CN119738236A - A biological detection chip, detection method and detection equipment - Google Patents

Abstract

The present invention discloses an integrated detection chip for separation and detection of biomolecules. The chip includes a substrate, a microfluidic separation unit made on the substrate, and used to separate biological samples to be detected; and a biosensor detection unit, which detects the biological samples to be detected after microfluidic separation treatment. The biosensor detection unit is a surface plasmon sensing unit. The microfluidic 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 connected in sequence. The particle diversion part is a multi-fork separation flow channel. The viscoelastic environment forming part is a cross orthogonal flow channel, or a triangular arrow flow channel. The flow rate control part is a smooth multi-bend multi-direction flow channel. The detection part has a detection cavity structure, and the detection cavity is in close contact with the biosensor detection unit.

Description

Biological detection chip, detection method and detection equipment

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a biological detection chip, a detection method and detection equipment.

Background

The rapid pretreatment of biological liquid samples is a key point for realizing effective clinical detection, and the biological liquid sample preparation method has the advantages of easy collection, no wound and the like, is relatively easy to obtain and sample, is mature in the practical use, and has less wound on human body during detection. The blood is one of the most widely used body fluids in the current clinical detection, contains various biomarkers for detection, and can realize diagnosis of various diseases by detecting the collected blood sample.

However, in the process of clinically detecting a specific biomolecule in a biological sample such as blood, large-size biological particles and small-size biological particles in biological fluid need to be separated by means of sample pretreatment, so that the viscosity is reduced, the detection accuracy is improved, and the situation of occupation or blockage is avoided. For example, in a blood sample, biological particles of different sizes, such as blood cells, protein molecules, nucleic acids, etc., may interfere with the detection of a particular biological molecule. Wherein large-sized biological particles may occupy detection sites, preventing binding of specific biomolecules to detection reagents, thereby reducing detection accuracy. Isolation of the biological particles may also provide a purer sample for subsequent detection steps. Furthermore, purification and enrichment of specific biomolecules are required in conventional detection methods. By pre-treatment to separate the biological particles, better starting materials can be provided for these subsequent steps, improving the sensitivity and reliability of the detection.

However, when facing complex samples such as blood samples, the existing rapid biomolecule detection technology lacks the capability of rapid sample processing, separation and sensing, and especially in ICU, operating room, emergency and other situations, the existing rapid biomolecule detection technology cannot meet the practical requirements, and the disadvantages include:

1. the sample processing capacity is insufficient. In the face of complex samples such as blood, the existing instant detection technology is difficult to perform rapid and effective sample processing. Impurities, interferents and the like in the sample cannot be sufficiently removed in a short time, and the accuracy of subsequent detection is affected. In particular, the process of individualizing different types of complex samples is not possible, and flexibility and adaptability are lacking.

2. The separation ability is poor. When processing blood samples, the separation speed of the different components is slow, and the requirement for rapid separation in emergency situations cannot be met. For example, components such as blood cells and plasma cannot be separated effectively in time, and detection of specific biomolecules is affected. If the separation effect is unstable, incomplete separation or erroneous separation may be caused, thereby affecting the reliability of the detection result.

3. Sensing capability is limited. The detection sensitivity of specific biomarkers in complex samples is not high enough, the low-concentration biomolecule signals are difficult to accurately capture, and the condition of missed detection or false detection is easy to occur. If the sensing response time is long, the detection result cannot be rapidly given in an emergency situation, and timely and effective support cannot be provided for clinical decision.

4. Lacks targeted application scenario adaptation capability. Under the key situations of ICU, operating room, first aid etc., the prior art can't be adjusted and optimized fast according to the requirement of special environment, is difficult to satisfy actual emergency medical demand. In particular, due to lack of integrity, the separation portion and the detection portion are separated from each other, and the manual operation amount is large, which is not suitable for environments such as an operating room. In particular, it does not seamlessly engage with other medical devices and procedures in these scenarios well, increasing the complexity and time costs of the operation. Moreover, the high-speed centrifugation technique requires the use of a centrifuge, which occupies a large area and requires special maintenance.

5. Errors increase during the detection process. Here, there is a problem of sample loss, and the sample has a certain break in centrifugation, transfer and detection, thus the requirement of initial sample size is correspondingly increased, and meanwhile, the error in the detection process is increased due to the sample loss. The operation steps are complicated, the pretreatment process is complex, the required time is long, and different devices and equipment are required for separation and detection, so that the detection cost is correspondingly increased.

Particularly, the conventional separation method needs to take more time, takes the participation of a large-scale instrument, and easily causes sample loss, so that the yield is reduced, and the detection result is affected, so that the method is difficult to be applied to the instant detection pretreatment in clinic. Although high-speed centrifugation is the current industry standard technique for biomolecule separation, the centrifugation has certain drawbacks including:

1) The separation process generally requires centrifuge equipment, occupies large area and cannot meet the detection requirement in operation;

2) The single-treatment sample size is large and is in the order of mL, and the application range is small;

3) The sample needs to be repeatedly replaced, so that continuous sample treatment and downstream detection cannot be realized, and interference is caused to medical staff.

Furthermore, in biomedical testing involving biological fluid samples, the sample to be analyzed obtained by pretreatment is required to be tested promptly and rapidly, or else the requirement of clinical immediate testing for rapid results cannot be met. In clinical practice, time is often critical. The rapid acquisition of the detection results can enable doctors to make diagnosis and treatment decisions in time, so that the treatment effect and survival rate of patients are improved. By rapidly detecting the corresponding biomarker levels in the biological fluid sample, the pathological state of the patient, such as inflammatory reaction, organ or tissue injury and the like, can be diagnosed in time. For example, in emergency situations, rapid detection may help a physician quickly determine a patient's condition and take appropriate emergency action. For detection of some infectious diseases, the rapid results can isolate patients in time, preventing spread of diseases. At present, the detection equipment in clinic cannot rapidly and directly analyze and detect the whole blood sample, and the complete medical detection process is complex and usually requires more time. For example, one of the common detection methods for biomolecules in biological fluid samples is Enzyme-linked immunosorbent assay (ELISA), but ELISA suffers from certain drawbacks, including:

1) The steps are complex, liquid needs to be repeatedly added, the whole detection process is long, and instant detection cannot be realized;

2) Since the detection requires multi-step biochemical reaction, the detection is easily influenced by external conditions such as temperature, time and the like;

3) The sensitivity is low, and the detection result is easy to be interfered.

Disclosure of Invention

One of the embodiments of the present disclosure is an integrated detection chip for biomolecule separation-detection. The chip comprises a substrate, and the chip manufactured on the substrate comprises:

the micro-channel separation unit is used for separating the biological sample to be detected;

And the biosensing detection unit is used for detecting the biological sample to be detected which is subjected to the micro-channel separation treatment.

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 diversion part, a flow speed control part and a detection part which are sequentially communicated.

The particle diversion part is a multi-fork separation flow channel.

The viscoelastic environment forming part is a cross-shaped orthogonal runner or a triangular arrow runner.

The flow speed control part is a smooth multi-bending multi-straight flow passage.

The detection part is provided with a detection cavity structure, and the detection cavity is in fit contact with the biosensing detection unit.

Drawings

The above, as well as additional purposes, features, and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description when read in conjunction with the accompanying drawings. Several embodiments of the present invention are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which:

FIG. 1 is a schematic diagram of particle bonding according to one embodiment of the present invention.

Fig. 2 is an exploded perspective view of a detection chip according to one embodiment of the present invention.

Fig. 3 is a schematic diagram of a micro-fluidic channel structure of a detection chip according to one embodiment of the present invention, in which shapes and positions of respective portions are labeled, 01, 02, and 03 are fluidic channel inlets, and 04, and 05 are fluidic channel outlets.

FIG. 4 is a schematic diagram of a partial force field formed for particle distribution according to one embodiment of the present invention.

Fig. 5 is a schematic structural diagram of each part of a micro flow channel according to one embodiment of the present invention.

FIG. 6 is a schematic diagram of a detection portion of a detection chip according to one embodiment of the present invention.

FIG. 7 is a flowchart of the operation of an integrated test chip according to one embodiment of the present invention.

Wherein 201-the microfluidic sensing chamber,

202-The SPR sensor chip,

203-Whole blood separation micro-flow control,

204-An integrated sensor chip,

205-Patient blood sample.

Detailed Description

Through the analysis of the prior art, in order to make up the defects of the traditional separation and detection modes, the method fuses a passive microfluidic separation technology and a surface plasmon label-free detection technology to prepare a separation-detection integrated chip, and is suitable for application scenes such as rapid and sensitive instant detection in operation. In addition, the separation-detection integrated detection chip greatly reduces additional human intervention and operation in the detection process, reduces non-standardized procedures and detection errors in the operation process, improves the detection sensitivity, shortens the detection time, is convenient for a clinician to operate, and can meet the requirements of bedside detection.

The purpose of the disclosure is to solve the defects of the traditional methods such as centrifugal separation technology, ELISA detection and the like in instant detection and medical diagnosis, and to provide a sensing method which uses a microfluidic technology as a sample separation and detection platform and is coupled with a surface plasma resonance technology at the same time, so as to realize integrated detection.

The centrifugal technology utilizes the strong centrifugal force generated when the object rotates at high speed to enable suspended particles in the rotating body to settle or float, so that some particles can be concentrated or separated from other particles. However, the existing centrifugation method has certain disadvantages:

1) The separation process generally requires centrifuge equipment, occupies large area and cannot meet the detection requirement in operation;

2) The single detection sample size is large and is of the order of mL, and the application range is small;

3) The sample needs to be repeatedly replaced, continuous detection cannot be realized, and interference is caused to medical staff.

ELISA is a fluorescent detection technique based on the immobilization of antigens or antibodies and the enzymatic labelling of antigens or antibodies. In detection, the test substance (antigen or antibody) in the sample binds to a receptor (antibody or antigen) immobilized on a 96-well plate. The non-specific binding substance is removed by washing the plate, and then an enzyme-labeled antigen or antibody is added, at which time the amount of enzyme label immobilized correlates with the amount of the substance to be detected in the sample. By adding a substrate for reaction with the enzyme and then developing the color, the content of the substance in the sample can be judged according to the color depth, and thus, qualitative or quantitative analysis can be performed. ELISA is suitable for biological samples of body fluid, and has the advantages of high sensitivity, wide detection range and the like. However, the equipment used in ELISA has a large floor space and requires complex pretreatment steps for detection, which makes it impractical for use in operating room detection.

Microfluidic separation is a recently emerging blood particle separation technique. It refers to a technique of treating or manipulating minute fluids using micro-pipes (having a size of several tens to several hundreds of μm), which is also called a Lab-on-a-Chip (Lab) or a micro-total analysis system due to its small occupied volume. Molecules of different sizes are separated by special microchannel structures. Microfluidic separation techniques can be divided into active separation techniques and passive separation techniques. Among them, the active technology realizes the separation of particles by the action of external forces such as photoacoustic electromagnetic, etc., but generally requires a long detection time and causes the breakage of cells, and these external forces also limit the application occasions, such as the conditions in surgery or the impossibility of biomolecules sensitive to the external forces. The passive separation method performs separation through differences in aspects of centrifugal force, inertial action, fluid dynamics and the like of particles, and is usually realized by using a flow channel structure design.

The Surface Plasmon Resonance (SPR) technology is a label-free, high-sensitivity and rapid biomolecule detection method based on micro-nano photonics, and the interaction between biomolecules is detected by detecting the resonance phenomenon of light and metal surface plasmon waves, so that the quantitative analysis of target biomolecules is realized. The core advantages of this technique are its label-free, high sensitivity of detection and faster detection times. When the target biomolecule is combined with the receptor on the surface of the plasma resonance medium, the refractive index of the surface changes and causes the optical parameters such as the intensity, the phase and the like of the reflected light to change obviously, and the concentration of the target biomolecule can be quantitatively detected by detecting the optical parameters such as the intensity, the phase and the like of the reflected light.

According to one or more embodiments, the separation-detection integrated detection biochip and detection system can realize integration of sample processing detection, have the advantages of rapidness, high efficiency and accuracy, and are suitable for instant detection application scenes with high timeliness requirements in surgery and the like.

As shown in fig. 1, the manner in which two different sizes of biological particles bind is illustrated. Among them, fig. 1A shows the smaller size of the biological particle binding. Each small circle may represent a biological particle that is bound to the underlying substrate in some way (possibly by chemical bonds, physical adsorption or other interactions between biological molecules). This binding approach is suitable for detection of single molecules or small molecule complexes where the particle size is small and high sensitivity detection means are required for identification. Fig. 1B shows the larger size of the biological particle binding. Here, two larger circles represent larger biological particles or particle complexes, which are also bound to the underlying substrate. This binding mode is used to detect larger biomolecules, such as cells, viruses or large protein complexes. The detection chip is designed according to biological particles with different sizes so as to realize detection of targets with different sizes. Because of the integrated design, sample processing and detection can be completed rapidly on the same chip, and the time for transferring samples between different devices is reduced. The method is particularly suitable for application scenes which need to obtain detection results quickly, such as quick diagnosis in operation.

For example, whole Blood contains various biological particles such as Red Blood Cells (RBC), white Blood Cells (WBC), platelets (PLT), small-sized biomolecules, and the like. In order to avoid mutual interference between molecular signals with excessively large size difference during detection of specific biomolecules, as shown in fig. 1, macromolecules such as erythrocytes and leukocytes are usually separated from biomolecules with smaller particle size by a sample pretreatment method such as high-speed centrifugation before detection, and then the separated samples are tested by a detection means such as an enzyme-linked immunosorbent assay (ELISA). Whereas in whole blood, small-sized biomolecules mainly include polypeptides, polysaccharides, amino acids, nucleotides, hormones, and some drugs and their metabolites. These small molecule biomolecules play an important role in organisms, including signaling, metabolic regulation, and maintenance of vital activities. In the detection of biological molecules, the small molecules and the large molecules such as red blood cells and white blood cells are required to be separated by a sample pretreatment method, so that signal interference is avoided, and the detection accuracy is ensured.

The embodiment of the disclosure is realized by preparing an integrated passive separation micro-channel and a detection cavity by using a micro-fluidic technology. After the liquid sample (whole blood) is introduced into the micro-channel chip from the inlet, the liquid sample is mixed and diluted with the buffer solution at the crossing structure to form a viscoelastic environment, and then the sample flows into the long straight channel, wherein all the species with different sizes are initially arranged near the side wall of the channel due to different elastic force, viscous resistance and inertial lift force of the particles with different sizes in the long channel. As they move along the channel, a magnitude-dependent force acts on the contained species, resulting in different lateral migration paths, creating a distribution of progressively decreasing particle diameters from the middle to the sides. The long direct current channel is tightly connected with particle shunt ports with different particle diameters, particles with different sizes are separately exported, wherein biological particles with target sizes are imported into the detection cavity, and plasmons are detected without marks. And after the phase change is measured, the corresponding inflammatory factor content can be judged by comparing the measured phase change with a standard curve, and the structural explosion diagram of the chip is shown in figure 2.

Fig. 2 shows a microfluidic chip system for detecting a blood sample of a patient, in which two drops of blood 205 are collected from the patient. A blood sample 205 is introduced into the microfluidic sensing chamber 201. The surface plasmon resonance (Surface Plasmon Resonance, SPR) sensor chip 202 is used to detect specific proteins, cells or other molecules in blood. The whole blood separation microfluidic assembly 203 is used to separate different components in whole blood, such as red blood cells, white blood cells, and platelets, in a microfluidic system. While integrated sensor chip 204 integrates the various units or components described above to enable detection or analysis of a variety of biomarkers in embodiments of the disclosure.

Thus, the workflow of a microfluidic chip system is such that a patient's blood sample is first introduced into a microfluidic chip, component separation is performed by a whole blood separation microfluidic assembly, and the separated components are continuously delivered to SPR sensing chambers or other types of sensing chips in the microfluidic chip for detection. The integrated sensor chip may be used for further analysis or data processing.

In fig. 3, the microfluidic chip composition is further illustrated. Fig. 3A is a body portion of a microfluidic chip, including channels and control structures for fluids. 01, 02, 03, 04, 05 in the figures represent different fluid inlets, outlets. FIG. 3B is a viscoelastic environment forming part for altering the viscoelastic properties of a fluid to affect the movement behavior of particles in the fluid. Fig. 3C is a particle distribution forming section for uniformly distributing particles in a fluid. Fig. 3D is a view of different particle size particle shunt ports allowing different sized particles to pass through different channels for particle size based separation. Fig. 3E is a flow rate control section for controlling the flow rate of a fluid. Fig. 3F is a detection section. The working process of the microfluidic chip comprises that a blood sample enters the chip through an inlet, the viscoelasticity of the blood sample fluid is adjusted at the position B, sample particles are uniformly distributed in the blood fluid at the position C, the blood sample fluid carries the particles through the position D, and the particles with different sizes are shunted to different channels. In section E the sample flow rate is controlled and finally in section F the sample is detected.

Therefore, the chip of the embodiment of the disclosure is composed of a viscoelastic environment forming part, a particle distribution forming part, particle shunt ports with different particle diameters, a flow rate control part and a detection part, and the whole flow channel is schematically shown in fig. 3A. The viscoelastic environment forming part is used for fully mixing the buffer solution and the sample to form a corresponding viscoelastic environment, so as to form a force field difference according to the particle size, as shown in fig. 3B. Here, the buffer is a mixed solution of 0.1% by volume prepared by dissolving PEO in 1xPBS solution, which has corresponding viscoelasticity. PEO (polyethylene oxide) is a nonionic water-soluble resin and has the properties of being completely water-soluble, soluble in many solvents, and nonionic. The 1xPBS solution, phosphate buffer (Phosphate Buffered Solution), is a buffer salt solution that is frequently used.

The particle distribution forming section, as shown in fig. 3C, is a section of linear micro-channel, and uses the principle of inertial force and fluid dynamics to cause the difference of elastic force, viscous resistance and inertial lifting force of particles with different sizes, as shown in fig. four, when entering the particle distribution forming section, particles with all sizes are initially arranged near the side wall of the channel. As they move along the channel, a magnitude dependent force acts on the particles, resulting in different lateral migration paths. Thereby forming particle distribution with gradually reduced particle sizes from the middle to the two sides, and facilitating the subsequent entry of biological particles with different particle diameters into the separation flow channel.

The stress condition of the particles in the linear flow channel is shown in fig. 4, the elastic force is F ₁, the elastic force is proportional to the third power of the particle radius, the viscous resistance position F ₂ is proportional to the particle radius, the inertial lifting force is F ₃, and the inertial lifting force 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 obviously smaller than the force in the flow channel plane, so that the second normal stress is ignored, and only the action of the three forces on the particles is considered.

Larger size particles experience greater lateral migration forces, and the lateral migration distance is far and faster than for smaller size particles.

The particle distribution forming part has a runner length of 25-35mm.

The different particle size collection ports, as shown in fig. 3D, are used to introduce different sized particles into different pathways, respectively, for subsequent detection or collection.

The flow rate control part is used for controlling the flow rate of the collecting ports with different particle diameters of macromolecules, enhancing the separation effect, relieving the pressure and preventing the flow passage from being blocked as shown in fig. 3E.

The detection moiety, as shown in FIG. 3F, is used to perform detection of a surface plasmon-based biomolecule.

Preferably, the microchannel width is 20 μm or more, such as 25 μm.

Preferably, the flow channel height is above 40 μm, such as 55 μm.

Preferably, the chip has a length of 60mm or more and a width of 50mm or more

Preferably, 01 is a sample inlet, 02, 03 is a buffer inlet, 04 is a large-sized particle outlet, and 05 is a detection chamber outlet. The large-size particle outlet can directly flow out waste liquid and can also be used for detection of other purposes.

Preferably, the viscoelastic environment forming portions may be orthogonal or angularly mixed, as shown in fig. 5A, B.

Preferably, the flow rate of the inlet 01 sample is 180-220 mu L/h, the flow rate of the inlet 02 buffer is 1900-2100 mu L/h, and the inlet 03 is the buffer which is introduced during detection and is used for forming a detection baseline, and the detection baseline is adjusted appropriately according to different detection objects.

After the sample flows through the particle distribution forming part, due to the difference of the elastic force, viscous resistance and inertial lifting force of the particles with different sizes, corresponding gaussian distribution is formed, namely, large-size particles are mainly located in the middle part, and small-size particles are located in the middle and on two sides, and the schematic diagram is shown in fig. 5C.

The collection ports with different particle sizes can be three channels or five channels as required, and can be adjusted according to different separation fineness during pretreatment, as shown in fig. 5d and 5 e.

The collection ports with different particle diameters are of a three-fork structure or a five-fork structure, the particle diameters of the collection ports gradually decrease from the middle to the two sides, the ratio of the width of the outlet is considered, the width of the middle outlet of the three-fork outlet is 30% -40% of the total width, and the middle outlet is the outlet of the needed particles. The proportion of the middle collecting flow passage in the three-fork structure to the total width is 30-40%, the proportion of the middle collecting flow passage in the five-fork structure to the total width is 30-50%, and the distance from the separating tip to the inlet of the collecting port with different particle diameters is 0.3-0.5mm.

Preferably, the flow rate control part is a 16-bend 32 straight flow passage, and the length of the straight part is 10-20mm.

Preferably, the separated biomolecules need to be diluted before entering the detection chamber, and the buffer solution inlet point can be before the collection ports with different particle sizes or before the detection chamber, as shown in fig. 5F.

Wherein the microfluidic mixing of fig. 5A employs orthogonal mixing in which two streams (particle-containing fluid and buffer) intersect at right angles (90 degrees). By orthogonal mixing, particles and buffer can be effectively mixed in the flow direction, but the uniformity of mixing can be affected by fluid velocity and fluid dynamics. Fig. 5B uses triangular mixing to form a triangular mixing region that provides a more uniform mixing effect than orthogonal mixing because the junction of the three fluid streams increases the contact area between the fluids.

Fig. 5C shows the distribution of the particles in the flow channel after mixing, and the distribution of two different sized particles (5 microns and 1 micron) in the flow channel, which exhibit a gaussian distribution shape. The gaussian distribution shows that the concentration of particles is highest in the center of the flow channel and gradually decreases towards both 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.

Figure 5D illustrates a three stage separation process, referring to the use of three different flow channels for distinguishing blood cells (greater than 1 micron in size) from sub-micron particles. Blood cells, due to their larger size, can be separated from smaller submicron particles by specific structures or obstructions in the microfluidic channel.

Fig. 5E shows a five-stage separation, a more complex separation process with five different flow channels for distinguishing blood cells, extracellular vesicles and biomolecules. Blood cells (orange) are first isolated due to their larger size. Extracellular vesicles (white) and biomolecules (grey) require finer separation techniques.

In fig. 5F, the position of the buffer used to form the baseline for the assay is shown.

Preferably, the detection part is a hexagonal detection cavity, and is connected with channels at two sides of the collection ports with different particle diameters through a right-angle bent flow channel, and the space structure of the detection cavity comprises 10-12mm in length, 2-4mm in width and 0.04-0.4mm in height.

Preferably, the integrated chip test sample size of the embodiments of the present disclosure is 200 μl.

Preferably, the integrated chip of the embodiments of the present disclosure requires dilution of the whole blood sample five times as much as it was, avoiding excessive viscosity.

Further, in practice, the detection chamber may be designed into an oval, circular, hexagonal or square structure according to needs, and the volume of the liquid contained therein may be adjusted according to the actual monitoring needs, for example, a microfluidic detection chamber of 50 μl, and the position of the sensing layer in the detection chamber may be a bottom surface, a top surface, or an intermediate layer of the chamber, as shown in fig. 6A, B. Taking the detection of inflammatory factors as an example, a sensing chip can be arranged on the bottom surface of the sensing cavity fluid, the surface is modified by specific biomolecules such as biotinylated antibodies, and the phase change is realized in the SPR sensor by the specific binding principle among molecules such as antigen antibodies, as shown in fig. 6C. Namely, SPR detection in the detection cavity needs to be provided with a thin film structure, and the thin film structure can be distributed at the bottom, the top and the middle of the cavity, so long as the thin film structure is combined with separated liquid.

Preferably, the glass sheet is combined with colloid, and the glass sheet part corresponding to the detection cavity part is plated with the surface plasmon resonance sensing material.

Fig. 6 illustrates the principle of biosensor detection based on antigen-antibody reactions, typically used to detect the presence of specific biomolecules. The three parts in the figure represent the sample introduction, the immobilization of the biological recognition element and the antigen-antibody binding process, respectively. Sample introduction of fig. 6A, and sample fixation of a biological recognition element, fig. 6B, the sample flows through a region with a surface to which the biological recognition element, such as an antibody, is immobilized. These biological recognition elements (shown as wavy lines in the figure) are specific, meaning that they bind only to a specific antigen.

Figure 6C shows antigen-antibody binding, where antigen molecules in the sample bind specifically to antibodies as they flow through the region where the antibodies are immobilized. The figure shows the binding of a plurality of antigen molecules (Y-shaped structures) to antibodies (inverted triangle structures). Such binding may result in the generation of a signal, such as an optical signal (color change), an electrical signal (current or voltage change), or other physical change, which may be detected and used to quantitatively analyze the concentration of antigen in the sample.

The sample liquid flow process described above (as shown in FIG. 7) is such that inlet 01-a split up-down triangular or square flow channel-is mixed with buffer at inlet 02 (FIG. 3B) -a particle distribution forming section (FIG. 3 c) -different particle size collection ports (03, 05 in FIG. 3; right side outlet in FIGS. 5D, 5E) -target size flows into the detection chamber (FIG. 3F).

Fig. 7 shows a flow of blood sample processing and analysis based on a microfluidic chip. The process comprises the following steps:

s701, adding a blood sample into a chip system;

S702, diluting the blood sample;

s703, introducing the diluted blood sample and a buffer solution into a microfluidic chip;

S704, in a chip, separating different components in a blood sample, such as cells, proteins, extracellular vesicles and the like, by a microfluidic technology;

S705, the separated sample components are guided to a detection area;

s706, quantitatively analyzing the target molecule based on the detection result to determine the concentration of the target molecule in the sample. S707, after the sample is processed, the waste liquid and the used chip need to be properly processed to avoid environmental pollution and cross contamination.

According to the embodiment of the disclosure, the integrated combination of passive biological particle separation microfluidic and plasmon label-free sensing detection is realized, separation is realized through the difference of elastic force, viscous resistance and inertial lift force of particles with different sizes, and the integration of separation detection is realized through the connection of a compact flow channel structure and a detection cavity, so that the problems of long separation time, easy cell breakage and low detection sensitivity, large occupied area, long detection time and high cost of the enzyme-linked immunosorbent assay technology of the traditional separation method are overcome. Not only meets the requirement of simultaneously completing whole blood separation and inflammatory factor detection on one chip, but also improves the operability of the product, and the accuracy and instantaneity of the result.

The embodiment of the disclosure has the advantages of good separation effect, good detection sensitivity, capability of drawing a standard curve of inflammatory factors and performing comparison, shortening the time of the whole detection process and reducing the equipment cost of inflammatory factor detection, wherein most of biological macromolecules such as red blood cells, white blood cells and the like are separated under a microscope in whole blood test. The biological detection chip of the embodiment of the disclosure can be applied to instant inflammatory factor detection in operation or long-time monitoring of the inflammatory factor level of a patient in clinic, can make up for the blank in the field of separation and detection integrated instant inflammatory factor detection, dynamically monitors the inflammatory factor level of the patient, judges the occurrence of acute tissue injury or inflammatory reaction, helps doctors to adjust treatment strategies in time, improves the survival rate of the patient and is beneficial to postoperative rehabilitation of the patient.

According to one or more embodiments, a separation detection integrated microfluidic chip, wherein the channel width on the chip is 20 μm or more and the channel height is 40 μm or more. The diameter of the inlet and outlet of the flow channel is 40-50 μm, and the design of the size is related to different inlet sample flow speed targets. To create the desired viscoelastic environment, the buffer was a 0.1% volume fraction mixed solution of PEO dissolved in 1xPBS solution, with the corresponding viscoelastic properties. The flow rate of the inlet 01 is 180-220 mu L/h, and the flow rate of the inlet 02 is 1900-2100 mu L/h. The viscoelastic environment forming part is formed by a sample flow channel in the middle and buffer flow channels at two sides, and the included angles between the two side flow channels and the middle flow channel can be right angles to form a square structure or acute angles to form a triangle structure. The length of a middle long runner formed by particle distribution is more than 32mm, and the corresponding Gaussian distribution is formed according to the difference of the radius to cause the difference of physical force fields. The middle ratio of the three-fork structure of the collecting ports with different particle diameters is 40% -50%, and the distance between the two parallel tips and the outlet of the long flow passage is 20-30 μm. The detection cavity structure is hexagonal or elliptic. Special glass sheets with plasma sensor material spray coating are used as the substrate of the chip. The blood sample needs to be diluted five times of the original sample, and the required sample size of the chip is 200-400 mu L.

The separation and detection integrated biochip disclosed by the invention is very widely applied to the field of sample detection. The chip is based on a surface plasmon label-free detection technology and is suitable for rapid analysis of blood samples of patients in operation. The chip comprises a specific micro-fluid channel structure, and can rapidly separate particles with different particle diameters in a blood sample of a patient in operation by utilizing a micro-fluid technology of viscoelasticity and particle inertial focusing. The surface plasmon sensor is integrated on the chip, and can perform label-free detection on specific biomolecules in the separated blood sample so as to meet the requirement of rapidly acquiring detection results in operation.

For example, in neurosurgery, specific proteins in blood are detected, and the brain nerve damage condition is estimated, so that a basis is provided for accurate operation of the operation. In heart operation, biomarkers such as myocardial enzymes in blood of a patient can be detected in real time, so that doctors can judge the myocardial injury degree and adjust an operation scheme in time. Aiming at the complexity of the blood sample of the patient in operation, the separation technology of the chip can effectively remove the interference objects and improve the detection accuracy. For another example, blood cells and plasma can be rapidly separated and specific biomarkers associated with surgery can be accurately detected. In the operation process, the time is crucial, and the integrated chip can process and detect the blood sample of a patient in a short time, so that timely and accurate information is provided for operation decision.

In summary, the separation and detection integrated microfluidic biochip disclosed by the disclosure is mainly characterized by comprising:

1. The structure of integration of separation-detection is adopted, and the structure integrates two independent steps of separation and detection on one chip traditionally, so that the detection efficiency and convenience are greatly improved. Meanwhile, the structure also comprises a specific micro-fluid channel layout for separating particles with different particle diameters, a sensor position for detecting biomolecules, a connection mode and the like, so that efficient collaborative operation of separation and detection functions is ensured.

2. The method can separate particles in complex samples such as blood and the like rapidly and accurately in a microfluidic environment, and provides a pure sample for subsequent detection. The protection separation method optimizes the utilization mode of particle inertia action, the control parameters of fluid flow in a micro-fluid channel and the like based on the characteristics of viscoelastic fluid, and ensures the stability and reliability of separation effect.

3. And the surface plasmon label-free detection is combined, so that the rapid and sensitive detection of specific biomolecules is realized. The detection method does not need a marker, reduces sample processing steps, and improves the accuracy and speed of detection.

The integrated plasma sensing chip for separation and detection is manufactured by a microfluidic preparation technology, can realize continuous separation and detection of a complex biological sample system, reduces equipment volume, reduces sample consumption and personnel operation, and realizes an integrated and microminiaturized high-separation-detection system. The separation part micro-flow channel adopts a passive biological particle separation micro-flow control technology, utilizes the principle of inertia force and fluid dynamics to cause the difference of the elastic force, viscous resistance and inertial lifting force of particles with different sizes, and when the particles enter a particle distribution forming part, all the particles with all the sizes are initially arranged near the side wall of the channel. As they move along the channel, a magnitude dependent force acts on the particles, resulting in different lateral migration paths. The particle diameter gradually decreases from the middle to two sides of the flow channel, and then the separation of particles is realized through a bifurcation structure. The detection part micro-flow channel disclosed by the disclosure utilizes the plasmon label-free sensing technology, utilizes the biotinylation modification of the detection chip surface in the detection cavity, realizes capturing through combination of specificity and biological particles, forms the change of optical phase, and realizes rapid detection of corresponding biological molecules after comparison with a standard curve.

Therefore, the beneficial effects of the present disclosure include:

1. Solves the problem of poor sample processing capability

The integrated detection chip can be used for rapidly and effectively processing and analyzing complex samples such as blood and the like through a coupling technology and an integrated design of micro-fluidic and biological sensing. The specific microfluidic structure and hydrodynamic mechanism inside the chip can remove impurities and interferents in the sample in a short time, so that the accuracy of subsequent detection is ensured. Meanwhile, the chip can be optimized and adjusted according to different types of complex samples, personalized sample processing and screening are achieved, and processing flexibility and adaptability are improved.

2. Improving the condition of weaker separating ability

In the processing of blood samples, the chip of the present disclosure enables rapid separation of different components in an efficient manner. The unique separation technology can meet the requirement of rapid separation under emergency conditions, effectively separate nanoscale components such as blood cells, cell vesicles and biomolecules in blood plasma in time, and create good conditions for detection of specific biomolecules. And the separation effect of the chip is stable and reliable, and the risk of incomplete separation or incorrect separation is greatly reduced, so that the reliability of the detection result is improved.

3. Improving the situation of limited sensing capability

For specific biomarkers in complex samples, the integrated detection chip of the present disclosure has high sensitivity biosensing capability. The method can accurately capture the low-concentration biomolecule signals and effectively avoid the condition of missing detection or false detection. Meanwhile, the sensing response time of the chip is extremely short, so that the real-time biomolecule monitoring can rapidly give out a detection result in an emergency situation, and timely and effective support is provided for clinical decision.

4. Enhancing application scenario adaptability

Under critical situations such as ICU, operating room, first aid, etc., the chip of the present disclosure exhibits excellent adaptability. The separation and detection functions are integrated, so that the manual operation amount is reduced, and the device is very suitable for environments such as an operating room. The chip can be in seamless connection with other medical equipment and processes in the scenes, and the complexity and time cost of operation are reduced. In addition, the integrated design enables the chip to occupy a small area, and an instrument which occupies a large area and needs maintenance is not needed like the traditional detection technology.

5. Reducing errors in detection

The chip disclosed by the disclosure can effectively reduce sample loss in the sample processing and detecting process. The optimized design and operation flow greatly reduce the breakage of the sample in the links of separation, transfer, detection and the like, thereby reducing the requirement on the initial sample size. At the same time, reducing the sample loss also means that the error in the detection process is reduced. In addition, the integrated design of the detection chip simplifies the detection steps, the pretreatment process is simpler and more efficient, and the detection time is shortened. In addition, the integrated design also reduces the detection cost and avoids the problem of high cost caused by using different devices and equipment.

It should be understood that, in the embodiment of the present invention, the term "and/or" is merely an association relationship describing the association object, which means that three relationships may exist. For example, A and/or B may mean that A alone, both A and B, and B alone are present. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.

It is to be understood that while the spirit and principles of the invention have been described in connection with several embodiments, it is to be understood that this invention is not limited to the specific embodiments disclosed nor does it imply that the features of these aspects are not combinable and that such is for convenience of description only. The invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims (10)

1. A bioassay chip, characterized in that the chip comprises a substrate, and the components fabricated on the substrate include: 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 , wherein the biological sensing detection unit is a surface plasmon sensing unit. 3 . The biological detection chip according to claim 1 , wherein the microfluidic separation unit comprises 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 diversion portion is a multi-branch separation 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 , wherein the detection portion has a detection cavity structure, and the detection cavity is in close contact with the biological sensing detection unit. 6 . The biological detection chip according to claim 2 , wherein the biological sensing 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 biological samples 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 comprises: 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.