfies the relationship: 0.1U ≤ A ≤ 0.5U. When the content A of phenylalanine dehydrogenase in the lyophilized reagent bulb 70 satisfies the above relationship, the lyophilized reagent bulb 70 has a full skeleton, without collapse or shrinkage, and can be reconstituted relatively quickly. In addition, the enzyme has high catalytic activity and a fast reaction rate. However, when the content of A is less than 0.1U, although the lyophilized reagent bulb 70 has good exfoliation and rapid reconstitution, the reaction rate is low due to the low catalytic activity of the enzyme. When the content of A is greater than 0.5U, the lyophilized reagent bulb 70 collapses and shrinks, and insoluble particles appear during reconstitution. In this embodiment, the content of A can be 0.1U, 0.2U, 0.3U, 0.4U, and 0.5U.

[0029] In some embodiments, the coenzyme component includes oxidized nicotinamide adenine dinucleotide. The content B of oxidized nicotinamide adenine dinucleotide in the lyophilized reagent spheres 70 satisfies the relationship: 10 nmol ≤ B ≤ 50 nmol. When B meets the above range, the lyophilized reagent spheres 70 are white, dense, hemispherical, with good strength, short reconstitution time, no visible particles, and no obvious side reactions caused by substrate or coenzyme excess. However, when B is less than 10 nmol, the lyophilized reagent spheres 70 are well-formed, with a slightly loose surface, longer reconstitution time, and clear solution, but due to NAD... + Insufficient quantity cannot support the complete conversion of high-concentration phenylalanine. When B is greater than 50 nmol, the lyophilized reagent spheres 70 exhibit a slight "collapse" phenomenon, and the surface of the spheres feels slightly sticky. The reconstitution time is too long. After the lyophilized reagent spheres 70 are completely dissolved, the solution is clear, but extremely fine non-specific particles can be seen after standing. In addition, the excessively high coenzyme content inhibits enzyme activity, reduces sensitivity, and results in poor reliability of low-concentration signals.

[0030] In some embodiments, the lyophilized reagent ball 70 further includes a protective layer located on the periphery of the enzyme catalytic component and the coenzyme component, and the protective layer is formed by lyophilization of a protectant.

[0031] Specifically, the protective layer protects the enzyme catalytic components and coenzyme components, providing better mechanical support to keep the lyophilized reagent balls 70 intact and less prone to breakage, preventing damage during transportation. Simultaneously, the protective layer acts as a physical barrier, encapsulating the easily inactivated enzymes and hygroscopically degradable coenzymes in the innermost layer, effectively isolating them from external oxygen, moisture, and light, greatly improving the stability of the reagent balls during storage and transportation.

[0032] In some embodiments, the protective agent includes at least trehalose, sucrose, bovine serum albumin, mannitol, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, and ethylenediaminetetraacetic acid (EDTA). The trehalose concentration is 2%-8% by weight (w / v), the sucrose concentration is 1%-3% by weight (w / v), the bovine serum albumin concentration is 0.1%-1% by weight (w / v), and the mannitol concentration is 1%-3% by weight (w / v). The concentration of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer in the protective agent is 20 mM-50 mM, and the concentration of the EDTA in the protective agent is 0.1 mM-1 mM. It should be noted that in this embodiment, the weight (w / v) concentration is expressed as % (w / v), defined as the number of grams of solute contained in 100 mL of solution (i.e., g / 100 mL).

[0033] Specifically, trehalose and sucrose form a protective glass matrix after freeze-drying, replacing water molecules in forming hydrogen bonds with proteins and maintaining the native conformation of the enzyme protein, thereby improving enzyme stability to some extent. Furthermore, bovine serum albumin, an inert protein, acts as a filler and protectant, preventing enzyme molecules from agglomerating and being adsorbed and lost during freeze-drying / reconstitution. Mannitol forms a microcrystalline structure after freeze-drying to support the spherical framework, preventing collapse and improving reconstitution speed. Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer provides a weakly alkaline environment before freeze-drying and facilitates the reconstitution of freeze-dried reagent spheres 70 in a weakly alkaline environment, protecting enzyme activity. Ethylenediaminetetraacetic acid (EDTA) is used to chelate heavy metal ions, preventing inhibition of enzyme activity.

[0034] On the other hand, the concentration of trehalose is chosen to be between 2% and 8% because when the concentration is below 2%, trehalose is difficult to form a glass matrix after freeze-drying, resulting in insufficient protection for the enzyme and causing significant enzyme inactivation. Simultaneously, the mechanical strength of the freeze-dried reagent spheres 70 is too low, making them prone to collapse. Conversely, when the concentration is above 8%, there is too much trehalose and too little water in the protein, preventing some trehalose from forming a glass matrix. Furthermore, the crystals formed by freeze-drying trehalose may cause physical damage to the enzyme, thus affecting its activity. In addition, if the trehalose concentration is too high, it will hinder water molecules from entering the interior of the freeze-dried reagent spheres 70 during reconstitution, leading to a slow dissolution rate. Moreover, a high trehalose concentration can also cause significant changes in the refractive index and absorbance of the reconstituted solution, ultimately resulting in larger detection errors. In a preferred embodiment, the concentration of trehalose can be selected as 5%, at which point the enzyme activity recovery rate can be greater than 96%, and the glass matrix formed by trehalose can effectively improve the structural strength of the lyophilized reagent ball 70.

[0035] When the sucrose concentration is between 1% and 3%, it can complement the glass matrix of trehalose, directly forming alternative hydrogen bonds with the enzyme protein to "lock in" the active conformation at extremely low moisture levels. In a preferred embodiment, the sucrose concentration is 2%, which perfectly fills the nanopores in the trehalose glass matrix, achieving maximum rigidity protection of the enzyme structure. Furthermore, when the sucrose concentration is below 1%, the filler concentration is insufficient, failing to compensate for the lost hydrated hydrogen bonds during drying, resulting in inadequate enzyme protection and incomplete reactions with high-concentration substrates. When the sucrose concentration is above 3%, supersaturated sucrose is prone to localized crystallization or microphase separation during freeze-drying, which not only disrupts the glass homogeneity and damages the enzyme structure but also significantly increases hygroscopicity and reconstitution viscosity, causing a double deterioration in background and variation.

[0036] Furthermore, the concentration of bovine serum albumin (BSA) is between 0.1% and 1%. Within this range, BSA provides a sufficient number of inert molecules to act as "molecular spacers" and "surface passivators," completely preventing enzyme loss. Moreover, within this concentration range, BSA itself does not cause any measurable spectral interference or physical property degradation. In a preferred embodiment, the concentration of BSA is 0.5%. When the concentration of BSA is less than 0.1%, the total protein amount is insufficient to completely cover the interfacial sites, resulting in significant enzyme adsorption and aggregation, leading to a sharp decrease in enzyme activity recovery. When the concentration of BSA is greater than 1%, the protein is severely excessive, forming a hard shell during freeze-drying, hindering water sublimation, prolonging reconstitution time, and causing localized enzyme inactivation. Simultaneously, it introduces exogenous impurities (such as trace amounts of phenylalanine or 340nm absorbers in BSA), directly raising the background and disrupting quantification in low-concentration regions, resulting in a double decrease in sensitivity and reliability.

[0037] Furthermore, when the mannitol concentration is selected within the range of 1% to 3%, the mannitol crystallizes completely into uniform, fine crystals that embed within the trehalose glass matrix, forming a composite framework that is both robust and hydrophilic. In a preferred embodiment, the mannitol concentration is 2%, at which the framework strength is optimal, resolution is fastest, enzymes are completely protected, and there is no chemical interference. However, when the mannitol concentration is less than 1%, the amount of mannitol is insufficient to form a continuous crystalline framework. During freeze-drying, amorphous components dominate, the structure collapses after sublimation, enzymes are exposed and damaged, the activity recovery rate drops sharply, and resolution is extremely slow. When the mannitol concentration is greater than 3%, excess mannitol undergoes supersaturated crystallization during rapid freezing, producing large needle-like crystals, which disrupts the microscopic uniformity of the freeze-dried cake, leading to local enzyme encapsulation and inactivation, a decrease in resolution rate, and the retention of crystal debris. At the same time, the side reactions of excess mannitol in the reaction system interfere, resulting in increased background and completely unreliable low-concentration signals.

[0038] It should be noted that the concentration of the tris(ol)methylaminomethane-hydrochloric acid buffer in the protectant is 20mM-50mM. This range is chosen because it maintains the pH of the protectant between 8.5 and 9 before lyophilization, preventing enzyme inactivation due to excessively low pH. When the concentration of the tris(ol)methylaminomethane-hydrochloric acid buffer is below 20mM, even trace amounts of acidic substances (such as dissolved CO2) can cause a significant pH shift, leading to partial enzyme inactivation during production. Concentrations above 50mM, however, will have excessively high ionic strength (total ionic strength after reconstitution > 80mM), directly inhibiting enzyme catalytic activity and causing problems such as salting out and phase separation.

[0039] On the other hand, when the concentration of ethylenediaminetetraacetic acid (EDTA) in the protective agent is between 0.1 mM and 1 mM, this range provides chelating equivalents several times greater than the total potential heavy metal pollution, forming an absolute protective barrier. Furthermore, it is completely soluble and amorphous during freeze-drying, without causing phase separation or spectral interference. However, when the concentration of EDTA in the protective agent is less than 0.1 mM, the chelating capacity is insufficient, especially at trace heavy metal pollution levels close to those in the real world (as low as 1–2 μM Cu²). + If this occurs, it will lead to an irreversible loss of more than 50% of enzyme activity, rendering the lyophilized reagent spheres 70 completely unusable. Even if no abnormalities are observed in a short period under ultrapure conditions, the gradual accumulation of metal contamination during long-term storage will slowly deactivate the enzyme. When the concentration of ethylenediaminetetraacetic acid (EDTA) in the protectant exceeds 1 mM, excess EDTA becomes the source of contamination itself. It begins to precipitate crystals, damaging the microstructure of the lyophilized reagent spheres 70, raising the UV end absorption background, non-selectively chelating trace amounts of stable divalent ions that maintain the higher conformation of the enzyme in the environment, and slightly interfering with the ion balance of the reaction system, leading to secondary inhibition of enzyme activity, reduced sensitivity, and failure of quantification at low concentrations. At the same time, the introduction of excessive acidic groups consumes part of the basic buffer pair in the reconstitution solution, causing the reaction pH to deviate slightly from the optimum point.

[0040] In some embodiments, the diameter R of the lyophilized reagent ball 70 satisfies the relationship: 1.3mm ≤ R ≤ 1.7mm. Specifically, the diameter of the lyophilized reagent ball 70 cannot be too large, for example, greater than 1.7mm. An excessively large diameter would make the lyophilized reagent ball 70 easily damaged during transportation. Furthermore, an excessively large diameter would make it difficult to adapt the lyophilized reagent ball 70 to the microfluidic detection chip 20, i.e., difficult to pre-install into the detection cavity 204 of the microfluidic detection chip 20. The diameter of the lyophilized reagent ball 70 should also not be too small, as a small diameter would result in excessively high manufacturing costs and make it difficult to control the freeze-drying time. In this embodiment, the diameter R of the lyophilized reagent ball 70 can be 1.3mm, 1.4mm, 1.5mm, 1.6mm, or 1.7mm.

[0041] In some embodiments, the water content of the lyophilized reagent beads 70 is less than or equal to 3%. It is known that when the water content of the lyophilized reagent beads 70 exceeds 3%, the enzyme protein within the lyophilized reagent beads 70 undergoes a slow hydrolysis reaction, peptide bond breakage, molecular weight reduction, and ultimately, loss of catalytic ability. Furthermore, the coenzyme within the lyophilized reagent beads 70 also undergoes hydrolysis in a humid environment; specifically, oxidized nicotinamide adenine dinucleotide hydrolyzes into nicotinamide and ADP-ribose, thereby losing its function as a hydrogen acceptor. To ensure that the residual moisture content of the lyophilized reagent beads 70 is strictly controlled to ≤3% and to verify long-term stability, a coulometric Karl Fischer moisture analyzer equipped with a Karl Fischer furnace can be used to detect trace moisture in the lyophilized reagent beads 70. This method is specifically designed for determining solid samples with low water content. The moisture inside the beads is released by heating, and then carried into the titration cell by a drying carrier gas to complete the coulometric determination. This effectively avoids electrode contamination by the lyophilized reagent bead 70 matrix and interference from environmental moisture.

[0042] In some embodiments, the phenylketonuria detection device further includes a heating component 60 disposed in the detection chamber, the heating component 60 being used at least to heat the liquid in the detection chamber 204 to a predetermined temperature.

[0043] Specifically, since the rate of enzymatic reactions is greatly affected by temperature, the heating component 60 can maintain the reaction system at the optimal temperature for phenylalanine dehydrogenase, significantly accelerating the reaction rate and shortening the detection time. In some specific embodiments, the heating component 60 heats the liquid in the detection chamber 204 to 37°C to increase the rate of the enzymatic reaction. In this embodiment, the heating component 60 can be a thin-film heating element, an infrared radiation heating module, or a metal heating element.

[0044] On the other hand, this application also provides a control method for a phenylketonuria (PKU) detection device. The control method controls the aforementioned PKU detection device and includes injecting a sample and reagent into a sample quantification chamber 201 and a reagent quantification chamber 202, respectively. Rotating the microfluidic detection chip 20 causes the sample to enter the mixing chamber 203 in a first predetermined volume under centrifugal force, and the reagent to enter the mixing chamber 203 in a second predetermined volume under centrifugal force, mixing with the sample to form a mixed liquid. Rotating the microfluidic detection chip 20 causes the mixed liquid in the mixing chamber 203 to enter the detection chamber 204, dissolving and mixing the lyophilized reagent bulb 70 into the mixed liquid. Heating the mixed liquid to a predetermined temperature and allowing it to stand for a predetermined time, irradiating the mixed liquid with light of a predetermined wavelength emitted by the light-emitting module 40, and detecting and analyzing the intensity of the light passing through the detection chamber 204 using the detection module 50.

[0045] In other words, the entire detection process consists of four steps: quantitative transfer, mixing, reaction, and optical detection. Each step can be automatically controlled by the equipment, eliminating the need for manual operation and thus improving the accuracy of the detection to a certain extent. Furthermore, thanks to the design of the sample quantification chamber 201 and reagent quantification chamber 202 of the microfluidic detection chip 20, the sample and reagent can enter the mixing chamber 203 in a first predetermined volume and a second predetermined volume, respectively, under centrifugal force, thereby forming a solution with a fixed ratio. In addition, after the lyophilized reagent bulb 70 dissolves, the heating component 60 can heat the mixed liquid to a predetermined temperature, thereby accelerating the enzymatic reaction in the detection chamber 204. Compared with related technologies, this application does not require specialized operating skills; ordinary personnel can operate it. Moreover, since quantification, mixing, reaction, and detection are all completed within the detection equipment, the sample and reagent will not be contaminated. In this application, the first and second predetermined volumes can be designed according to actual needs, and this application does not impose specific limitations.

[0046] In some embodiments, the step after the mixed liquid in the mixing chamber 203 enters the detection chamber 204 further includes rotating the microfluidic detection chip 20 forward at a predetermined speed for a predetermined time, and then rotating the microfluidic detection chip 20 in reverse at a predetermined speed for a predetermined time, and then alternating between forward and reverse rotation multiple times.

[0047] In other words, the oscillations formed by repeated alternating forward and reverse rotations can promote the reconstitution of the lyophilized reagent ball 70 on the one hand, and further mix the reconstituted solution and the mixed solution on the other hand, so that the enzymatic reaction can proceed synchronously and improve the accuracy of the detection results.

[0048] In one specific embodiment, after the sample and reagent are added to the sample quantification chamber 201 and reagent quantification chamber 202 respectively, the rotation speed of the microfluidic detection chip 20 is first adjusted to 5000 rpm, and then centrifuged for 60 seconds. During this time, the blood in the sample quantification chamber 201 is separated into plasma and red blood cells. Subsequently, the rotation speed of the microfluidic detection chip 20 is adjusted to 3000 rpm, and the plasma is transferred to the mixing chamber 203 in a first predetermined volume, and the reagent is transferred to the mixing chamber 203 in a second predetermined volume. Then, the rotation speed of the microfluidic detection chip 20 is adjusted to 1000 to 1500 rpm to ensure thorough mixing of the plasma and reagent. Subsequently, the rotation speed of the microfluidic detection chip 20 is adjusted to 3000 rpm to transfer the mixed liquid to the detection chamber 204. After a predetermined time, the rotation is stopped. The heating component 60 is activated to heat the mixed liquid. Then, the microfluidic detection chip 20 is rotated 30 times in both directions at 1500 rpm, with a 1-second interval between each rotation, to complete the mixing vibration. Subsequently, the light-emitting module 40 emits light of a predetermined wavelength to irradiate the mixed liquid, and the detection module 50 detects and analyzes the intensity of the light passing through the detection cavity 204.

[0049] In addition, this application provides some specific embodiments to verify the effect of enzyme content in lyophilized reagent beads 70 on detection, see appendix. Figure 4 and Figure 5 As shown, attached Figure 4 The dashed line represents the standard curve, and the dotted line represents the curve obtained from the experiment.

[0050] Example 1: The lyophilized reagent beads 70 contain 0.20 U of enzyme. The lyophilized reagent beads 70 have a full skeleton, without collapse or shrinkage, exhibiting excellent "flash dissolution" characteristics (reconstitution time less than 3 seconds). Within the set test window, the reaction kinetic curve is stable, and the linear period perfectly covers the detection range. Experiments show that within the linear range of 30-1200 μmol / L, the correlation coefficient R... 2 The coefficient of variation (CV) for all high, medium, and low concentration quality control samples was ≥0.998, with a value of <3.5%, and the enzyme activity recovery rate remained above 92% after lyophilization. This parameter balances the excipient requirements of the lyophilization process with the accuracy of clinical quantification, and was therefore set as the final process production value.

[0051] Comparative Example 1: The lyophilized reagent spheres 70 contain 0.05 U of enzyme. At this dosage, the lyophilized reagent spheres 70 exhibit good excipient formation and rapid reconstitution. However, due to insufficient catalytic activity, the reaction rate is significantly reduced. When the concentration of L-phenylalanine in the sample exceeds 400 μmol / L, the reaction fails to reach a plateau within the linear period, and the standard curve prematurely bends at the high concentration end (correlation coefficient R). 2 <0.95); meanwhile, samples below 30 μmol / L cannot be effectively distinguished from background noise, resulting in a severe deterioration of the detection limit. This concentration cannot meet the clinical needs for quantification of high- and extremely low-value samples.

[0052] Comparative Example 2: The enzyme content in lyophilized reagent sphere 70 is 1.00 U. Although the curve at this concentration corresponds well with the standard curve, it still deviates from the standard curve in the low concentration region. Simultaneously, the physical appearance of lyophilized reagent sphere 70 is damaged at this concentration. This is because the high concentration of protein introduces impurities and protective agents, altering the eutectic point of the system. This results in insufficient internal skeletal support during the drying process, leading to severe vitrification collapse and shrinkage. Furthermore, reconstitution is accompanied by adherence to the walls and insoluble particles.

[0053] In addition, to verify the different NAD values ​​in lyophilized reagent beads 70 + For the impact of content on detection, please refer to the appendix. Figure 6 , Figure 6 The dashed line represents the standard curve, and the dotted line represents the curve obtained from experimental data. This application provides the following specific embodiments: Example 2: NAD + Content 20 nmol / sphere, lyophilized reagent sphere 70 appearance and reconstitution: Lyophilized reagent sphere 70 is a white, dense hemispherical shape with good strength. The reconstitution time is <20 seconds, and the solution is clear and transparent with no visible particles. Actual reaction performance: The standard curve shows excellent linearity throughout the 0-2000 μM range, R0. 2 =1. Linearity validation: Residual analysis was used, and the relative deviation at each calibration point was less than 2.5%. Recovery: Low concentration (50 μM) recovery rate 97.8%-102.3%; medium concentration (800 μM) 98.5%-101.4%; high concentration (2000 μM) 98.1%-100.9%. Sensitivity (limit of detection): Calculated at 3 times the blank standard deviation, LOD is 8 μM, LOQ is 24 μM. Precision: Intra-batch CV is 2.1% at 50 μM, 1.3% at 1000 μM, and 1.5% at 2000 μM (n=20 each). Accelerated stability (37℃ for 10 days): No significant change in linear range and recovery rate, absorbance drift <5%. Conclusion: 20 nmol / sphere NAD + The loading capacity ensured that the highest concentration of substrate (2000 μM phenylalanine, with 10 μL of sample corresponding to 20 nmol of substrate) was fully converted, without any obvious side reactions caused by substrate or coenzyme overload. The linear range, accuracy, and precision of the entire system fully met the detection requirements of 0-2000 μM, making it the preferred solution.

[0054] Comparative Example 3: NAD + Content 5 nmol / sphere, lyophilized reagent sphere 70 appearance and reconstitution: Lyophilized reagent sphere 70 is intact with a slightly loose surface; reconstitution time is approximately 25 seconds, and the solution is clear. Actual reaction performance: Within the phenylalanine concentration range of 0-500 μM, the absorbance shows a good linear relationship with concentration, R... 2 =0.9931, when the phenylalanine concentration in the sample exceeds 500 μM, the increase in absorbance at the reaction endpoint drops sharply. The measured absorbance of the 1000 μM sample is only equivalent to 62% of the theoretical complete conversion value; the absorbance of the 2000 μM sample plateaus and is not significantly different from that of the 1500 μM sample (p > 0.05), making them indistinguishable. Standard curve throughout (0-2000 μM) R 2 The value dropped to 0.8399, exhibiting a typical "linear at the beginning, plateau at the end" pattern. The recoveries for samples with concentrations above 500 μM were only 51%-76%, while the recovery rate for low concentrations (50 μM) was 98%. Intra-batch CV: 4.2% for low concentrations (50 μM), and increased to 18.7% for high concentrations (1500 μM) due to increased absorbance variation. Conclusion: 5 nmol / sphere NAD... +The loading capacity is insufficient to support the complete conversion of high-concentration phenylalanine (>500μM) and cannot cover the linear range requirement of 0-2000μM. This loading capacity is only suitable for narrow-range detection scenarios and requires strict limitation of the upper limit of sample concentration.

[0055] Comparative Example 4: NAD + Content 70 nmol / sphere; Lyophilized appearance and reconstitution: Lyophilized reagent spheres 70 exhibited a slight "collapse" phenomenon, and the surface of the spheres felt slightly sticky. The reconstitution time was extended to approximately 45 seconds; after complete dissolution, the solution was clear, but extremely fine non-specific particles were visible after standing. Actual reaction performance: Shows NAD... + In the solid state, trace degradation produces impurities capable of absorbing up to 340 nm, and this interference is amplified at high concentrations. Intra-batch CV is as high as 9.8%-13.5% at low concentrations (50-200 μM), while it remains at 3%-5% at medium to high concentrations. Recovery: Recovery is acceptable at medium to high concentrations (97%-103%), but at low concentrations (80 μM), the recovery is only 82%-115%, showing significant fluctuations. Enzyme inhibition: Parallel enzyme activity assays revealed that free NAD in the system after reconstitution at 70 nmol / sphere was... + At a concentration of approximately 0.35 mM, the relative specific activity of phenylalanine dehydrogenase decreased to 78% of its maximum activity, indicating mild coenzyme over-inhibition. This led to a decrease in reaction rate and a reduced endpoint signal. Lyophilized storage stability: After 14 days at 37°C, the absorbance of the 70 nmol group further increased to 0.22, and the linear dispersion of the standard curve at the high concentration end increased, while the 20 nmol group showed no significant change. Conclusion: The 70 nmol / sphere concentration resulted in severe coenzyme over-inhibition, leading to three problems: 1. High blank background, limiting low-concentration detection; 2. Inhibited enzyme activity, resulting in decreased sensitivity and poor reliability of low-concentration signals; 3. Decreased lyophilized reagent form and storage stability. Overall, it cannot meet the requirements for accurate quantification over a wide linear range.

[0056] For ease of description, spatial relative terms such as "above," "on top of," "on the upper surface of," "above," etc., are used herein to describe the spatial positional relationship of a device or feature as shown in the figures to other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations in use or operation beyond the orientation of the device as described in the figures. For example, if the device in the figures were inverted, a device described as "above" or "on top of" other devices or structures would subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0057] Furthermore, it should be noted that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be construed as limiting the scope of protection of this application.

[0058] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A device for detecting phenylketonuria, characterized in that, include: The main body (10) is provided with a detection chamber; A microfluidic detection chip (20) is detachably installed in the detection chamber. The microfluidic detection chip (20) is provided with a sample quantification chamber (201), a reagent quantification chamber (202), a mixing chamber (203), and a detection chamber (204). The sample quantification chamber (201) is connected to the mixing chamber (203), the reagent quantification chamber (202) is connected to the mixing chamber (203), and the mixing chamber (203) is connected to the detection chamber (204). Along the microfluidic detection chip (201)... From the center of the sample quantification chamber (201), the mixing chamber (203), and the detection chamber (204) to the center of the microfluidic detection chip (20) gradually increase the distance from the center of the sample quantification chamber (201), the mixing chamber (203), and the detection chamber (204) to the center of the microfluidic detection chip (20). The distance from the reagent quantification chamber (202), the mixing chamber (203), and the detection chamber (204) to the center of the microfluidic detection chip (20) gradually increases. The detection chamber (204) is provided with a lyophilized reagent ball (70) that undergoes an enzymatic reaction with phenylalanine in the sample. A drive assembly (30) is disposed on the main body (10) and is used to drive the microfluidic detection chip (20) to rotate within the detection chamber. A light-emitting module (40) is disposed in the detection chamber and is used to emit light of a specific wavelength into the detection cavity (204); A detection module (50) is disposed on the main body (10) and is used to detect and analyze the intensity of light passing through the detection cavity (204); The lyophilized reagent ball (70) includes at least an enzyme catalytic component and a coenzyme component. The enzyme catalytic component is used to catalyze the enzymatic reaction of phenylalanine in the sample, and the coenzyme component is used as a hydrogen acceptor to cooperate with the enzyme catalytic component and participate in the enzymatic reaction of phenylalanine.

2. The detection device for phenylketonuria according to claim 1, characterized in that, The enzyme catalytic component includes phenylalanine dehydrogenase; Wherein, the specific activity of the phenylalanine dehydrogenase is greater than or equal to 6 U / mg; and / or, The content A of phenylalanine dehydrogenase in the lyophilized reagent ball (70) satisfies the following relationship: 0.1U≤A≤0.5U.

3. The detection device for phenylketonuria according to claim 1, characterized in that, The coenzyme component includes oxidized nicotinamide adenine dinucleotide; The content B of the oxidized nicotinamide adenine dinucleotide in the lyophilized reagent ball (70) satisfies the following relationship: 10 nmol ≤ B ≤ 50 nmol.

4. The detection device for phenylketonuria according to claim 1, characterized in that, The lyophilized reagent ball (70) further includes a protective layer located on the periphery of the enzyme catalytic component and the coenzyme component, and the protective layer is formed by lyophilization of a protectant.

5. The phenylketonuria detection device according to claim 4, characterized in that, The protective agent includes at least trehalose, sucrose, bovine serum albumin, mannitol, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer, and ethylenediaminetetraacetic acid; The mass-volume percentage concentration of trehalose is 2%-8%. The mass-volume percentage concentration of sucrose is 1%-3%; The mass-volume percentage concentration of bovine serum albumin is 0.1%-1%. Mannitol has a mass-volume percentage concentration of 1%-3%; The concentration of the tris(hydroxymethyl)aminomethane-hydrochloric acid buffer in the protective agent is 20 mM-50 mM; The concentration of ethylenediaminetetraacetic acid in the protective agent is 0.1 mM-1 mM.

6. The detection device for phenylketonuria according to any one of claims 1 to 5, characterized in that, The diameter R of the lyophilized reagent spheres (70) satisfies the following relationship: 1.3 mm ≤ R ≤ 1.7 mm; and / or, The water content of the freeze-dried reagent balls (70) is less than or equal to 3%.

7. The detection device for phenylketonuria according to any one of claims 1 to 5, characterized in that, The phenylketonuria detection device further includes a heating component (60), which is disposed in the detection chamber and is used at least to heat the liquid in the detection chamber (204) to a predetermined temperature.

8. A control method for a detection device for phenylketonuria, characterized in that, The control method is used to control the phenylketonuria detection device according to any one of claims 1 to 7, the control method comprising: The sample and reagent are injected into the sample quantification chamber (201) and the reagent quantification chamber (202) respectively. The microfluidic detection chip (20) is rotated so that the sample enters the mixing chamber (203) with a first predetermined volume under the action of centrifugal force, and the reagent enters the mixing chamber (203) with a second predetermined volume under the action of centrifugal force, and mixes with the sample to form a mixed liquid. Rotate the microfluidic detection chip (20) to allow the mixed liquid in the mixing chamber (203) to enter the detection chamber (204), thereby dissolving and mixing the lyophilized reagent ball (70) into the mixed liquid; The mixed liquid is heated to a predetermined temperature and left to stand for a predetermined time. Then, the light-emitting module (40) emits light of a predetermined wavelength to irradiate the mixed liquid, and the detection module (50) detects and analyzes the intensity of the light passing through the detection cavity (204).

9. The control method for the phenylketonuria detection device according to claim 8, characterized in that, The steps following the introduction of the mixed liquid in the mixing chamber (203) into the detection chamber (204) further include: After rotating the microfluidic detection chip (20) forward at a predetermined speed for a predetermined time, rotate the microfluidic detection chip (20) in reverse at a predetermined speed for a predetermined time, and repeat the forward and reverse rotations multiple times.

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