Microfluidic protein expression purification and affinity detection chip, system and method

Abstract

The application discloses a kind of microfluidic protein expression purification and affinity detection chip, system and method, the chip includes the sample loading area, protein expression area, magnetic bead purification area, phase transfer transition area, LSPR detection area and waste collection area in turn in the direction of fluid travel Serially arranged.The system includes chip, motor drive module, temperature control module, optical spectrum detection module, magnetic field control module and data processing module.The method includes reagent loading, cell-free protein expression, magnetic bead purification, magnetic bead oil phase transfer and local surface plasmon resonance, protein and ligand binding and affinity kinetics detection and the like steps.The application is integrated by digital microfluidic droplet control, magnetic bead enrichment purification and local surface plasmon resonance detection technology, and can obtain the kinetic parameter such as binding rate constant, dissociation rate constant and equilibrium dissociation constant of target molecule in real time.

Description

Technical Field

[0001] This invention relates to the field of biochip and microfluidics technology, specifically to a microfluidic protein expression, purification, and affinity detection system, chip, and method. Background Technology

[0002] Characterizing the affinity of proteins for biomolecules such as antibodies is a core step in drug discovery and biotherapy development. Currently, commonly used molecular interaction analysis techniques include surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR), and biolayer interferometry (BLI). Among these, LSPR technology has advantages such as no need for fluorescent labeling, fast detection speed, low sample consumption, and ease of chip integration, making it a promising area for protein interaction analysis.

[0003] However, existing LSPR detection platforms typically require target protein expression, centrifugation purification, concentration, and sample transfer before introducing the purified protein into the detection chip for binding analysis. This entire process involves multiple independent instruments and manual steps, which is not only time-consuming but also prone to sample loss, contamination, and batch-to-batch variability, thus affecting the accuracy and reproducibility of the results. Furthermore, cell-free protein expression technology can rapidly obtain target proteins in vitro, avoiding the complexity and long cycles of cell culture. However, existing cell-free expression platforms lack effective integration with subsequent affinity detection equipment, and the expressed products still require manual transfer and processing, making automated continuous detection difficult.

[0004] Digital microfluidics is a technology that uses the electrowetting effect to manipulate discrete droplets, enabling the generation, transport, mixing, splitting, and reaction control of droplets. It offers advantages such as low reagent consumption, high automation, and ease of integration. In recent years, digital microfluidics has been applied in fields such as nucleic acid detection, immunoassay, and cell experiments; however, integrated solutions covering the entire process of protein expression, sample purification, and affinity detection remain relatively rare.

[0005] Therefore, there is an urgent need to develop a digital microfluidic system that can integrate protein expression, magnetic bead purification, and LSPR affinity detection into the same platform, so as to realize continuous automated processing of target proteins from expression, purification to detection, thereby improving detection efficiency, reducing sample consumption, and enhancing the consistency and reliability of experimental results. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the independence of protein expression, sample purification and affinity detection, complex operation procedures, low degree of automation and large sample loss, and to provide a microfluidic protein expression purification and affinity detection chip, system and method.

[0007] To achieve the above objectives, the present invention provides a microfluidic protein expression, purification, and affinity detection chip in a first aspect, comprising: An upper electrode plate and a lower electrode plate, wherein a droplet operating space is formed between the upper electrode plate and the lower electrode plate; An electrode array disposed on the lower electrode plate; The electrode array is arranged sequentially along the droplet flow direction as a sample loading area, a protein expression area, a magnetic bead purification area, a phase transition area, an LSPR detection area, and a waste liquid collection area.

[0008] Furthermore, the protein expression region is used for cell-free protein expression in an oil-sealed environment; The magnetic bead purification zone is used to capture target proteins using functionalized magnetic beads; The phase transition region is provided with a magnetic bead cross-phase transfer pathway, which is used to drive magnetic beads carrying the target protein through the oil-water interface into the clean aqueous phase. The LSPR detection area is equipped with a localized surface plasmon resonance sensing structure. Furthermore, the LSPR detection region includes a glass substrate, a gold reflective layer, a second dielectric layer, a surface modification layer, and a gold nanostructure array; Furthermore, a thermal isolation structure is provided between the protein expression region and the LSPR detection region to reduce thermal interference of the protein expression region on the LSPR detection region; Furthermore, the thermal insulation structure includes one or more of the following: thermal insulation groove, air gap layer, and low thermal conductivity material layer.

[0009] Preferably, the surface modification layer is used to immobilize ligand molecules and realize the detection of binding between the target protein and the ligand.

[0010] In a second aspect, the present invention provides a microfluidic protein expression, purification, and affinity detection system, the system comprising: the chip as described above, and further comprising a motor drive module, a temperature control module, a magnetic field control module, a spectral detection module, and a data processing module; The motor drive module is electrically connected to the electrode array of the chip and is used to control the movement of the droplet between the functional areas; The temperature control module is thermally coupled to the protein expression region of the chip and is used to control the working temperature of the protein expression region; the magnetic field control module corresponds spatially to the magnetic bead purification region and the phase transition region of the chip and is used to control the magnetic bead capture and cross-phase transfer process. The spectral detection module is positioned above the LSPR detection area of ​​the chip and is used to acquire the spectral signal of the LSPR detection area in real time. The data processing module is electrically connected to the spectral detection module, the motor drive module, the temperature control module, and the magnetic field control module, and is used to analyze the detection results. The data processing module controls the ligand fixation endpoint according to the LSPR detection signal and controls the subsequent affinity detection process.

[0011] In some embodiments, the temperature control module includes a temperature controller and a heating unit, with a temperature control range of 20–45°C and a temperature control accuracy of ±0.5°C.

[0012] In some embodiments, the data processing module stops the ligand fixation process when the LSPR response signal reaches a preset fixed amount threshold, and the data processing module controls multiple analytes of different concentrations to be delivered sequentially to the LSPR detection area to perform single-cycle kinetic detection.

[0013] In a third aspect, this invention provides a method for protein expression purification and affinity detection, the method comprising the following steps: S1. Add protein expression-related reagents to the sample loading area; S2. Complete the expression of the target protein in the protein expression region; S3. Functionalized magnetic beads are used in the magnetic bead purification zone to capture the target protein and complete the purification. S4. In the phase transition region, magnetic beads carrying the target protein are transferred to the LSPR detection region via the magnetic bead cross-phase transfer pathway. S5. Ligand fixation is completed in the LSPR detection area; S6. Detect the binding process between the target protein and the ligand in the LSPR detection region; S7. Obtain the binding kinetic parameters between the target protein and the ligand.

[0014] In some embodiments, a cell-free protein expression system is used for protein expression in step S2.

[0015] In some embodiments, functionalized magnetic beads are used in step S3 to capture target proteins with affinity tags.

[0016] In some embodiments, step S6 uses a single-cycle kinetic detection mode to continuously inject samples of different concentrations and obtain the binding rate constant, dissociation rate constant, and equilibrium dissociation constant between the target protein and the ligand.

[0017] Compared with the prior art, the technical solution disclosed in this invention has the following beneficial effects: This invention integrates protein expression, sample purification, ligand immobilization, and affinity detection into a single digital microfluidic chip, enabling continuous processing from sample preparation to result output and reducing intermediate transfer steps. It utilizes an electrode array to automate droplet generation, transport, mixing, and reaction control, reducing errors from manual operation and improving experimental repeatability. Employing a micro-to-nano-level droplet reaction system significantly reduces the consumption of protein samples, reagents, and ligands, lowering experimental costs. Protein expression products, after purification with magnetic beads, can be directly transferred to the LSPR detection area for analysis, eliminating the need for offline processing and manual transfer, thus shortening the overall detection time. The sensitive response of the LSPR nanostructure to local refractive index changes enables real-time monitoring of the target protein-ligand binding process. By setting up a thermal isolation structure, heat conduction between the protein expression region and the detection region is reduced, improving the stability and accuracy of affinity detection results. The magnetic bead purification and transfer mechanism achieves target protein enrichment and impurity removal, improving the quality of subsequent detection signals. It can acquire the binding and dissociation processes between the target protein and ligand in real time, obtaining kinetic parameters such as binding rate constant, dissociation rate constant, and equilibrium dissociation constant. Attached Figure Description

[0018] Exemplary embodiments of the present invention can be more fully understood by referring to the accompanying drawings. The drawings are provided to further illustrate the embodiments of this application and form part of the specification. They are used together with the embodiments of this application to explain the present invention and do not constitute a limitation thereof. In the drawings, the same reference numerals generally represent the same components or steps.

[0019] Figure 1 This is a block diagram illustrating the module connections and control principle of the microfluidic protein expression, purification, and affinity detection system provided in Embodiment 1 of the present invention. Figure 2 This is a schematic diagram of the microscopic layered stacking structure of the chip in the vertical direction provided in Embodiment 2 of the present invention; Figure 3 This is a schematic diagram of the planar functional partitioning and droplet transport path of the chip provided in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the vertical microstructure and surface functionalization arrangement of the micro / nano enhanced sensing chip inside the LSPR detection region provided in Embodiment 2 of the present invention. Figure 5 This is a scatter plot showing the correlation trend of KD between the chip platform of this invention and the commercial SPR detection platform in Embodiment 4 of this invention.

[0020] Figure Labels 1-Chip; 2-Motor drive module; 3-Temperature control module; 4-Spectroscopic detection module; 5-Magnetic field control module; 6-Data processing module; 101-Sample loading area; 102-Protein expression area; 103-Magnetic bead purification area; 104-Phase transition area; 105-LSPR detection area; 106-Waste liquid collection area; 11-Upper electrode; 12-Fluid layer; 13-Lower electrode; 131-Bottom layer; 132-Substrate layer; 133-Electrode layer; 134-First dielectric layer; 19-Gold nanostructure array; 20-Surface modification layer; 21-Second dielectric layer; 22-Gold reflective layer; 23-Glass substrate. Detailed Implementation

[0021] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the present application.

[0022] Those skilled in the art will understand that the terms "first," "second," etc., used in the embodiments of this application are only used to distinguish different steps, devices, or modules, and do not represent any specific technical meaning, nor do they indicate a necessary logical order between them. It should also be understood that in the embodiments of this application, "multiple" can refer to two or more, and "at least one" can refer to one, two, or more. Furthermore, the term "and / or" in this application is merely a description of the relationship between related objects, indicating that three relationships can exist. Techniques, methods, and devices known to those skilled in the art are not discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification.

[0023] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "digital microfluidics (DMF)" refers to microfluidics technology that uses the principle of electro-wetting on dielectric (EWOD) to apply voltage to an electrode array on the surface of a chip to drive nano- to micro-level droplets for generation, routing, merging, splitting, and mixing.

[0024] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "cell-free protein expression (CF)" refers to a technique that uses DNA or mRNA as a template to directly synthesize target proteins in an in vitro reconstructed transcription-translation system without the participation of living cells, and the reaction volume can be compressed to nanoliters, significantly shortening the turnaround time compared to cell culture protocols.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "local surface plasmon resonance (LSPR)" refers to the resonant absorption phenomenon generated by the collective oscillation of local surface plasmons in a metal nanostructure (such as a gold nanostructure array) under incident light irradiation. The resonance wavelength is extremely sensitive to the refractive index change near the sensing surface and can be used for real-time, label-free monitoring of biomolecule binding-dissociation dynamics.

[0026] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "single-cycle kinetics (SCK)" refers to a detection mode in which multiple analyte solutions of increasing concentrations are sequentially introduced in a single experimental run, without a sensor surface regeneration step between each concentration, and finally, a global fitting model is used to extract all kinetic parameters at once. Compared with the traditional multi-cycle regeneration mode, SCK eliminates the cumulative damage to the sensor surface caused by the regeneration step, while shortening the effective detection time.

[0027] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "closed-loop ligand fixation control" refers to using the real-time spectral detection signal of the LSPR sensing region as feedback input to determine the fixation density of the ligand on the sensing surface in real time. When the fixation density reaches a preset condition (the response value reaches a predetermined percentage of the target value or the response value growth rate is lower than a preset slope threshold), the routing of ligand droplets to the sensing region is automatically stopped, thereby achieving adaptive and highly uniform control of the ligand fixation density.

[0028] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "thermal isolation structure" refers to a thermal insulation structure (such as a thermal isolation groove or thermal insulation gap) disposed between the protein expression region (approximately 37°C) and the LSPR detection region (approximately 25°C), used to block or significantly reduce heat conduction between the two regions, reduce temperature crosstalk in the LSPR detection region, and thereby reduce baseline drift.

[0029] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the term "magnetic bead transfer through oil phase" refers to the use of the magnetic field of an external permanent magnet array to pull surface-functionalized magnetic particles (magnetic beads) to carry the captured target protein from the oil phase encapsulation environment across the oil-water phase interface into a pre-placed clean aqueous buffer solution, thereby achieving a contactless, low-residue medium switching technology for reaction products from the oil phase system to the all-aqueous phase detection environment.

[0030] It should also be understood that, for ease of description, the dimensions of the various parts shown in the accompanying drawings are not drawn to actual scale. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this application or its application or use. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0031] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0033] Example 1 refer to Figures 1 to 3 This embodiment provides a microfluidic protein expression purification and affinity detection system. The system takes chip 1 as its core and integrates microfluidic control, physical field regulation and high-precision label-free optical detection to achieve fully automated control of the entire process from protein synthesis to molecular dynamics analysis.

[0034] like Figure 1 As shown, the microfluidic protein expression purification and affinity detection system of the present invention is integrated by the following functional modules: Motor drive module 2: Electrically connected to electrode layer 133 of chip 1, responsible for controlling the precise distribution and mixing of droplets. Motor drive module 2 adopts a high-voltage multiplexer circuit and an AC drive source, with the output voltage preferably being an AC drive signal of 15-40V, used to precisely control the generation, movement, merging, splitting, and high-frequency reciprocating oscillation of aqueous droplets inside the chip.

[0035] Temperature control module 3: Thermally coupled to the heating and temperature sensing elements at the bottom of chip 1, it is used to maintain the optimal temperature environment for local biochemical reactions. In this embodiment, temperature control module 3 includes a micro feedback controller and a thin-film microheater, achieving a temperature control range of 20–45°C and a temperature control accuracy of ±0.5°C.

[0036] Spectral detection module 4: Located above the local surface plasmon resonance (LSPR) sensing area of ​​chip 1, it integrates a broadband white light source, a high-resolution reflective fiber-coupled probe, a miniature spectrometer, and a signal acquisition circuit. It is used to dynamically acquire the reflection spectrum changes at the sensing point and extract the wavelength shift signal of the characteristic resonance valley.

[0037] Magnetic field control module 5: Located below chip 1, it includes a high-energy permanent magnet array (such as NdFeB strong magnet) and a vertical / horizontal precision electric displacement stage connected to it. It realizes the loading, removal and movement of local magnetic fields through programmed physical displacement, thereby realizing the capture, aggregation, cleaning and cross-regional transfer of on-chip paramagnetic beads.

[0038] Data Processing Module 6: As the central processing and control core of the entire system, it is electrically connected to the spectral detection module 4, motor drive module 2, temperature control module 3, and magnetic field control module 5. Data Processing Module 6 embeds self-developed fluid-coordinated control and optical data analysis software, responsible for real-time background correction of the reflectance spectrum, reference channel correction, centroid tracking algorithm, online fitting of dynamic curves, and automatic calculation of the equilibrium dissociation constant KD. It also issues timing commands to each control module based on real-time feedback data.

[0039] refer to Figure 2 and Figure 3 By regularly arranging and planning the paths of the electrode array in the electrode layer 133 within the chip, the space inside the chip 1 for droplet movement is divided into six interconnected functional areas on a plane: Sample loading area 101: Located on the far left of chip 1, it contains multiple large-area liquid storage electrodes and sample introduction channels, which are used to introduce and store DNA templates, cell-free expression system reagents, reaction buffers, magnetic bead suspensions, elution buffers, and multiple series of gradient antigen samples, respectively.

[0040] Protein expression region 102: Adjoining the right side of sample loading region 101, this region is pre-filled with oil phase. The substrate below protein expression region 102 integrates a thin-film microheater and a temperature sensor to maintain this local area at a constant temperature of 37±0.5℃, thereby providing an extremely stable biosynthetic microenvironment for cell-free protein expression reactions.

[0041] Magnetic bead purification zone 103: Located to the right of protein expression zone 102, its electrode surface corresponds vertically to the magnetic field control module 5 below. Through the combined control of alternating electrical signals driving the driving electrode and an external magnetic field, this zone is used to complete the specific capture of the target tagged protein by paramagnetic magnetic beads, washing and removing non-specific impurities, and elution.

[0042] Phase transition region 104: This phase transition region 104 is physically positioned as a thermal isolation structure between the protein expression region 102 and the detection region on the right. An air isolation groove (preferably 1 mm wide and 500 μm deep) is formed in the chip substrate layer of this phase transition region 104 to cut off the lateral heat conduction path and prevent heat from the 37°C expression region from diffusing into the detection region. The phase transition region 104 also includes a magnetic bead transfer pathway for driving the transfer of magnetic beads encapsulating the target protein across the boundary in a fully enclosed oil phase environment.

[0043] In other embodiments of the present invention, the thermal isolation structure may also employ an air gap layer. Specifically, the air gap layer is a lateral air gap layer formed between the upper electrode 11 and the lower electrode 13, located between the protein expression region 102 and the LSPR detection region 105. This air gap layer, by etching a recessed cavity structure of a certain depth on the upper surface of the substrate layer 132, increases the thickness of the fluid layer 12 in this local area. Utilizing the low thermal conductivity of air, the lateral thermal resistance is significantly increased, thereby effectively blocking the conduction of heat from the protein expression region 102 to the LSPR detection region 105. Compared to thermal isolation trenches, the air gap layer, by increasing the cross-sectional area of ​​the air-filled region, can provide higher thermal isolation efficiency while maintaining the integrity of the chip structure.

[0044] In some embodiments of the present invention, the thermal isolation structure may further employ a low thermal conductivity material layer, which is a low thermal conductivity solid material layer disposed on the upper surface of the substrate layer 132 between the protein expression region 102 and the LSPR detection region 105, or embedded within the substrate layer 132. The low thermal conductivity material may be selected from one or more of polyimide (PI), polydimethylsiloxane (PDMS), SU-8 photoresist, or silica aerogel.

[0045] The three thermal isolation structures mentioned above (thermal isolation groove, air gap layer, and low thermal conductivity material layer) can be used individually or in combination depending on the chip fabrication process conditions and specific requirements for thermal isolation performance. All of them can effectively reduce the thermal interference of the protein expression region to the LSPR detection region.

[0046] LSPR detection region 105: Located on the right side of chip 1, it is the core label-free optical sensing point. This region integrates a periodic micro / nano metallic configuration, with a specific trapping layer on the surface, to monitor molecular binding and dissociation dynamics by exciting local surface plasmon resonance effects.

[0047] Waste liquid collection area 106: Located at the far right edge of the chip, it consists of a large-size hydrophilic electrode and a waste discharge channel, and is used to promptly and forcibly absorb and lock all waste aqueous droplets generated during the entire purification, washing and detection process.

[0048] like Figure 2As shown, the entire chip 1 adopts a multi-layer stacked dual-plate composite configuration in the vertical direction. Its specific micro-multilayer stacked structure from top to bottom is as follows: Upper electrode 11: Located at the top layer, it uses high-transmittance optical-grade quartz glass or polymethyl methacrylate (PMMA) as a cover plate. A thin layer of indium tin oxide (ITO) is sputtered onto the lower surface of the upper electrode 11 as a common reference electrode grounding layer, and a perfluorinated hydrophobic layer is spin-coated onto the ITO surface. In this embodiment, the perfluorinated hydrophobic layer uses either Teflon AF or CYTOP film, with a thickness ranging from 50 to 100 nm.

[0049] Fluid layer 12: Located in the gap formed between the upper and lower electrodes arranged in parallel, the spacing of which is precisely limited to 50-300 μm by a control gasket, preferably 150 μm in this embodiment. The entire gap space is filled with a low-viscosity, highly biocompatible oil phase. In this embodiment, fluorinated oil FC-40 or HFE-7500 is used. Aqueous droplets are suspended in a closed manner within this space, effectively isolating them from external cross-contamination and eliminating droplet evaporation.

[0050] The lower electrode 13 includes a substrate layer 132 and an electrode layer 133 formed on the upper surface of the substrate layer, as well as a first dielectric layer 134 covering the electrode layer 133. The electrode array is formed in the electrode layer 133. The bottom surface of the lower electrode 13 is also provided with a bottom plate layer 131. In some embodiments, the bottom plate layer 131 integrates a micro heater and a temperature sensor.

[0051] The first dielectric layer 134 covers the electrode layer 133 and serves as an insulating dielectric layer to prevent electrical breakdown. The first dielectric layer 134 is prepared by chemical vapor deposition (CVD) and is made of one or more of SiO2, Si3N4, Parylene C, and SU-8. In this embodiment, a Parylene C film with a thickness of 2.0–2.5 μm is used, which possesses excellent density, high dielectric constant, and electrical breakdown resistance. A perfluorinated hydrophobic layer is uniformly coated on the uppermost surface of the first dielectric layer 134 to significantly reduce fluid slip resistance.

[0052] Electrode layer 133: Fabricated on the upper surface of substrate layer 132, it is made of chromium / gold (Cr / Au) or metallic copper (Cu) using microelectronic photolithography and acid etching processes to form an independent driving electrode array with a thickness of 3μm and a jigsaw staggered edge. The spacing between adjacent electrodes is controlled at 30μm to ensure the smoothness and continuous control of the droplet when it moves across the electrodes.

[0053] Substrate layer 132: As the framework supporting the micro-nano structures of each layer of the lower electrode plate, it is made of FR-4 epoxy resin printed circuit board or soda-lime glass substrate with high insulation and excellent surface flatness.

[0054] The substrate layer 131 is bonded and integrated on the bottom surface of the substrate layer 132. Within the local physical space of the corresponding protein expression region 102, it integrates a serpentine-arranged thin-film platinum (Pt) resistance microheater and a high-sensitivity thermistor temperature sensor through microfabrication. It directly forms an electrothermal closed-loop feedback loop with the temperature control module 3 through an external cable.

[0055] Example 2 refer to Figure 4 The LSPR detection region 105, located on the right side of chip 1, integrates a high-sensitivity, label-free optical plasma-enhanced sensing chip on the bottom side of the fluid layer 12. Its internal components, from bottom to top, include: Gold reflective layer 22: Densely deposited on the surface of glass substrate 23, with its thickness precisely controlled between 80 and 120 nm. The highly dense reflective gold film not only serves as the bottom reflective optical mirror, significantly enhancing the reception efficiency of backscattered / reflected spectra, but also acts as a physical support and electric field enhancer.

[0056] The second dielectric layer 21 is deposited on top of the gold reflective layer 22 and has a thickness of 20–100 nm. This layer acts as a uniform optical medium transition layer and a barrier layer, effectively tuning the distribution of the near-field electromagnetic field and optimizing the line shape and full width at half maximum (FWHM) of the local surface plasmon resonance absorption peak in the reflection spectrum.

[0057] Gold nanostructure array 19: Regularly distributed on the second dielectric layer 21 using electron beam lithography (EBL) or high-precision nanoimprinting (NIL) techniques. The geometric dimensions of a single gold nanostructure array 19 are precisely controlled as follows: diameter 100–150 nm, height 30–50 nm, and array period 200–300 nm. Under vertical illumination with white light of a specific wavelength, this periodic nanodisk structure can excite an extremely strong localized surface plasmon resonance (LSPR) electromagnetic field. This near-field electromagnetic wave is extremely confined to a space of tens of nanometers in proximity to the disk surface, exhibiting extreme sensitivity to changes in the refractive index of the external medium.

[0058] Surface modification layer 20: A tightly self-assembled modification on the metallic outer surface of the gold nanostructure array 19. A dense monolayer with carboxyl group (-COOH) terminals is constructed on the nanosurface by spontaneously forming a stable Au-S covalent bond between an alkyl thiol (such as 11-mercaptoundecanoic acid) and the gold surface. This self-assembled monolayer is treated with EDC / NHS-activated droplets before detection for covalent coupling with Protein A / G or antibodies, achieving selective capture of target molecules. When a biorecognition reaction occurs, the microscopic refractive index of the near-field surrounding the gold nanostructure array 19 shifts, resulting in a clear redshift of the resonance characteristic valley. This shift signal is rapidly read by the upper spectral detection module 4.

[0059] Example 3 This embodiment, based on the microfluidic protein expression, purification, and affinity detection system described in Embodiments 1 and 2, details the on-chip fully automated microfluidic and biochemical operation process using the in-situ synthesis and purification of His-tagged nanobodies (VHH-His protein) in a cell-free wheat germ / E. coli expression system as an example: Fully automated in situ expression: Data processing module 6 and motor drive module 2 output a 30V AC drive signal. At the edge of the independent reservoir corresponding to sample loading area 101, the generation of plasmid DNA template droplets, cell-free system reaction droplets, and reaction buffer droplets with a volume of 200-500 nL is controlled.

[0060] The motor drive module 2 drives the three types of droplets to be transferred to the protein expression region 102 through the cross matrix electrode channel according to the preset timing sequence, and spontaneously merges into a single reaction droplet under the coating of the oil phase (FC-40 fluorinated oil) (total volume controlled at 600-1200 nL).

[0061] Temperature control module 3 activates the local microheater in the substrate layer 131, locking the core temperature of the protein expression region 102 at 37±0.5℃ via the substrate layer 132. The reaction droplets are incubated under this constant temperature condition for 2–4 hours, efficiently transcribing and translating the target VHH-His protein. During this process, due to the physical insulation of the phase transition region 104 (air isolation chamber), the high temperature of 37℃ will not conduction affect the LSPR detection region 105 on the right (maintained at room temperature of 25℃).

[0062] Magnetic bead mixing and on-chip magnetic separation and capture: After the synthesis reaction is completed, the temperature control is turned off. The motor-driven module 2 guides a certain concentration of Ni-NTA (nickel-nitrogen triacetic acid) paramagnetic magnetic bead suspension droplets from the sample loading area 101 to the magnetic bead purification area 103.

[0063] Simultaneously, the product droplets after expression are expelled from the expression area and sent to the purification area where they spontaneously fuse with the magnetic bead droplets to form a large droplet. Subsequently, the motor drive module 2 applies an alternating electrical signal to drive the mixed droplet to perform high-frequency reciprocating oscillation between four adjacent electrodes. The strong shear force generated within the fluid layer 12 ensures that the paramagnetic magnetic beads and expression solution are thoroughly mixed and suspended. The efficient reaction lasts for 15–30 minutes, allowing the VHH-His protein to fully chelate with Ni2+ on the surface of the magnetic beads through the His tag.

[0064] After the reaction is complete, the magnetic field control module 5 drives the permanent magnet below to rise and approach the bottom surface of the lower electrode plate. Under the confinement of the strong magnetic field, the magnetic beads dispersed within the droplet rapidly condense and lock onto the electrode layer 133 at the center of the specific driving electrode within 30 seconds. Keeping the magnetic field stationary, the motor drive module 2 drives an external force to stretch and split the droplet, separating the supernatant droplet containing no His tag and cell-free expression impurities, and expelling it to the waste liquid collection area 106 on the right for sealing and discharge.

[0065] Multiple rounds of washing and elution: Maintaining the magnetic field to lock the magnetic bead bundle, the motor-driven module 2 sequentially delivers three washing buffer droplets (each with a volume of 200–500 nL) from the sample loading area 101. Each time the washing droplet reaches the magnetic bead, the magnetic field control module 5 controls the magnet to move downwards (removing the magnetic field), and the motor-driven module 2 again performs high-frequency electrode oscillation, causing the magnetic beads to be completely resuspended within the washing droplet. Subsequently, the magnet rises again to gather the magnetic beads and displace the washing waste liquid to the waste liquid collection area 106. This "wash-gathering-discharge" process is automatically repeated three times to thoroughly remove physically adsorbed impurities.

[0066] After washing, a strong elution droplet of glycine or imidazole is introduced into the cleaned magnetic bead bundle. The magnetic field is then removed and the mixture is shaken for 5 minutes to allow the target VHH protein to efficiently detach from the magnetic beads and be released into the elution droplet. Finally, the magnet is raised again to lock the empty magnetic beads, allowing the elution droplet rich in the purified target nanobody protein to split and separate independently.

[0067] To verify the feasibility of the digital microfluidic magnetic bead purification process of this invention, a purification verification experiment was conducted using VHH-His protein as a model protein.

[0068] Five independent replicate experiments were conducted using the same expression system, with each group following the above-described "magnetic bead capture - three-round washing - elution and recovery" process.

[0069] The total amount of target protein in the expression product and the amount of target protein recovered in the eluent were determined using the BCA protein quantification method, and the protein recovery rate was calculated according to the following formula: Protein recovery rate (%) = Amount of target protein obtained by elution / Total amount of target protein in the expression product × 100%.

[0070] The experimental results are shown in Table 1: Table 1 1 5.2 2.8 53.9 2 5.6 3.0 53.6 3 5.4 2.9 53.7 4 5.5 3.1 56.4 5 5.8 3.2 55.2 As shown in Table 1, when using the microfluidic protein expression purification and affinity detection system of the present invention to purify VHH-His protein on magnetic beads, the protein recovery rate of the five independent replicate experiments remained above 53%, with the highest reaching 56.4%.

[0071] Statistical analysis showed that the average protein recovery rate was 54.56%, the standard deviation was 1.16%, and the relative standard deviation (RSD) was 2.13%, indicating that the system of the present invention has good repeatability and stability.

[0072] Experimental results show that this invention, through a digital microfluidic-driven magnetic bead capture, automated washing, and automated elution process, can achieve efficient enrichment and purification of target proteins. Compared with traditional centrifugal column purification or manual magnetic bead manipulation, this invention eliminates the need for manual pipetting and sample transfer, enabling automated purification of protein expression products within the chip, effectively reducing sample loss and human error.

[0073] Meanwhile, the purified protein samples can meet the requirements for sample purity and concentration for subsequent LSPR affinity detection, providing a reliable basis for realizing integrated automated analysis of protein expression, purification and affinity detection.

[0074] The above results verify that the digital microfluidic on-chip magnetic bead purification process proposed in this invention has good feasibility, stability and application value.

[0075] Example 4 This embodiment illustrates the combined application capability and testing reliability of the integrated system of the present invention in the initial screening of multiple varieties and high-throughput mutant antibody drugs.

[0076] Multichannel parallel fluid manipulation: For a candidate nanobody targeting a specific tumor biomarker, researchers constructed eight different mutant variants (numbered VHH-Mut1 to VHH-Mut8) using genetic engineering. The plasmid DNA templates of these eight variants were loaded into different parallel, equidistant reservoirs in the sample loading area 101.

[0077] Under the unified clock control of the data processing module 6, the driving electrode array on the chip is divided into 8 parallel fluid manipulation paths in physical space, which synchronously and in parallel execute the fluid logic behavior described in Example 3: that is, cell-free protein synthesis is synchronously implemented in the protein expression region 102, high-purity automatic washing and purification based on paramagnetic magnetic beads is synchronously implemented in the magnetic bead purification region 103, and finally, a drop of purified antibody mutant elution solution is independently output in the phase transition region 104.

[0078] Serial high-throughput sensing analysis: The purified antibody droplets of the eight mutants are sequentially and rapidly introduced into different independent physical sensing points in the LSPR detection area 105 by the motor-driven module 2 (or by using a single-channel batch rapid replacement serial test). Then the system automatically executes the single-cycle kinetic five-step concentration antigen delivery process described in Example 3. The spectral detection module 4 acquires eight clear characteristic peak shift kinetic sensing curves in a multi-threaded synchronous manner.

[0079] Screening, ranking, and cross-validation on a commercial SPR platform: After the experiment, the system's data analysis software automatically completed curve fitting and background removal within 1 minute, calculated the equilibrium dissociation constant values ​​of each of the 8 mutant molecules against the target antigen, and automatically quantified their performance in order of affinity from strong to weak.

[0080] To rigorously verify the detection accuracy of the nanoscale integrated chip system of this invention, the industry-recognized "gold standard"—the commercial large-scale surface plasmon resonance platform (Biacore 8K)—was used to perform standard affinity tests on the same eight mutant antibodies (expressed in a conventional large mammalian cell system and purified by column chromatography). Table 2 compares the absolute KD values ​​and throughput ranking results obtained from the two independent platforms (Comparison of KD and affinity ranking for antibody mutants between the chip platform of this invention and the commercial SPR platform). Table 2 VHH-Mut1 <![CDATA[2.3×10 -9 ]]> <![CDATA[1.9×10 -9 ]]> Extremely strong integration ability VHH-Mut2 <![CDATA[1.5×10 -8 ]]> <![CDATA[1.1×10 -8 ]]> General ability to combine VHH-Mut3 <![CDATA[9.1×10 -8 ]]> <![CDATA[8.5×10 -8 ]]> Poor bonding ability VHH-Mut4 <![CDATA[6.8×10 -9 ]]> <![CDATA[5.4×10 -9 ]]> Strong integration ability VHH-Mut5 <![CDATA[2.4×10 -7 ]]> <![CDATA[3.1×10 -7 ]]> Weak binding ability VHH-Mut6 <![CDATA[1.0×10 -6 ]]> <![CDATA[1.5×10 -6 ]]> No obvious binding VHH-Mut7 <![CDATA[4.3×10 -8 ]]> <![CDATA[3.8×10 -8 ]]> Moderate integration ability VHH-Mut8 <![CDATA[7.6×10 -7 ]]> <![CDATA[6.8×10 -7 ]]> Weak binding ability like Figure 5 As shown, a bivariate statistical correlation analysis was performed on the logarithms of molecular affinity constants measured by the two independent detection platforms. The calculated Spearman rank correlation coefficient was greater than 0.75 (the actual data in this group reached 0.82, showing a highly significant correlation).

[0081] This data powerfully demonstrates that although the present invention reduces the micro-scale reaction system and cumbersome manual purification process required by traditional experiments to a fully automated nano-scale closed-loop control, the qualitative ranking of its output kinetic curves and the absolute quantitative parameters are highly consistent with those of large, expensive commercial clinical-grade optical equipment.

[0082] In summary, the microfluidic protein expression purification and affinity detection system and chip proposed in this invention successfully overcomes the traditional industry bottlenecks in molecular biology and interaction analysis that rely on complex centrifugation, manual column chromatography purification, and large sample volume consumption. It truly achieves one-stop, fully automated, closed-loop, label-free detection from "genotype (DNA)" to "phenotype functional analysis (affinity kinetic parameters)." This invention demonstrates significant industrial application value and scalability in cutting-edge pharmaceutical scenarios such as high-throughput screening of innovative antibody drugs, directed evolutionary optimization of protein engineering, in-situ quality control of synthetic biology end products, and rapid discovery of targeted drugs.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application.

Claims

1. A microfluidic protein expression, purification, and affinity detection chip, characterized in that, include: An upper electrode plate and a lower electrode plate, wherein a droplet operating space is formed between the upper electrode plate and the lower electrode plate; An electrode array disposed on the lower electrode plate; The electrode array divides the space inside the chip for droplet movement into six interconnected functional areas on a plane, namely, a sample loading area, a protein expression area, a magnetic bead purification area, a phase transition area, an LSPR detection area, and a waste liquid collection area, which are arranged in series along the droplet flow direction. The protein expression region is used for cell-free protein expression in an oil-sealed environment; The magnetic bead purification zone is used to capture and purify target proteins with affinity tags using functionalized magnetic beads. The phase transition region is provided with a magnetic bead cross-phase transfer pathway, which is used to drive magnetic beads carrying the target protein through the oil-water interface into the clean aqueous phase. A thermal isolation structure is provided between the protein expression region and the LSPR detection region to reduce thermal interference of the protein expression region to the LSPR detection region; The LSPR detection region is equipped with a localized surface plasmon resonance sensing structure, which includes a glass substrate, a gold reflective layer, a second dielectric layer, a surface modification layer, and a gold nanostructure array, for real-time monitoring of molecular binding and dissociation processes.

2. The microfluidic protein expression, purification, and affinity detection chip according to claim 1, characterized in that: The thermal insulation structure includes one or more of the following: thermal insulation groove, air gap layer, and low thermal conductivity material layer.

3. The microfluidic protein expression, purification, and affinity detection chip according to claim 1, characterized in that: The surface modification layer is used to immobilize ligand molecules and enable the detection of binding between the target protein and the ligand.

4. A microfluidic protein expression, purification, and affinity detection system, characterized in that, The microfluidic protein expression, purification, and affinity detection chip according to any one of claims 1 to 3 further includes a motor drive module, a temperature control module, a magnetic field control module, a spectral detection module, and a data processing module; The motor drive module is electrically connected to the electrode array of the chip and is used to control the movement of the droplet between the functional areas; The temperature control module is thermally coupled to the protein expression region of the chip and is used to control the working temperature of the protein expression region. The magnetic field control module corresponds to the magnetic bead purification region and phase transition region of the chip, and is used to control the magnetic bead capture and cross-phase transfer process. The spectral detection module is positioned above the LSPR detection area of ​​the chip and is used to acquire the spectral signal of the LSPR detection area in real time. The data processing module is electrically connected to the spectral detection module, the motor drive module, the temperature control module, and the magnetic field control module, and is used to analyze the detection results. The data processing module controls the ligand fixation endpoint according to the LSPR detection signal and controls the subsequent affinity detection process.

5. The microfluidic protein expression, purification, and affinity detection system according to claim 4, characterized in that: The temperature control module includes a temperature controller and a heating unit, with a temperature control range of 20℃~45℃ and a temperature control accuracy of ±0.5℃.

6. The microfluidic protein expression, purification, and affinity detection system according to claim 4, characterized in that: The data processing module stops the ligand fixation process when the LSPR response signal reaches a preset fixed amount threshold. The data processing module controls multiple analytes of different concentrations to be delivered sequentially to the LSPR detection area to perform single-cycle kinetic detection.

7. A method for protein expression, purification, and affinity detection, characterized in that, The microfluidic protein expression, purification, and affinity detection system according to claim 6 includes the following steps: S1. Add protein expression-related reagents to the sample loading area; S2. Complete the expression of the target protein in the protein expression region; S3. Functionalized magnetic beads are used in the magnetic bead purification zone to capture the target protein and complete the purification. S4. In the phase transition region, magnetic beads carrying the target protein are transferred to the LSPR detection region via the magnetic bead cross-phase transfer pathway. S5. Ligand fixation is completed in the LSPR detection area; S6. Detect the binding process between the target protein and the ligand in the LSPR detection region; S7. Obtain the binding kinetic parameters between the target protein and the ligand.

8. The method for protein expression purification and affinity detection according to claim 7, characterized in that: In step S2, a cell-free protein expression system is used for protein expression.

9. The method for protein expression purification and affinity detection according to claim 7, characterized in that: In step S3, functionalized magnetic beads are used to capture target proteins with affinity tags.

10. The protein expression, purification, and affinity detection method according to claim 7, characterized in that: Step S6 uses a single-cycle kinetic detection mode to continuously inject samples of different concentrations and obtains the binding rate constant, dissociation rate constant, and equilibrium dissociation constant between the target protein and the ligand.

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