A fully passive microfluidic whole blood sample preparation device and method

Abstract

The application provides a full-passive microfluidic whole blood sample preparation device, which comprises, from bottom to top, a bottom support layer, a bottom hydrophilic layer, a bottom adhesive layer, a middle main body layer, a liquid outlet channel layer, an upper surface hydrophilic layer of the liquid outlet channel, a first intermediate adhesive layer, an upper layer main body layer, a second intermediate adhesive layer, a top cover layer and a product collection and storage cavity sealing patch which are laminated and assembled in sequence. The application does not need any external driving force or active surface treatment, and only relies on the material properties and ingenious structure design, so that the plasma / serum can be automatically and efficiently separated from trace whole blood, and the application has the advantages of simple structure, low cost, convenient operation, one-time use, instant detection application and the like.

Description

Technical Field

[0001] This invention belongs to the field of real-time detection technology, and in particular relates to a fully passive microfluidic whole blood sample preparation device and method. Background Technology

[0002] Rapid and efficient separation of blood samples is a key prerequisite for point-of-care testing and point-of-care diagnostics. Traditional centrifugation methods rely on large equipment, are cumbersome to operate, and require large sample volumes, making them unsuitable for on-site testing. In recent years, microfluidic technology has provided a new approach for the separation of small amounts of blood. However, existing technologies rely on external vacuum chambers or pretreatment to alter channel wettability (such as plasma treatment), making them not truly passive. Furthermore, commonly used PDMS materials are easily deformable and adsorb biomolecules, while rigid plastics such as PMMA, although stable, lack sufficient surface hydrophilicity, making it difficult to achieve spontaneous capillary aspiration of blood. In addition, existing passive microfluidic separation devices still face challenges in controlling the separation process (such as ensuring sufficient settling time) and driving the complete transfer of separated products to the collection area. While simple hydrophilic channel designs can drive sample injection, it is difficult to precisely control the residence time of blood in the separation zone, and at the connection between the channel and the chamber, capillary force interruption caused by abrupt changes in cross-section often leads to fluid stagnation, making it impossible to ensure complete product recovery. Therefore, there is an urgent need in this field for a truly passive microfluidic whole blood sample pretreatment device that requires no external power, has a stable structure, and can intelligently control the separation process and ensure complete product transfer. Summary of the Invention

[0003] In view of this, the present invention aims to overcome the defects in the prior art and proposes a fully passive microfluidic whole blood sample preparation device and method to solve the technical problems in the prior art that microfluidic blood separation devices require external driving force or complex surface treatment, cannot passively and accurately control the separation sequence, and have incomplete recovery due to fluid resistance during the product transfer stage.

[0004] To achieve the above objectives, the technical solution created by this invention is implemented as follows: A fully passive microfluidic whole blood sample preparation device, comprising the following structural layers laminated and assembled sequentially from bottom to top: Bottom support layer; The bottom hydrophilic layer has its hydrophilic surface facing upwards. The bottom adhesive layer is made of double-sided pressure-sensitive adhesive, with liquid inlet channels cut out on it; The middle main layer is made of polymethyl methacrylate sheet, which has filter grooves and product collection and storage chambers. The liquid outlet channel layer is made of double-sided pressure-sensitive adhesive, with liquid outlet channels cut out on it; A hydrophilic layer on the upper surface of the liquid outlet channel covers the liquid outlet channel; The first intermediate adhesive layer is composed of double-sided pressure-sensitive adhesive; The upper main layer is made of polymethyl methacrylate (PMMA) sheets; The second intermediate adhesive layer is composed of double-sided pressure-sensitive adhesive. The top cover layer is made of polymethyl methacrylate sheet, which is provided with a sample inlet, a filter groove exhaust hole and a collection groove, with the filter groove exhaust hole corresponding to the filter groove. The product collection and storage chamber sealing sticker is made of double-sided pressure-sensitive adhesive and covers the upper surface of the top cover layer; The bottom adhesive layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the upper main body layer, the second intermediate adhesive layer, and the top cover layer are all provided with filtration ports corresponding to the filtration trench. The liquid inlet channel is connected to the filtration port on the bottom adhesive layer, and the liquid outlet channel is connected to the filtration port on the liquid outlet channel layer. The bottom adhesive layer, the intermediate main body layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the second intermediate adhesive layer, and the upper main body layer are all provided with sample loading ports corresponding to the sample inlet. The bottom adhesive layer, the intermediate main body layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the upper main body layer, and the second intermediate adhesive layer are all provided with collection ports corresponding to the collection tank.

[0005] Furthermore, the thickness of the polymethyl methacrylate sheet in the intermediate main layer is 2-5 mm.

[0006] Furthermore, the double-sided pressure-sensitive adhesive used in the bottom adhesive layer, the liquid outlet channel layer, the first intermediate adhesive layer, the second intermediate adhesive layer, and the product collection and storage cavity sealing sticker is selected from ARcare 90106 NB or ARcare 8939.

[0007] Furthermore, the double-sided pressure-sensitive adhesive used in the bottom adhesive layer, the liquid outlet channel layer, the first intermediate adhesive layer, the second intermediate adhesive layer, and the product collection and storage cavity sealing sticker has a thickness of 100-200μm.

[0008] Furthermore, both the bottom hydrophilic layer and the upper surface hydrophilic layer of the liquid outlet channel are made of single-sided hydrophilic pressure-sensitive adhesive, and the single-sided hydrophilic pressure-sensitive adhesive used in both is ARflow 93049.

[0009] Furthermore, the bottom support layer, the upper main body layer, and the top cover layer are all made of polymethyl methacrylate (PMMA) sheets.

[0010] Furthermore, the thickness of the bottom support layer, the upper main body layer, and the top cover layer is 0.1-0.5 mm.

[0011] A method for fabricating a fully passive microfluidic whole blood sample preparation device includes the following steps: S1. Each structural layer is processed separately by laser cutting; S2. The cut layers are laminated and assembled on a prefabricated alignment base in the order described in claim 1. S3. During the lamination assembly process, a portion of the hydrophilic layer on the upper surface of the liquid outlet channel is laser-cut to form a partially exposed hydrophobic valve structure and a guide plate extending into the product collection and storage cavity. S4. Use a roller laminator to pressurize the assembly and complete the encapsulation.

[0012] Furthermore, in step S4, the lamination pressure is 50-70 psi, the lamination temperature is room temperature, and the lamination direction is bidirectional, with one press each time.

[0013] Compared with existing technologies, the present invention has the following advantages: This invention utilizes a rigid framework consisting of a middle main layer, a bottom support layer, an upper main layer, and a top cover layer made of polymethyl methacrylate (PMMA) sheets to ensure the dimensional stability of core structures such as the filtration trench and the product collection and storage chamber. Within this framework, a bottom adhesive layer and an outlet channel layer, both composed of double-sided pressure-sensitive adhesive, define the paths for the inlet and outlet channels. The hydrophilic walls formed by the bottom hydrophilic layer and the hydrophilic layer on the upper surface of the outlet channel, through strong capillary force, sequentially drive whole blood samples to automatically fill the filtration trench and, after separation, drive plasma to flow out through the outlet channel. By partially exposing a micro-hydrophobic valve formed by hydrophobic pressure-sensitive adhesive at the inlet of the outlet channel, combined with the air pressure sealing system formed by the sealing patch of the product collection and storage chamber, the settling time of blood in the filtration trench can be passively controlled to ensure sufficient separation. After removing the sealing patch, plasma can enter the product collection and storage chamber under capillary force. The overall structure is stable, simple to operate, requires no external equipment, and is low in cost, making it suitable for point-of-care testing scenarios. Attached Figure Description

[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 A schematic diagram of the structure created by this invention; Figure 2 A schematic diagram showing the invention after removing the bottom support layer and the top cover layer; Figure 3 This invention provides a schematic diagram of the layered structure. Figure 4 A schematic diagram illustrating the hydrophilic layer created on the upper surface of the liquid channel in this invention; Figure 5 A schematic diagram illustrating the liquid channel layer created for this invention; Figure 6 A schematic diagram illustrating the bottom adhesive layer of this invention; Figure 7 A schematic diagram illustrating the intermediate main body layer of this invention; Figure 8 A schematic diagram illustrating the top cover layer created for this invention. Detailed Implementation

[0015] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0016] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on this invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, features defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0017] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0018] The invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0019] A fully passive microfluidic whole blood sample preparation device, such as Figures 1 to 8 As shown, the structure comprises the following layers laminated together from bottom to top: Bottom support layer; The bottom hydrophilic layer 1 has its hydrophilic surface facing upwards; The bottom adhesive layer 2 is made of double-sided pressure-sensitive adhesive, and liquid inlet channels 10 are cut on it. The intermediate main layer 3 is made of polymethyl methacrylate sheet, and has filter grooves 11 and product collection and storage chamber 12 on it; The liquid outlet channel layer 4 is made of double-sided pressure-sensitive adhesive, and the liquid outlet channel 13 is cut on it. A hydrophilic layer 5 is formed on the upper surface of the liquid outlet channel, covering the liquid outlet channel. The first intermediate adhesive layer 6 is composed of double-sided pressure-sensitive adhesive; The upper main layer 7 is made of polymethyl methacrylate sheet; The second intermediate adhesive layer 8 is composed of double-sided pressure-sensitive adhesive; The top cover layer 9 is made of polymethyl methacrylate sheet, and has an inlet 14, a filter groove exhaust hole 15 and a collection groove 16 on it. The filter groove exhaust hole is set in correspondence with the filter groove. The product collection and storage chamber sealing sticker is made of double-sided pressure-sensitive adhesive and covers the upper surface of the top cover layer; The bottom adhesive layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the upper main body layer, the second intermediate adhesive layer, and the top cover layer are all provided with a filtration port 17 corresponding to the filtration trench. The liquid inlet channel is connected to the filtration port on the bottom adhesive layer, and the liquid outlet channel is connected to the filtration port on the liquid outlet channel layer. The bottom adhesive layer, the intermediate main body layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the second intermediate adhesive layer, and the upper main body layer are all provided with a sample loading port 18 corresponding to the sample inlet. The bottom adhesive layer, the intermediate main body layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the upper main body layer, and the second intermediate adhesive layer are all provided with a collection port 19 corresponding to the collection tank.

[0020] This device uses a rigid framework consisting of a middle main layer, a bottom support layer, an upper main layer, and a top cover layer made of polymethyl methacrylate (PMMA) sheets. This ensures the dimensional accuracy of core functional structures such as the filtration trench and the product collection and storage chamber, as well as the overall mechanical stability of the device, laying a physical foundation for reliable fluid control. Within this rigid framework, the bottom adhesive layer and the outlet channel layer, made of double-sided pressure-sensitive adhesive, not only firmly bond the layers together, but also form the fluid flow path through the inlet and outlet channels created by laser cutting. The bottom hydrophilic layer and the upper surface hydrophilic layer of the outlet channel constitute the bottom wall of the inlet channel and the top wall of the outlet channel, respectively. When their exposed hydrophilic surfaces come into contact with blood, they generate capillary forces. This force sequentially drives the whole blood sample to automatically fill the filtration trench through the inlet channel, and after separation, it drives the plasma to flow through the outlet channel to the product collection and storage chamber.

[0021] To ensure thorough separation, a passive control unit is installed at the connection between the filter trench and the outlet channel. Combined with the gas pressure sealing system formed by the product collection and storage chamber sealing strip, this effectively prevents fluid from prematurely entering the downstream during the separation stage. After sedimentation, removing the product collection and storage chamber sealing strip breaks the equilibrium, triggering plasma transfer. The entire device uses double-sided pressure-sensitive adhesive layers, such as the first and second intermediate adhesive layers, to bond all the functional layers together under pressure at room temperature, making the manufacturing process extremely simple. This invention integrates a rigid structure, adhesive channel, active capillary drive material, and passive control structure in a vertical direction, synergistically achieving a process where high-purity plasma samples are obtained solely through gravity and capillary force, requiring only sample addition and removal of the sealing strip. It offers advantages such as structural stability, simple operation, low cost, and suitability for single-use point-of-care testing applications.

[0022] In an optional embodiment, the hydrophilic layer on the upper surface of the outlet channel is partially cut off at the corresponding outlet channel position to form a guide groove 20. The guide groove is connected to the filter port on the hydrophilic layer on the upper surface of the outlet channel, so that the hydrophobic pressure-sensitive adhesive of the adjacent outlet channel layer is partially exposed at the inlet end of the outlet channel, forming a micro hydrophobic valve, which is used to prevent fluid from entering the outlet channel during the sample filling and separation stages. The sample loading port on the hydrophilic layer on the upper surface of the outlet channel has an uncut portion, which forms a guide piece 21 that extends into the product collection reservoir to guide the separated serum to overcome the flow resistance caused by the sudden change in spatial size and smoothly enter and fill the product collection reservoir. The hydrophilic layer on the upper surface of the outlet channel is composed of a single-sided hydrophilic pressure-sensitive adhesive, the outlet channel layer is composed of a hydrophobic double-sided pressure-sensitive adhesive, and the micro hydrophobic valve is composed of the exposed hydrophobic pressure-sensitive adhesive surface.

[0023] Specifically, at the inlet of the liquid outlet channel (i.e., the connection between the filter trench and the liquid outlet channel), a small section (approximately 0.3-0.5 mm wide) of the hydrophilic pressure-sensitive adhesive (i.e., the "hydrophilic layer on the upper surface of the liquid outlet channel") that should completely cover the channel is selectively cut off. This cutting action exposes the hydrophobic double-sided pressure-sensitive adhesive material (such as ARcare 90106 NB), which originally formed the sidewall or bottom wall of the liquid outlet channel, directly inside the channel, becoming a small section of surface along the fluid flow path. This exposed hydrophobic pressure-sensitive adhesive surface has a large contact angle (>90°) with blood or plasma, exhibiting hydrophobic properties. According to the Yang-Laplace equation, when fluid flows through a region where the surface suddenly changes from hydrophilic to hydrophobic, a large capillary resistance (i.e., the "capillary valve" effect) is generated. This resistance can effectively prevent blood from spontaneously flowing into the liquid outlet channel before separation is complete.

[0024] After the blood sample is injected and enters the filtration trench, the red blood cells begin to settle. At this point, the inlet of the downstream outflow channel is blocked by this "microhydrophobic valve," preventing blood from passing through. This passively ensures that the red blood cells have sufficient time (e.g., 5 minutes) to completely settle in the filtration trench, achieving the separation of the upper serum layer from the lower blood cell layer. When the separation is complete and it is necessary to transfer the upper serum (plasma) to the collection chamber, the resistance of this hydrophobic valve can be overcome by removing the sealing patch above the product collection reservoir, changing the air pressure balance, and applying a slight disturbance (such as a light tap). Because serum has fewer components and slightly different surface properties than whole blood, and the capillary force (from the hydrophilic channels before and after) and pressure difference driving its flow are dominant, the serum can smoothly pass through this area into the downstream.

[0025] The intermediate main body layer, made of polymethyl methacrylate (PMMA), has a thickness of 2-5 mm. This thickness range ensures that the filter trenches have sufficient depth (typically consistent with the thickness of the sheet) to accommodate a sufficient volume of blood sample (approximately 45 μL) and provide the necessary settling space and time for blood cells, forming the physical basis for efficient gravity sedimentation separation. Simultaneously, this thickness of PMMA sheet provides sufficient structural rigidity and overall support, preventing bending or deformation of the device during operation or lamination. This ensures the geometric stability of the internal microchannels (such as inlet and outlet channels), which is crucial for the reliability of capillary flow that relies on precise channel dimensions.

[0026] The bottom adhesive layer, the outlet channel layer, the first intermediate adhesive layer, the second intermediate adhesive layer, and the product collection reservoir sealing sticker all use double-sided pressure-sensitive adhesives selected from ARcare 90106 NB or ARcare 8939. The use of medical-grade ARcare series PSAs ensures good biocompatibility and avoids non-specific adsorption or introduction of interfering substances to biomarkers in blood samples. The double-sided pressure-sensitive adhesives used in the bottom adhesive layer, the outlet channel layer, the first intermediate adhesive layer, the second intermediate adhesive layer, and the product collection reservoir sealing sticker have a thickness of 100-200 μm. Standardizing the thickness of each adhesive layer and sealing sticker ensures that the channels (such as inlet and outlet channels) and cavities (such as the product collection reservoir) formed by these PSA layers are uniform and consistent in height during multi-layer stacking assembly. This is crucial for generating stable and repeatable capillary driving force. Especially for the outlet channel layer, the height of the channel formed by its cutting directly affects the magnitude of capillary force and flow velocity. These PSA materials achieve strong adhesion at room temperature with the application of pressure alone, without the need for heating or chemical treatment. This simplifies the manufacturing process and allows the entire device to be assembled through a fast, low-cost lamination process, making it ideal for mass production.

[0027] Both the bottom hydrophilic layer and the upper hydrophilic layer of the outlet channel are made of single-sided hydrophilic pressure-sensitive adhesive, specifically ARflow 93049. Polymethyl methacrylate (PMMA) itself has poor hydrophilicity (water contact angle 70-80 degrees), making it difficult to drive spontaneous blood flow. ARflow 93049, a specially designed hydrophilic PSA, has a surface water contact angle of less than 30 degrees and exhibits extremely strong hydrophilicity. By placing it at the bottom of the inlet channel and the top of the outlet channel, the key walls along the fluid flow path are composed of highly hydrophilic materials. When a blood sample comes into contact with the ARflow 93049 at the bottom of the inlet channel, strong capillary forces automatically draw the sample in and fill the filter trench. Similarly, the separated plasma can spontaneously flow to the collection chamber under the drive of ARflow 93049 at the top of the outlet channel. If the wall of the microfluidic channel is made of polymethyl methacrylate sheet itself, the wall is hydrophobic. Therefore, this solution cleverly avoids the need for complex hydrophilic surface treatment (such as plasma treatment) of the PMMA channel wall, which is the fundamental guarantee for achieving the goal of eliminating the need for external pumps or vacuum drive.

[0028] The bottom support layer, upper main body layer, and top cover layer are all made of polymethyl methacrylate (PMMA) sheet. The thickness of these layers is 0.1-0.5 mm. Using PMMA ensures material consistency between these outer layers and the middle main body layer, avoiding interlayer stress or deformation that may be caused by differences in thermal expansion coefficients. The 0.1-0.5 mm thickness design allows for a thinner and lighter device while providing necessary encapsulation protection, mechanical support, and overall rigidity. The thinner top cover layer facilitates light transmission, enabling visual or optical monitoring of the sample separation process inside the device. Simultaneously, the thinner sheet material also improves the precision and efficiency of laser cutting and reduces overall material costs.

[0029] The following is a method for fabricating a fully passive microfluidic whole blood sample preparation device, including the following steps: S1. Each structural layer is processed separately by laser cutting. This step fully utilizes the high precision and flexibility of laser processing, and can accurately form the complex contours and internal cavities on the middle main layer, bottom support layer, upper main layer and top cover layer of PMMA sheet in one go, as well as cut out fine patterns defining the liquid inlet and outlet channels from the double-sided pressure-sensitive adhesive material, ensuring the dimensional consistency of all functional components and laying the foundation for subsequent seamless assembly. S2. The cut layers are laminated and assembled on a prefabricated alignment base in the order described in claim 1. During the lamination and assembly process, the principle of "applying only soft materials to hard materials" is followed, and the release paper is removed, aligned, and pressed layer by layer. The process of laminating and assembling the cut layers on a prefabricated alignment base in the order described in claim 1 is crucial for successful manufacturing. The prescribed order ensures that the hydrophilic surface of the bottom hydrophilic layer faces upward to drive sample injection, and the hydrophilic layer on the upper surface of the liquid outlet channel covers the liquid outlet channel to drive product transfer. The liquid outlet channel layer and the bottom adhesive layer, etc., are bonded together while precisely forming the height of the fluid path. The prefabricated alignment base ensures the precise alignment of more than ten layers, making the filter trenches, channels, and chambers perfectly connected in the vertical direction. S3. During the lamination assembly process, a portion of the hydrophilic layer on the upper surface of the liquid outlet channel is laser-cut to form a partially exposed hydrophobic valve structure and a guide plate extending into the product collection and storage cavity. This laser-cutting of a portion of the hydrophilic layer on the upper surface of the liquid outlet channel during lamination assembly is an integrated functional step that simultaneously manufactures the passive valve during the assembly process: by removing a small section of the hydrophilic layer at the inlet end of the liquid outlet channel, the underlying hydrophobic double-sided pressure-sensitive adhesive is exposed, thus forming a micro-hydrophobic valve in situ to control the separation sequence; simultaneously, the remaining hydrophilic layer naturally extends into the product collection and storage cavity, forming a guide plate that guides the complete transfer of the product. This "subtractive" operation cleverly achieves both fluid blocking and guiding functions in a single process, without the need for additional components or complex surface modifications. S4. Pressurize the assembly using a roller laminator to complete the encapsulation. This step uses controlled pressure (e.g., 50-70 psi) to ensure a strong and uniform bond between each double-sided pressure-sensitive adhesive layer and the adjacent PMMA and hydrophilic PSA layers, forming a well-sealed integrated device. This also avoids damage to material properties (especially hydrophilicity) that may be caused by high temperature or chemical treatment.

[0030] This method features a continuous process flow of "precise laser prefabrication—sequential layering and alignment—laser in-situ functionalization—roll pressing at room temperature encapsulation." During laser cutting, a portion of the hydrophilic layer on the upper surface of the outlet channel is retained. This preserved portion of the single-sided hydrophilic pressure-sensitive adhesive covers the outlet channel at its front end, while its rear end extends forward and hovers above the product collection reservoir. When the separated plasma flows out of the narrow outlet channel under capillary force and is about to enter the suddenly enlarged product collection reservoir, the capillary driving force weakens or even disappears sharply due to the abrupt change in cross-section, according to the Young-Laplace equation, causing the fluid to easily stagnate at this point. The guide plate designed in this invention, with its inherently strong hydrophilic surface, extends from the channel outlet all the way into the collection cavity, continuously providing capillary traction force to guide plasma droplets smoothly across the "gap" between the channel and the cavity. This ensures that the plasma is completely and efficiently introduced and fills the entire collection cavity, thereby avoiding product residue and loss and improving sample recovery rate.

[0031] In step S4, the lamination pressure is 50-70 psi, the lamination temperature is room temperature, and the lamination direction is bidirectional, with one press in each direction. This ensures a strong, uniform, and bubble-free bond between the adhesive layers (such as the bottom adhesive layer, the liquid outlet channel layer, and the intermediate adhesive layers) and the adjacent PMMA sheets (such as the intermediate main layer and the support layer), as well as the single-sided hydrophilic pressure-sensitive adhesive layer. This forms well-sealed microchannels and chambers, preventing liquid leakage or gas infiltration from interfering with capillary flow. This pressure is below the threshold that would cause plastic deformation or crushing of the PMMA sheet, thus protecting the geometric integrity of the fine filtration grooves, inlet channels, and outlet channels formed by laser cutting.

[0032] The lamination temperature is room temperature, avoiding any potential damage to the inherent hydrophilicity of single-sided hydrophilic pressure-sensitive adhesive materials (such as ARflow 93049). High-temperature processing can cause changes in the chemical composition of the adhesive layer or failure of hydrophilic functional groups, thereby weakening its core ability to drive fluid flow. Room temperature operation also simplifies the process and reduces energy consumption and equipment requirements. The lamination direction is bidirectional, with one press in each direction, ensuring uniform pressure distribution within the assembly plane. This effectively eliminates air that may be trapped during layer stacking, and in particular, ensures a tight fit between the hydrophilic layer on the upper surface of the liquid outlet channel and the lower structure. This allows the guide plate portion to extend precisely and be positioned above the product collection and storage chamber, while ensuring clear boundaries and reliable function of the micro-hydrophobic valve area (i.e., the exposed hydrophobic adhesive surface after the cut-off portion).

[0033] To ensure precise alignment of each layer in the fully passive microfluidic device of this invention during lamination assembly, thereby guaranteeing the vertical connectivity of fluid channels, chambers, and functional areas, all structural layers of the device are laser-cut with lamination alignment holes 22 at the four corners or specific positioning points. The specific assembly and fabrication steps are as follows: First, based on the overall size of the device and the location of the calibration holes, an assembly platform is prepared by 3D printing or machining. The platform is equipped with four assembly pillars that perfectly match the size of the calibration holes in each layer. The height of the assembly pillars should be slightly higher than the cumulative thickness of all the laminated materials to ensure that they can penetrate all structural layers during the assembly process.

[0034] During lamination assembly, the operator first places the bottom PMMA support layer (layer 1) on a flat workbench, then places the assembly platform on it, with the four assembly pillars passing through the four calibration holes in the support layer. This fixes the positions of the assembly platform and the support layer.

[0035] Next, following a predetermined bottom-up sequence, each layer of material is processed sequentially. Before assembling a new layer, the release paper on its back is removed (for the PSA layer), and then its calibration holes are precisely fitted onto the already positioned assembly column. Since the calibration holes of all layers are made using the same laser cutting program, the positional accuracy is extremely high. Through the physical constraints of the assembly column, it can be ensured that key structures such as the liquid inlet channel, filter trench, liquid outlet channel, product collection and storage chamber, micro-hydrophobic valve area, and hydrophilic layer guide plate area of ​​each layer are perfectly aligned in three-dimensional space.

[0036] After each layer is positioned and placed in place through the calibration holes, a manual roller is used to roll it from the center outwards to initially bond it to the material below. Once all structural layers are stacked, aligned, and initially pressed together, the entire device is lifted upwards to separate it from the assembly column of the assembly platform, ultimately forming a prefabricated component with strictly aligned structural layers.

[0037] The device of this invention performs localized laser ablation of the hydrophilic layer at the inlet of the liquid outlet channel, exposing the underlying hydrophobic PSA layer. This creates a microhydrophobic valve in situ that passively controls blood retention time. The retained hydrophilic layer naturally extends to the product collection and storage chamber, forming a guide plate that guides complete plasma transfer. Combined with the pressure control provided by the sealing patch covering the collection chamber, the device achieves fully automated processing from whole blood sampling and blood cell sedimentation to plasma collection and transfer, relying solely on capillary forces and gravity, without requiring any external pumps, valves, or active surface treatments. The manufacturing method employs a continuous process of "precise laser cutting - sequential alignment lamination - laser in-situ functionalization - room temperature roll forming encapsulation," ensuring the reliability and consistency of the device. The entire solution boasts significant advantages, including structural stability, extremely simple operation, low manufacturing cost, small sample requirements, and the complete elimination of the need for external power.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fully passive microfluidic whole blood sample preparation device, characterized in that, The following structural layers are laminated and assembled from bottom to top: Bottom support layer; The bottom hydrophilic layer has its hydrophilic surface facing upwards. The bottom adhesive layer is made of double-sided pressure-sensitive adhesive, with liquid inlet channels cut out on it; The middle main layer is made of polymethyl methacrylate sheet, which has filter grooves and product collection and storage chambers. The liquid outlet channel layer is made of double-sided pressure-sensitive adhesive, with liquid outlet channels cut out on it; A hydrophilic layer on the upper surface of the liquid outlet channel covers the liquid outlet channel; The first intermediate adhesive layer is composed of double-sided pressure-sensitive adhesive; The upper main layer is made of polymethyl methacrylate (PMMA) sheets; The second intermediate adhesive layer is composed of double-sided pressure-sensitive adhesive. The top cover layer is made of polymethyl methacrylate sheet, which is provided with a sample inlet, a filter groove exhaust hole and a collection groove, with the filter groove exhaust hole corresponding to the filter groove. The product collection and storage chamber sealing sticker is made of double-sided pressure-sensitive adhesive and covers the upper surface of the top cover layer; The bottom adhesive layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the upper main body layer, the second intermediate adhesive layer, and the top cover layer are all provided with filtration ports corresponding to the filtration trench. The liquid inlet channel is connected to the filtration port on the bottom adhesive layer, and the liquid outlet channel is connected to the filtration port on the liquid outlet channel layer. The bottom adhesive layer, the intermediate main body layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the second intermediate adhesive layer, and the upper main body layer are all provided with sample loading ports corresponding to the sample inlet. The bottom adhesive layer, the intermediate main body layer, the liquid outlet channel layer, the hydrophilic layer on the upper surface of the liquid outlet channel, the first intermediate adhesive layer, the upper main body layer, and the second intermediate adhesive layer are all provided with collection ports corresponding to the collection tank.

2. The fully passive microfluidic whole blood sample preparation device according to claim 1, characterized in that: The thickness of the polymethyl methacrylate sheet for the intermediate main layer is 2-5 mm.

3. The fully passive microfluidic whole blood sample preparation device according to claim 1, characterized in that: The bottom adhesive layer, the liquid outlet channel layer, the first intermediate adhesive layer, the second intermediate adhesive layer, and the product collection and storage cavity sealing sticker all use double-sided pressure-sensitive adhesives selected from ARcare 90106 NB or ARcare 8939.

4. The fully passive microfluidic whole blood sample preparation device according to claim 3, characterized in that: The bottom adhesive layer, the liquid outlet channel layer, the first intermediate adhesive layer, the second intermediate adhesive layer, and the product collection and storage cavity sealing sticker all use double-sided pressure-sensitive adhesive with a thickness of 100-200μm.

5. The fully passive microfluidic whole blood sample preparation device according to claim 1, characterized in that: Both the bottom hydrophilic layer and the upper surface hydrophilic layer of the liquid outlet channel are made of single-sided hydrophilic pressure-sensitive adhesive, and the single-sided hydrophilic pressure-sensitive adhesive used in both is ARflow 93049.

6. The fully passive microfluidic whole blood sample preparation device according to claim 1, characterized in that: The bottom support layer, the upper main body layer, and the top cover layer are all made of polymethyl methacrylate (PMMA) sheets.

7. The fully passive microfluidic whole blood sample preparation device according to claim 6, characterized in that: The thickness of the bottom support layer, the upper main body layer, and the top cover layer is 0.1-0.5mm.

8. A method for manufacturing a fully passive microfluidic whole blood sample preparation device as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Each structural layer is processed separately by laser cutting; S2. The cut layers are laminated and assembled on the prefabricated alignment base. S3. During the lamination assembly process, a portion of the hydrophilic layer on the upper surface of the liquid outlet channel is laser-cut to form a partially exposed hydrophobic valve structure and a guide plate extending into the product collection and storage cavity. S4. Use a roller laminator to pressurize the assembly and complete the encapsulation.

9. The method according to claim 8, characterized in that: In step S4, the lamination pressure is 50-70 psi, the lamination temperature is room temperature, and the lamination direction is bidirectional, with one press in each direction.

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