Biological detection chip, detection method and detection equipment

Abstract

The invention discloses an integrated detection chip for separation and detection of biological molecules. The chip comprises a substrate and a micro-channel separation unit manufactured on the substrate, and the micro-channel separation unit is used for separating a biological sample to be detected; and the biological sensing detection unit is used for detecting the biological sample to be detected after the micro-channel separation treatment. And the biosensing detection unit is a surface plasmon sensing unit. The micro-channel separation unit comprises a viscoelastic environment forming part, a particle distribution forming part, a particle shunting part, a flow speed control part and a detection part which are communicated in sequence. And the particle shunting part is a multi-fork separation runner. And the viscoelastic environment forming part is a cross orthogonal flow channel or a triangular arrow flow channel. And the flow rate control part is a smooth multi-bend multi-straight flow channel. The detection part is provided with a detection cavity structure, and the detection cavity is in fit contact with the biosensing detection unit.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a biological detection chip, detection method, and detection equipment. Background Technology

[0002] Rapid pretreatment of biological fluid samples is crucial for effective clinical testing. Biological fluids such as blood, saliva, and urine offer numerous advantages, including ease of collection and non-invasiveness. Sample acquisition is relatively straightforward in practice, and the techniques for obtaining these samples are well-established. Furthermore, the testing process causes minimal trauma to the human body. Blood, as one of the most widely used bodily fluids in current clinical testing, contains a variety of detectable biomarkers. Testing collected blood samples can enable the diagnosis of various diseases.

[0003] However, in the clinical testing of specific biomolecules in biological samples such as blood, sample pretreatment is necessary to separate large-sized and small-sized biological particles in the body fluids. This reduces viscosity, improves detection accuracy, and prevents blockage or clogging. For example, in blood samples, biological particles of different sizes, such as blood cells, protein molecules, and nucleic acids, may interfere with the detection of specific biomolecules. Large-sized biological particles may occupy detection sites, preventing the binding of specific biomolecules to detection reagents, thus reducing detection accuracy. Separating biological particles also provides a purer sample for subsequent testing steps. Furthermore, traditional detection methods require the purification and enrichment of specific biomolecules. Pretreatment to separate biological particles provides better starting materials for these subsequent steps, improving detection sensitivity and reliability.

[0004] However, existing rapid biomolecular detection technologies lack the ability to rapidly process, separate, and sense complex samples, especially in scenarios such as ICUs, operating rooms, and emergency rooms, and therefore cannot meet practical requirements. Their drawbacks include:

[0005] I. Insufficient Sample Processing Capacity. Current point-of-care testing technologies struggle to process complex samples, such as blood, quickly and effectively. They cannot adequately remove impurities and interfering substances from samples within a short time, affecting the accuracy of subsequent tests. In particular, they lack the flexibility and adaptability to provide personalized processing for different types of complex samples.

[0006] Second, the separation capability is inadequate. When processing blood samples, the separation speed of different components is slow, failing to meet the demand for rapid separation in emergency situations. For example, it cannot effectively separate blood cells from plasma and other components in a timely manner, affecting the detection of specific biomolecules. Furthermore, unstable separation results may lead to incomplete or erroneous separation, thus affecting the reliability of the test results.

[0007] Third, limited sensing capabilities. The sensitivity for detecting specific biomarkers in complex samples is insufficient, making it difficult to accurately capture low concentrations of biomolecular signals, leading to missed or false detections. Furthermore, the long sensor response time prevents the rapid delivery of results in emergency situations, hindering timely and effective support for clinical decision-making.

[0008] Fourth, there is a lack of adaptability to specific application scenarios. In critical situations such as ICUs, operating rooms, and emergency care, existing technologies cannot be quickly adjusted and optimized to meet the requirements of specific environments, making it difficult to meet actual emergency medical needs. In particular, due to a lack of integration, the separation and detection components are separate, resulting in a large amount of manual operation and making it unsuitable for environments such as operating rooms. Furthermore, it cannot seamlessly integrate with other medical equipment and processes in these scenarios, increasing operational complexity and time costs. Moreover, high-speed centrifugation technology requires the use of centrifuges, which occupy a large area and require special maintenance.

[0009] Fifth, increased errors during the detection process. This includes sample loss, as samples are inevitably damaged during centrifugation, transfer, and detection, thus increasing the initial sample volume required; simultaneously, sample loss leads to increased errors during the detection process. Furthermore, the operation steps are cumbersome, the pretreatment process is complex and time-consuming; separation and detection require different devices and equipment, consequently increasing detection costs.

[0010] It is particularly important to note that traditional separation methods not only consume a significant amount of time and require large-scale instruments, but also easily lead to sample loss, reducing yield and consequently affecting test results. Therefore, they are difficult to apply to point-of-care testing pretreatment in clinical settings. While high-speed centrifugation is currently the industry standard for biomolecular separation, it has certain drawbacks, including:

[0011] 1) The separation process usually requires centrifuge equipment, which occupies a large area and cannot meet the needs of intraoperative testing;

[0012] 2) Typically, the sample volume processed in a single session is large, on the order of mL, and the applicable range is small;

[0013] 3) Repeated sample replacement is required, making continuous sample processing and downstream testing impossible, which interferes with medical staff.

[0014] Furthermore, in biomedical testing of biological fluid samples, the pre-processed samples need to be tested rapidly and immediately; otherwise, the clinical demand for rapid results cannot be met. In clinical practice, time is often critical. Rapidly obtaining test results allows doctors to make timely diagnostic and treatment decisions, thereby improving patient treatment outcomes and survival rates. By rapidly detecting the levels of corresponding biomarkers in biological fluid samples, the pathological state of patients, such as inflammatory responses, organ or tissue damage, can be diagnosed promptly. For example, in emergency situations, rapid testing can help doctors quickly determine the patient's condition and take appropriate emergency measures. For the detection of some infectious diseases, rapid results can promptly isolate patients and prevent the spread of disease. Currently, clinical testing equipment cannot rapidly and directly analyze whole blood samples, and the complete medical testing process is complex and usually requires considerable time. For instance, one of the common methods for detecting biomolecules in biological fluid samples is enzyme-linked immunosorbent assay (ELISA), but ELISA has certain drawbacks, including:

[0015] 1) The process is complex, requiring repeated addition of liquid, and the entire testing process is time-consuming, making real-time testing impossible.

[0016] 2) Because the detection requires multiple biochemical reactions, it is easily affected by external conditions such as temperature and time;

[0017] 3) Low sensitivity, which can easily interfere with the detection results. Summary of the Invention

[0018] One embodiment of this disclosure discloses an integrated detection chip for biomolecule separation and detection. The chip includes a substrate, and components fabricated on the substrate comprising:

[0019] Microchannel separation unit is used to separate biological samples to be tested;

[0020] The biosensing detection unit detects biological samples that have undergone microfluidic separation.

[0021] The biosensing detection unit is a surface plasmon resonance sensing unit.

[0022] The microchannel separation unit includes a viscoelastic environment forming section, a particle distribution forming section, a particle diversion section, a flow rate control section, and a detection section connected in sequence.

[0023] The particle splitting section is a multi-branched separation channel.

[0024] The viscoelastic environment forming section is a cross-shaped positive flow channel or a triangular arrow flow channel.

[0025] The flow rate control unit is a smooth, multi-bend, multi-direction channel.

[0026] The detection unit has a detection cavity structure, which is in close contact with the biosensor detection unit. Attached Figure Description

[0027] The above and other objects, features, and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of the invention are illustrated in the drawings by way of example, not limitation, in which:

[0028] Figure 1 A schematic diagram of particle binding according to one embodiment of the present invention.

[0029] Figure 2 One embodiment of the present invention is an exploded three-dimensional view of a detection chip.

[0030] Figure 3 According to one embodiment of the present invention, a schematic diagram of the microchannel structure of a detection chip is provided, in which the shape and position of each part are marked, with 01, 02 and 03 as channel inlets and 04 and 05 as channel outlets.

[0031] Figure 4 One embodiment of the present invention is a schematic diagram of the partial force field formed by particle distribution.

[0032] Figure 5 One embodiment of the present invention is a schematic diagram of the structure of each part of the microchannel.

[0033] Figure 6 A schematic diagram of the detection section in a detection chip is shown in one embodiment of the present invention.

[0034] Figure 7 An operation flowchart of an integrated detection chip according to one embodiment of the present invention.

[0035] Among them, 201—microfluidic sensing cavity,

[0036] 202—SPR sensor chip,

[0037] 203—Whole Blood Separation Microfluidics

[0038] 204—Integrated sensor chip,

[0039] 205—Patient's blood sample. Detailed Implementation

[0040] Based on the analysis of existing technologies, this disclosure addresses the shortcomings of traditional separation and detection methods by integrating passive microfluidic separation technology with label-free surface plasmon resonance (SPR) detection technology to create an integrated separation-detection chip. This chip is suitable for applications such as rapid and sensitive point-of-care testing during surgery. Furthermore, the integrated separation-detection chip significantly reduces additional human intervention and operation during the detection process, lowers non-standardized procedures and detection errors, improves detection sensitivity, shortens detection time, facilitates operation by clinicians, and meets the needs of point-of-care testing.

[0041] The purpose of this disclosure is to address the shortcomings of traditional methods such as centrifugation and ELISA in point-of-care testing and medical diagnosis, and to propose the use of microfluidic technology as a sample separation and detection platform, coupled with sensing methods such as surface plasmon resonance technology, to achieve integrated detection.

[0042] Centrifugation technology utilizes the powerful centrifugal force generated when an object rotates at high speed, causing suspended particles placed in the rotating body to settle or float, thereby concentrating certain particles or separating them from other particles. However, existing centrifugation methods have certain drawbacks:

[0043] 1) The separation process usually requires centrifuge equipment, which occupies a large area and cannot meet the needs of intraoperative testing;

[0044] 2) The sample volume for a single test is large, in the mL range, limiting its applicability;

[0045] 3) Repeated sample replacement is required, making continuous testing impossible and causing interference for medical staff.

[0046] ELISA is a fluorescent detection technique based on the immobilization of antigens or antibodies and enzyme labeling of antigens or antibodies. During detection, the analyte (antigen or antibody) in the sample binds to the receptor (antibody or antigen) immobilized on a 96-well plate. Non-specific binding is removed by washing the plate, and then enzyme-labeled antigen or antibody is added. At this point, the amount of immobilized enzyme labeling is related to the amount of the analyte in the sample. After adding a substrate that reacts with the enzyme, color development occurs, and the intensity of the color indicates the concentration of the substance in the sample, allowing for qualitative or quantitative analysis. ELISA is suitable for bodily fluid samples and has advantages such as high sensitivity and a wide detection range. However, the equipment used for ELISA occupies a large area, and the detection requires complex pretreatment steps, which prevents its application in operating room testing.

[0047] Microfluidic separation is a recently emerging blood particle separation technology. It refers to the technology of processing or manipulating tiny fluids using microchannels (tens to hundreds of μm in size). Due to its small size, it is also known as a lab-on-a-chip or micro total analysis system. It separates molecules of different sizes through special microchannel structures. Microfluidic separation technology can be divided into active and passive separation technologies. Active technologies achieve particle separation through external forces such as photoacoustics and electromagnetism, but these usually require long detection times and can cause cell damage. These external forces also limit its application, such as during surgery or for biomolecules sensitive to these forces. Passive separation methods separate particles based on differences in centrifugal force, inertia, and fluid dynamics, typically achieved through channel structure design.

[0048] Surface plasmon resonance (SPR) is a label-free, highly sensitive, and rapid biomolecule detection method based on micro-nano photonics. It detects the interaction between biomolecules by sensing the resonance phenomenon between light and plasmon waves on a metal surface, thereby enabling quantitative analysis of target biomolecules. The core advantages of this technology lie in its label-free, highly sensitive detection capability and fast detection time. When a target biomolecule binds to an acceptor on the surface of a plasmon resonance medium, the surface refractive index changes, causing significant alterations in the intensity, phase, and other optical parameters of the reflected light. By detecting these optical parameters, the concentration of the target biomolecule can be quantitatively determined.

[0049] According to one or more embodiments, a separation-detection integrated biochip and detection system can realize the integration of sample processing and detection, and has the advantages of being fast, efficient and accurate. It is suitable for point-of-care testing application scenarios with high timeliness requirements, such as during surgery.

[0050] like Figure 1 The diagram illustrates how two different sizes of biological particles combine. Among them, Figure 1 Display A shows the binding of smaller-sized biological particles. Each small circle can represent a biological particle that binds to the substrate at the bottom through some means (such as chemical bonding, physical adsorption, or other interactions between biomolecules). This binding method is suitable for the detection of single molecules or small molecule complexes where the particle size is small and requires highly sensitive detection methods for identification. Figure 1Section B illustrates the binding of larger-sized biological particles. Here, two larger circles represent larger biological particles or particle complexes, which also bind to the substrate at the bottom. This binding method is used to detect larger biomolecules, such as cells, viruses, or large protein complexes. The detection chip disclosed herein is designed for biological particles of different sizes to achieve the detection of targets of varying sizes. Due to its integrated design, sample processing and detection can be rapidly completed on the same chip, reducing the time required to transfer samples between different devices. It is particularly suitable for applications requiring rapid detection results, such as rapid diagnosis during surgery.

[0051] For example, whole blood contains various biological particles, including red blood cells (RBCs), white blood cells (WBCs), blood platelets (PLTs), and small biomolecules. When detecting specific biomolecules, to avoid interference between signals from molecules with significantly different sizes, such as… Figure 1 As shown, sample pretreatment methods such as high-speed centrifugation are typically used to separate large molecules, such as red blood cells and white blood cells, from smaller biomolecules before testing. The separated samples are then tested using detection techniques such as enzyme-linked immunosorbent assay (ELISA). In whole blood, small biomolecules mainly include polypeptides, polysaccharides, amino acids, nucleotides, hormones, and some drugs and their metabolites. These small biomolecules play important roles in the body, including signal transduction, metabolic regulation, and maintaining vital functions. When performing biomolecule detection, sample pretreatment methods are necessary to separate these small molecules from large molecules such as red blood cells and white blood cells to avoid signal interference and ensure detection accuracy.

[0052] This embodiment is implemented as follows: An integrated passive separation microchannel and detection chamber are fabricated using microfluidic technology. A liquid sample (whole blood) is introduced into the microchannel chip through the inlet and mixed and diluted with buffer solution at the cross-structure to create a viscoelastic environment. The sample then flows into a long, direct current channel. Here, due to the different elastic forces, viscous drag, and inertial lift experienced by particles of different sizes in the long channel, all species initially align near the channel sidewalls. As they move along the channel, size-dependent forces act on the contained species, resulting in different lateral migration paths and a distribution pattern where particle diameter gradually decreases from the center to the sides. The long, direct current channel is connected to a particle splitter port for different particle sizes, separating and exporting particles of different sizes. Biological particles of the target size are introduced into the detection chamber for label-free plasmon detection. The phase change is measured and compared with a standard curve to determine the corresponding inflammatory factor content. An exploded view of the chip structure is shown below. Figure 2 As shown.

[0053] Figure 2 A microfluidic chip system for patient blood sample testing is illustrated. The two blood drops 205 in the figure are blood samples collected from a patient. The blood sample 205 is introduced into the microfluidic sensing cavity 201. A surface plasmon resonance (SPR) sensor chip 202 is used to detect specific proteins, cells, or other molecules in the blood. A whole blood separation microfluidic component 203 is used to separate different components in whole blood, such as red blood cells, white blood cells, and platelets, within the microfluidic system. An integrated sensor chip 204 integrates the above-mentioned units or components to achieve the detection or analysis of multiple biomarkers in this embodiment of the disclosure.

[0054] Therefore, the workflow of a microfluidic chip system is as follows: a patient's blood sample is first introduced into the microfluidic chip, where it is separated into components using a whole blood separation microfluidic component. The separated components are then continuously delivered to the SPR sensing cavity within the microfluidic chip or to other types of sensing chips for detection. The integrated sensing chip may be used for further analysis or data processing.

[0055] exist Figure 3 The composition of the microfluidic chip was further demonstrated. Figure 3 A is the main body of the microfluidic chip, containing the fluid channels and control structures. In the diagram, 01, 02, 03, 04, and 05 represent different fluid inlets and outlets. Figure 3 B is the viscoelastic environment formation component, used to alter the viscoelastic properties of the fluid to influence the motion behavior of particles within the fluid. Figure 3 C is the particle distribution forming part, which uniformly distributes particles in the fluid. Figure 3 D is a particle diversion port for different particle sizes, allowing particles of different sizes to pass through different channels, thus achieving particle size-based separation. Figure 3 E is the flow rate control section, used to control the flow rate of the fluid. Figure 3 F represents the detection section. The operation of this microfluidic chip includes: a blood sample enters the chip through the inlet; at position B, the viscoelasticity of the blood sample fluid is adjusted; in section C, sample particles are uniformly distributed in the blood fluid; the blood sample fluid carries the particles through section D, where particles of different sizes are diverted to different channels; in section E, the sample flow rate is controlled; and finally, in section F, the sample is detected.

[0056] Therefore, the chip in this embodiment of the present disclosure consists of a viscoelastic environment forming part, a particle distribution forming part, a particle diversion port with different particle sizes, a flow rate control part, and a detection part. The overall flow channel schematic diagram is shown below. Figure 3 As shown in Figure A. The viscoelastic environment forming portion, such as... Figure 3As shown in Figure B, this is used to thoroughly mix the buffer solution with the sample to create a viscoelastic environment, facilitating the formation of force field differences based on particle size. Here, the buffer solution is a 0.1% (v / v) mixture of PEO dissolved in 1xPBS solution, exhibiting the corresponding viscoelasticity. PEO (polyethylene oxide) is a nonionic, water-soluble resin, possessing complete water solubility, solubility in many solvents, and the characteristics of a nonionic polymer. 1xPBS solution, or phosphate buffered solution, is a commonly used buffer salt solution.

[0057] The particle distribution forms a portion, such as Figure 3 As shown in Figure C, this is a straight microchannel. The differences in elastic force, viscous drag, and inertial lifting force for particles of different sizes are created using the principles of inertial force and fluid dynamics. Figure 4 As shown, when entering the particle distribution forming section, particles of all sizes initially align near the channel sidewalls. As they move along the channel, size-dependent forces act on the particles, resulting in different lateral migration paths. This creates a particle distribution where particle size gradually decreases from the center to the sides, facilitating the subsequent entry of biological particles of different sizes into the separation channel.

[0058] The force situation of particles in a straight flow channel is as follows Figure 4 As shown, the elastic force is F1, which is proportional to the cube of the particle radius; the viscous drag force is F2, which is proportional to the particle radius; and the inertial lifting force is F3, which is proportional to the fourth power of the particle radius. The second normal stress generated on the upper and lower surfaces of the flow channel is significantly smaller than the force in the flow channel plane, so it is ignored, and only the above three forces are considered to affect the particles.

[0059] Larger particles experience greater lateral migration forces, and compared to smaller particles, they migrate laterally over greater distances and at higher speeds.

[0060] The particle distribution forms a flow channel with a length of 25-35 mm.

[0061] The collection ports with different particle sizes, such as Figure 3 As shown in D, it is used to introduce particles of different sizes into different pathways for subsequent detection or collection.

[0062] The flow rate control section, such as Figure 3 As shown in E, it is used to control the flow rate of the collection ports of different particle sizes of macromolecules, enhance the separation effect, relieve pressure, and prevent the flow channel from clogging.

[0063] The detection section, such as Figure 3 As shown in F, it is used for the detection of biomolecules based on surface plasmon resonances.

[0064] Preferably, the width of the microchannel is 20 μm or more, such as 25 μm.

[0065] Preferably, the flow channel height is 40 μm or more, such as 55 μm.

[0066] Preferably, the chip has a length of 60mm or more and a width of 50mm or more.

[0067] Preferably, 01 is the sample inlet, 02 and 03 are the buffer solution inlets, 04 is the large particle outlet, and 05 is the detection chamber outlet. The large particle outlet can either directly discharge waste liquid or be used for detection for other purposes.

[0068] Preferably, the viscoelastic environment forming portion can be a mixture of orthogonal and angular structures, such as... Figure 5 As shown in A and B.

[0069] Preferably, the sample flow rate at inlet 01 is 180-220 μL / h, the buffer flow rate at inlet 02 is 1900-2100 μL / h, and inlet 03 is the buffer solution introduced during detection to form a detection baseline. The flow rate can be adjusted appropriately according to different analytes.

[0070] After the sample flows through the particle distribution area, due to the differences in elastic force, viscous drag, and inertial lifting force of particles of different sizes, a corresponding Gaussian distribution will form. That is, large-sized particles are mainly located in the middle part, and small-sized particles are located in the middle and on both sides, as shown in the schematic diagram. Figure 5 As shown in C.

[0071] The collection ports for different particle sizes can be configured as three or five channels as needed, and can be adjusted according to the required fineness of separation during pretreatment. Figure 5 As shown in D, 5E.

[0072] The collection ports for different particle sizes are either three-pronged or five-pronged structures, with the particle diameter gradually decreasing from the middle to the sides. Considering the proportion of the outlet width, the middle outlet width of the three-pronged outlet is 30%-40% of the total width, and this middle outlet is the outlet for the desired particles. That is, in the three-pronged structure, the middle collection channel occupies 30-40% of the total width, and in the five-pronged structure, the middle collection channel occupies 30-50% of the total width. The distance from the separation tip to the inlet of the collection port for different particle sizes is 0.3-0.5 mm.

[0073] Preferably, the flow rate control section is a flow channel with 16 bends and 32 straight lines, and the length of the straight section is 10-20mm.

[0074] Preferably, the separated biomolecules need to be diluted before entering the detection chamber. The buffer solution can be introduced before the collection ports of different particle sizes or before the detection chamber, such as... Figure 5 As shown in F.

[0075] in, Figure 5 The microfluidic mixing in A employs orthogonal mixing, in which two fluid streams (a fluid containing particles and a buffer solution) intersect at a right angle (90 degrees). Orthogonal mixing allows for efficient mixing of particles and buffer solution in the flow direction; however, the uniformity of the mixture can be affected by fluid velocity and hydrodynamic characteristics. Figure 5 B employs triangular mixing, forming a triangular mixing region, which can provide a more uniform mixing effect than orthogonal mixing because the convergence of three fluid flows increases the contact area between the fluids.

[0076] Figure 5 C illustrates the distribution of the mixed particles in the flow channel, showing the distribution of two different particle sizes (5 micrometers and 1 micrometer) exhibiting a Gaussian distribution. This Gaussian distribution indicates that the particle concentration is highest at the center of the flow channel and gradually decreases towards the sides. This distribution is ideal for many analytical and detection applications because it allows for high-concentration detection at the center of the flow channel.

[0077] Figure 5 D illustrates a three-stage separation process, referring to the use of three different flow channels to distinguish blood cells (larger than 1 micrometer) from submicron particles. Due to their larger size, blood cells can pass through specific structures or barriers within the microfluidic channels, thus separating them from the smaller submicron particles.

[0078] Figure 5 E demonstrates a five-stage separation process, a more complex procedure with five distinct flow channels to distinguish between blood cells, extracellular vesicles, and biomolecules. Blood cells (orange), due to their larger size, are separated first. Extracellular vesicles (white) and biomolecules (gray) require more sophisticated separation techniques.

[0079] Figure 5 F shows the location of the buffer solution used to form the baseline for detection.

[0080] Preferably, the detection section is a hexagonal detection cavity, which is connected to the channels on both sides of the collection port with different particle sizes through a right-angle curved flow channel. The spatial structure of the detection cavity includes a length of 10-12 mm, a width of 2-4 mm, and a height of 0.04-0.4 mm.

[0081] Preferably, the sample size of the integrated chip detection in this embodiment of the present disclosure is 200 μL.

[0082] Preferably, the integrated chip in this embodiment of the present disclosure needs to be diluted to five times the original amount for whole blood samples to avoid excessive viscosity.

[0083] Furthermore, in practice, the detection chamber can be designed in various shapes such as elliptical, circular, hexagonal, or square, and the volume of liquid it can hold can be adjusted according to actual monitoring needs. For example, in a 50μL microfluidic detection chamber, the sensing layer can be located at the bottom, top, or middle of the chamber. Figure 6 As shown in A and B. Taking the detection of inflammatory factors as an example, a sensor chip can be placed on the bottom surface of the fluid in the sensing cavity, and its surface can be modified with specific biomolecules such as biotinylated antibodies. Through the principle of specific binding between molecules such as antigens and antibodies, phase changes can be realized in the SPR sensor, such as... Figure 6 As shown in C, SPR detection in the detection chamber requires a thin membrane structure, which can be distributed at the bottom, top, or middle of the chamber, as long as it binds to the separated liquid.

[0084] Preferably, a glass slide is used in combination with a colloid, and the glass slide portion corresponding to the detection cavity portion is coated with a surface plasmon resonance sensing material.

[0085] Figure 6 This diagram illustrates the detection principle of a biosensor based on antigen-antibody reactions, typically used to detect the presence of specific biomolecules. The three parts in the diagram represent the sample introduction, the immobilization of the biorecognition element, and the antigen-antibody binding process, respectively. Figure 6 Sample introduction of A, Figure 6 B represents the immobilization of biometric elements. The sample flows through an area whose surface is immobilized with biometric elements, such as antibodies. These biometric elements (shown as wavy lines in the figure) are specific, meaning they can only bind to specific antigens.

[0086] Figure 6 Figure C illustrates antigen-antibody binding, where antigen molecules in a sample specifically bind to antibodies as they flow through areas immobilized with those antibodies. The figure shows multiple antigen molecules (Y-shaped structures) binding to antibodies (inverted triangular structures). This binding can lead to the generation of signals, such as optical signals (color changes), electrical signals (current or voltage changes), or other physical changes, which can be detected and used to quantitatively analyze the antigen concentration in the sample.

[0087] The above sample liquid flow process (e.g.) Figure 7 As shown): Inlet 01 - splits the upper and lower triangular or square flow channels - mixes with the buffer solution at inlet 02 ( Figure 3 B) - Particle distribution formation part ( Figure 3 c) - Collection ports with different particle sizes Figure 3 03 and 05 in the middle; Figure 5 (Right output port in D, 5E) - Target size flows into the detection cavity ( Figure 3 F).

[0088] Figure 7 A blood sample processing and analysis workflow based on a microfluidic chip was demonstrated. The workflow includes the following steps:

[0089] S701, adds blood samples to the chip system;

[0090] S702, diluted blood sample;

[0091] S703 introduces the diluted blood sample and buffer solution into the microfluidic chip;

[0092] The S704 chip uses microfluidic technology to separate different components in a blood sample, such as cells, proteins, and extracellular vesicles.

[0093] S705, the separated sample components are guided to the detection area;

[0094] S706. Based on the detection results, quantitative analysis of the target molecules is performed to determine their concentration in the sample. S707. After sample processing, waste liquid and used chips need to be properly disposed of to avoid environmental pollution and cross-contamination.

[0095] This disclosure integrates passive biological particle separation microfluidics and plasmonic label-free sensing detection. Separation is achieved by leveraging the differences in elastic force, viscous drag, and inertial lift of particles of varying sizes. The compact flow channel structure and the connection of the detection chamber integrate separation and detection, overcoming the limitations of traditional separation methods such as long separation times and cell damage associated with centrifugation, and traditional detection methods such as enzyme-linked immunosorbent assay (ELISA) which suffer from low detection sensitivity, large footprint, long detection time, and high cost. This not only meets the need to simultaneously perform whole blood separation and inflammatory factor detection on a single chip but also improves product operability, as well as the accuracy and immediacy of results.

[0096] The embodiments disclosed herein have the following advantages: They exhibit excellent separation effects, with most red blood cells, white blood cells, and other biomolecules visible under a microscope during whole blood testing; they possess good detection sensitivity, with a standard curve of inflammatory factors already plotted and available for comparison; they shorten the overall detection process time; and they reduce the equipment cost for inflammatory factor detection. The bio-detection chip of this disclosure can be applied to intraoperative, real-time inflammatory factor detection or long-term clinical monitoring of patient inflammatory factor levels. It fills the gap in the field of integrated separation and detection for real-time inflammatory factor detection, dynamically monitoring patient inflammatory factor levels, determining the occurrence of acute tissue damage or inflammatory responses, helping doctors adjust treatment strategies in a timely manner, improving patient survival rates, and facilitating postoperative recovery.

[0097] According to one or more embodiments, an integrated microfluidic chip for separation and detection is provided, wherein the channel width on the chip is greater than 20 μm, and the channel height is greater than 40 μm. The diameter of the channel inlet and outlet is 40-50 μm, and this size is designed to be related to different inlet sample flow rates. To form the required viscoelastic environment, the buffer solution is a 0.1% (v / v) mixture of PEO dissolved in 1xPBS solution, which has corresponding viscoelasticity. The flow rate of inlet 01 is 180-220 μL / h, and the flow rate of inlet 02 is 1900-2100 μL / h. The viscoelastic environment is formed by a central sample channel and two buffer channels on both sides. The angle between the two side channels and the central channel can be a right angle to form a square structure, or an acute angle to form a triangular structure. The length of the central long channel formed by particle distribution is greater than 32 mm, and a corresponding Gaussian distribution is formed according to the different physical force fields caused by the difference in radius. The three-pronged structure of the collection ports for different particle sizes accounts for 40%-50% of the total volume, with the two parallel tips 20μm-30μm away from the long flow channel outlet. The detection chamber structure is hexagonal or elliptical. A special glass slide coated with plasma sensing material serves as the chip substrate. Blood samples need to be diluted five times; the required sample volume for this chip is 200-400μL.

[0098] This disclosed integrated biochip for separation and detection has wide applications in sample testing. Based on surface plasmon resonance (SPR) label-free detection technology, it is suitable for rapid analysis of intraoperative patient blood samples. The chip includes a specific microfluidic channel structure, enabling rapid separation of particles of different sizes in intraoperative patient blood samples using viscoelastic and particle inertial focusing microfluidic technology. An integrated SPR sensor allows for label-free detection of specific biomolecules in the separated blood sample, meeting the need for rapid intraoperative results.

[0099] For example, in neurosurgery, detecting specific proteins in the blood can assess brain nerve damage, providing a basis for precise surgical procedures. In cardiac surgery, real-time detection of biomarkers such as myocardial enzymes in the patient's blood can help doctors determine the extent of myocardial damage and adjust the surgical plan accordingly. Addressing the complexity of intraoperative patient blood samples, the chip's separation technology can effectively remove interfering substances, improving detection accuracy. Furthermore, it can rapidly separate blood cells and plasma, accurately detecting specific biomarkers related to the surgery. Time is crucial during surgery; the integrated chip of this invention can process and test patient blood samples in a short time, providing timely and accurate information for surgical decisions.

[0100] In summary, the main features of the microfluidic biochip for separation and detection disclosed herein include:

[0101] 1. It adopts an integrated separation-detection structure, which integrates the traditionally separate separation and detection steps onto a single chip, greatly improving detection efficiency and convenience. Simultaneously, this structure also includes a specific microfluidic channel layout for separating particles of different sizes, and the sensor positions and connection methods for detecting biomolecules, ensuring efficient and coordinated operation of separation and detection functions.

[0102] 2. This method employs viscoelastic and particle inertial focusing microfluidic technology to separate particles of different sizes. It enables rapid and accurate separation of particles from complex samples such as blood in a microfluidic environment, providing pure samples for subsequent testing. This protective separation method, based on the properties of viscoelastic fluids, optimizes the utilization of particle inertia and the control parameters of fluid flow in the microfluidic channel, ensuring the stability and reliability of the separation effect.

[0103] 3. Combining label-free surface plasmon resonance detection enables rapid and sensitive detection of specific biomolecules. This detection method eliminates the need for labeling, reduces sample processing steps, and improves detection accuracy and speed.

[0104] The overall concept of this disclosure is to fabricate an integrated plasma sensing chip for separation and detection using microfluidic preparation technology. This enables continuous separation and detection of complex biological sample systems, reducing equipment size, sample volume, and personnel operation, achieving a highly integrated and miniaturized separation-detection system. The separation microchannel of this disclosure employs passive biological particle separation microfluidic technology. Utilizing the principles of inertial force and fluid dynamics, it creates differences in elastic force, viscous drag, and inertial lifting force for particles of different sizes. When entering the particle distribution formation section, particles of all sizes initially align near the channel sidewalls. As they move along the channel, size-dependent forces act on the particles, resulting in different lateral migration paths. This forms a distribution state where the particle diameter gradually decreases from the center to the sides of the channel, followed by particle separation through a bifurcation structure. The detection microchannel of this disclosure utilizes plasmon label-free sensing technology. By modifying the surface of the detection chip in the detection chamber with biotinylation, it captures biological particles through specific binding, creating changes in optical phase. After comparison with a standard curve, it achieves rapid detection of corresponding biomolecules.

[0105] Therefore, the beneficial effects of this disclosure include:

[0106] I. Addressing the problem of inadequate sample processing capabilities

[0107] This disclosed integrated detection chip, through the coupling technology of microfluidics and biosensing and its integrated design, enables rapid and effective sample processing and analysis of complex samples such as blood. The specific microfluidic structure and hydrodynamic mechanism within the chip can remove impurities and interfering substances from the sample in a short time, ensuring the accuracy of subsequent detection. Simultaneously, the chip can be optimized and adjusted according to different types of complex samples, achieving personalized sample processing and screening, thus improving the flexibility and adaptability of the processing.

[0108] II. Improving the situation of weak separation ability

[0109] In blood sample processing, the chip disclosed herein can achieve rapid and efficient separation of different components. Its unique separation technology can meet the need for rapid separation in emergency situations, effectively separating nanoscale components such as blood cells, cell vesicles, and biomolecules in plasma, creating favorable conditions for the detection of specific biomolecules. Furthermore, the chip's separation effect is stable and reliable, greatly reducing the risk of incomplete or erroneous separation, thereby improving the reliability of the detection results.

[0110] III. Limited Improvement in Sensing Capabilities

[0111] For specific biomarkers in complex samples, the integrated detection chip disclosed herein possesses highly sensitive biosensing capabilities. It can accurately capture low concentrations of biomolecular signals, effectively avoiding missed or false detections. Simultaneously, the chip's extremely short sensing response time enables real-time biomolecular monitoring, providing rapid test results in emergency situations and offering timely and effective support for clinical decision-making.

[0112] IV. Enhancing the adaptability to application scenarios

[0113] In critical scenarios such as ICUs, operating rooms, and emergency care, this disclosed chip demonstrates exceptional adaptability. It integrates isolation and detection functions, reducing manual intervention and making it ideal for environments like operating rooms. The chip seamlessly integrates with other medical devices and processes in these scenarios, reducing operational complexity and time costs. Furthermore, its integrated design results in a small footprint, eliminating the need for bulky, maintenance-intensive instruments required by traditional detection technologies.

[0114] V. Reduce errors during the testing process

[0115] This disclosed chip effectively reduces sample loss during sample processing and detection. Its optimized design and operation significantly reduce sample loss during separation, transfer, and detection, thereby reducing the initial sample volume required. Simultaneously, reduced sample loss also means reduced errors during the detection process. Furthermore, the integrated design of the detection chip simplifies the detection steps, making the preprocessing process more concise and efficient, and shortening the detection time. In addition, the integrated design reduces detection costs, avoiding the high costs associated with using different components and equipment.

[0116] It should be understood that in the embodiments of the present invention, the term "and / or" is merely a description of the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Furthermore, the character " / " in this document generally indicates that the preceding and following associated objects have an "or" relationship.

[0117] It is worth noting that although the spirit and principles of this invention have been described with reference to several specific embodiments, it should be understood that this invention is not limited to the disclosed specific embodiments, and the division of aspects does not imply that the features in these aspects cannot be combined; such division is merely for the convenience of description. This invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. A biological detection chip, characterized in that: The chip includes a substrate, and the following components are fabricated on the substrate: A microfluidic separation unit, used for separating biological samples to be tested; The biosensor detection unit detects the biological sample to be detected after being separated and processed by the microfluidic channel.

2. The biological detection chip according to claim 1, characterized in that: The biosensing detection unit is a surface plasmon sensing unit.

3. The biological detection chip according to claim 1, characterized in that: The microchannel separation unit includes a viscoelastic environment forming part, a particle distribution forming part, a particle diversion part, a flow rate control part and a detection part which are connected in sequence.

4. The biological detection chip according to claim 3, characterized in that: The particle flow dividing part is a multi-branch separation flow channel, Preferably, the viscoelastic environment forming part is a cross-orthogonal or triangular cross-flow channel for mixing the sample to be separated and the buffer solution. Preferably, the flow rate control portion is a smooth multi-bend and multi-direction channel.

5. The biological detection chip according to claim 3, characterized in that: The detection part has a detection cavity structure, and the detection cavity is in close contact with the biosensor detection unit.

6. The biological detection chip according to claim 2, characterized in that: The biosensing detection unit is an optical sensing unit based on the surface plasmon resonance principle.

7. The biological detection chip according to claim 3, characterized in that: The microfluidic separation unit further comprises a first inlet for collecting a biological sample to be detected, which is arranged before the flow channel of the viscoelastic environment forming part. Preferably, the viscoelastic environment forming portion has a second inlet, and the second inlet is used for the second buffer solution to flow in. Preferably, the flow rate control unit has a third inlet for the inflow of a third buffer solution.

8. A biological sample detection method, characterized in that: Adding a biological sample to the biological detection chip according to any one of claims 1 to 7; Obtaining a test result of the biological sample.

9. The biological sample detection method according to claim 8, characterized in that: The detection results obtained from the biological detection chip are quantitatively analyzed.

10. A biological detection device, characterized in that: The device includes, A biochip carrier, used for loading the biodetection chip according to any one of claims 1 to 7; The controller is used to control the biological detection device to detect the biological sample to be detected through the biological detection chip.

Images