Full-automatic in-situ X-ray microstructure characterization device

Abstract

The invention discloses a full-automatic in-situ X-ray microstructure characterization device. The device comprises an automatic liquid pretreatment module, an in-situ solution condition screening module, a high-flux X-ray microstructure characterization module, an automatic sampling and motion control module and a system control module, a control module of the system is electrically connected with other modules, collaborative program regulation and control of liquid sample configuration and an X-ray channel sample loading mechanical arm can be achieved, signal interaction and linkage control among the modules can be achieved, and full-process automatic operation of the device from sample pretreatment to structural characterization is completed. Through an integrated technical scheme of automatic liquid pretreatment, in-situ condition screening and high-flux X-ray scattering characterization, full-process automatic closed operation is realized, the experiment efficiency is greatly improved, the scattering data quality and batch consistency are also improved, and the method is suitable for large-scale popularization and application. The requirements of multiple fields on high-flux and automatic in-situ characterization of soft substances and biological macromolecular systems can be met.

Description

Technical Field

[0001] This invention relates to the field of automated characterization of X-ray scattering experiments and analysis of soft matter nanostructures, specifically to a fully automated in-situ X-ray microstructure characterization device. Background Technology

[0002] Soft matter systems are complex material systems that lie between traditional solids and ideal liquids. They encompass proteins, nucleic acids, liposomes, micelles, polymer solutions, biomacromolecule complexes, and their self-assembled structures. The microstructure of these systems is concentrated in the nanometer to hundred-nanometer range, with weak intermolecular interactions and high sensitivity to the external solution environment. Their microstructural arrangement and molecular assembly behavior can be significantly altered by minute changes in solution concentration, pH, ionic strength, temperature, and additive composition. Therefore, achieving in-situ, reproducible, and highly consistent nanoscale structural characterization of soft matter systems in solution environments close to those used in practical applications or physiological conditions is a core technological requirement in soft matter physics, biological structural physics, biomedicine, materials research, and other related application fields.

[0003] Currently, structural characterization of biological and soft matter systems still primarily relies on X-ray crystallography, cryo-electron microscopy, and dynamic light scattering (DLS). X-ray crystallography can obtain high-resolution static crystal structures of molecules, cryo-electron microscopy is suitable for resolving the fine spatial conformations of biomacromolecule complexes, and DLS can rapidly determine the average particle size and particle size distribution of nanoparticles. While these techniques have significant advantages in their respective applications, they all suffer from a core limitation: they are difficult to adapt to in-situ characterization in solution phases. These techniques typically require non-in-situ treatments such as crystallization and cryogenic freezing, failing to reflect the true structural characteristics and dynamic behavior of soft matter systems in physiological or practical solution environments. Their applicability is further severely limited for highly flexible, conformationally diverse, or weakly interacting soft matter systems.

[0004] Small-angle X-ray scattering (SAXS) has become a key technique for studying the nanoscale structure of soft matter systems because it eliminates the need for sample crystallization, allows testing in solution at room temperature and pressure, and can accurately acquire information on the overall size, molecular shape, and assembly of the system. The basic components of an existing SAXS experimental system include an X-ray source, a sample cell / flow cell, a scattering signal detector, and a data acquisition and analysis system. The typical experimental procedure is as follows: first, sample pretreatment steps such as solution preparation, concentration adjustment, filtration, and centrifugation are completed on a separate experimental device or operating table; then, the pretreated sample is manually transferred to the sample cell of the SAXS system for scattering testing; finally, the detector collects the scattering signal and transmits it to a computer terminal for subsequent structural analysis.

[0005] Although SAXS technology has inherent advantages in the solution phase characterization of soft matter systems, existing SAXS experimental systems and supporting experimental procedures still have several significant shortcomings in meeting the actual testing needs of soft matter systems, making it difficult to satisfy the requirements of high-throughput, in-situ, and standardized structural characterization. Specifically, these shortcomings include:

[0006] Sample pretreatment relies on manual operation, making it difficult to guarantee experimental consistency: Soft matter systems are mostly in a thermodynamically metastable state, prone to aggregation, sedimentation, or phase separation, requiring extremely high consistency in pretreatment operations such as solution preparation and transfer. However, existing sample pretreatment processes are all performed manually, with cumbersome steps and prone to introducing human error. In batch testing with multiple samples and under multiple conditions, batch consistency cannot be guaranteed, directly leading to a decrease in the reliability and comparability of scattering data.

[0007] Lack of high-throughput multi-condition in-situ screening capability: The structural study of soft matter systems often requires systematic analysis of structural change patterns under different concentrations, buffer systems, additive compositions, etc. However, the existing SAXS experimental process mainly relies on single-sample, single-condition testing, and the switching of conditions depends entirely on manual intervention, resulting in extremely low experimental efficiency. It cannot support large-scale solution condition screening or parameter space exploration, and it is difficult to meet the multi-condition optimization needs in biomedical and materials research and development.

[0008] The separation of pretreatment and characterization processes can easily lead to distortion of sample structure: In the existing technology, sample pretreatment and X-ray scattering testing belong to different experimental stages and need to be completed on different equipment. The sample needs to be transferred manually multiple times and wait for a long time. This process can easily lead to structural evolution, degradation or contamination of soft matter systems. In particular, it is impossible to achieve in-situ characterization of transient structures and weakly interacting driven systems, making it difficult to obtain true structural data.

[0009] Insufficient data throughput and time resolution make it unsuitable for studying dynamic processes: Soft matter systems commonly exhibit rapid molecular conformational changes, self-assembly, and phase transitions, placing high demands on the time resolution of measurement techniques. However, existing SAXS devices mostly employ a single-injection, sequential testing mode, which cannot achieve high-time-resolution continuous data acquisition or parallel testing capabilities, severely limiting the systematic study of the dynamic structural evolution processes of soft matter systems.

[0010] Low automation levels hinder support for AI-assisted R&D models: As AI methods are increasingly applied to the prediction of soft matter and biomolecular structures, the demand for high-quality, standardized, and batch-acquired experimental data is becoming more urgent. However, existing SAXS experimental setups suffer from deficiencies in automated operation, standardized data acquisition, and batch data acquisition efficiency, resulting in an inability to stably output scattering data with uniform experimental condition labels. This restricts the effective integration of experimental data and intelligent algorithms.

[0011] In summary, existing small-angle X-ray scattering experimental devices and supporting processes have significant shortcomings in terms of automated sample pretreatment, in-situ control of solution conditions, high-throughput testing under multiple conditions, and standardization of experimental data, in order to meet the actual needs of biomedicine, synthetic biology, and new materials research and development for high-throughput, in-situ, and highly consistent characterization of nanoscale structures of soft matter systems.

[0012] Therefore, it is necessary to develop an experimental device that can integrate liquid sample pretreatment, solution condition control and small-angle X-ray scattering characterization processes and achieve fully automated operation to meet the in-situ characterization needs of nanoscale spatial structures in the research and application development of nanoscale structures of soft matter systems. Summary of the Invention

[0013] The purpose of this invention is to provide a fully automated in-situ X-ray microstructure characterization device, thereby solving the technical problems of existing X-ray scattering experiments on soft matter and biomacromolecule systems, such as reliance on manual sample pretreatment, low accuracy and batch consistency of solution preparation, lack of in-situ multi-condition screening capability, easy structural changes caused by sample transfer, insufficient precision in sample loading and delivery, and the disconnect between solution preparation, sample loading, and detection without coordinated automated control, resulting in low experimental throughput, poor data reliability and consistency, and the inability to achieve high-throughput, in-situ nanoscale microstructure characterization.

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0015] A fully automated in-situ X-ray microstructure characterization device is provided, the device comprising an automated liquid pretreatment module, an in-situ solution condition screening module, a high-throughput X-ray microstructure characterization module, an automated sample introduction and motion control module, and a system control module;

[0016] The automated liquid pretreatment module includes a multi-channel liquid workstation, which includes a robotic arm, a high-precision liquid delivery unit, a sample tray and a reagent tray. It is also equipped with a liquid preparation structure consisting of a reagent bottle, polytetrafluoroethylene tubing, a mixing chamber and a high-precision flow meter, for completing the automatic sampling, preparation, dilution and transfer of liquid samples.

[0017] The in-situ solution condition screening module includes a porous array reaction cell for parallel processing of samples under different solution conditions to form a multivariate solution condition combination.

[0018] The high-throughput X-ray microstructure characterization module integrates a small-angle X-ray scattering (SAXS) detection unit and / or a wide-angle X-ray scattering (WAXS) detection unit for in-situ microstructure characterization of samples after pretreatment and condition screening.

[0019] The automatic sample introduction and motion control module includes at least one three-axis cantilever motion mechanism, a high-precision lifting mechanism and a microfluidic sample loading structure, which are used to realize the automatic transfer, precise positioning and accurate loading of samples between various functional modules.

[0020] The system control module is electrically connected to the automatic liquid pretreatment module, the in-situ solution condition screening module, the high-throughput X-ray microstructure characterization module, and the automatic sample introduction and motion control module. This enables coordinated program control of liquid sample preparation and X-ray path sample loading robotic arm, as well as signal interaction and linkage control between modules, completing the fully automated operation of the device from sample pretreatment to structural characterization.

[0021] Preferably, the multi-channel liquid workstation of the automated liquid pretreatment module is further equipped with a temperature control and mixing unit. This unit works in conjunction with the robotic arm and the high-precision liquid delivery unit to maintain a set temperature and uniformly mix the sample during sample pretreatment. The high-precision liquid delivery unit is an injection pump, plunger pump, or droplet dispenser, which can achieve quantitative liquid distribution with a precision ranging from micro-level to nano-level. The mixing chamber is equipped with a magnetic stir bar or an ultrasonic oscillation module. The system control module can drive multiple precision injection pumps or solenoid valve groups through a preset ratio scheme by the main control unit to draw the solvent and solute into the mixing chamber in proportion and complete the homogenization process, achieving a precise ratio with a component error of less than 0.1%.

[0022] Preferably, the in-situ solution condition screening module further includes a condition control unit and a status monitoring unit. The condition control unit is connected to the porous array reaction cell and the automatic liquid pretreatment module, and can automatically adjust the pH value, ionic strength, additive concentration or component ratio of the sample in the porous array reaction cell. The porous array reaction cell adopts an equivalent optical path and structural isolation design, and is composed of multiple independently isolated reaction holes, each of which is used to independently hold samples with different solution conditions. The status monitoring unit is a dynamic light scattering detection component or other equivalent optical detection means.

[0023] Preferably, the high-precision lifting mechanism of the automatic sample introduction and motion control module is a Z-axis vertical motion mechanism driven by a precision lead screw stepper motor, equipped with a loading platform and an origin sensor; after receiving the signal from the origin sensor, the motor drives the loading platform to rise at a constant speed, causing the liquid container to be tested to perform lifting and lowering motion, so that the end of the sample injection needle of the loading capillary is immersed to a preset depth below the liquid surface, thereby achieving high-precision liquid surface positioning of the container lifting and lowering.

[0024] Preferably, the microfluidic sample loading structure of the automatic sample loading and motion control module includes a peristaltic pump, a sample loading capillary, and a capillary sample cell. The capillary sample cell is located at the center of the X-ray optical path, and the sample loading capillary is made of quartz glass. The peristaltic pump is connected to the main control unit via an RS485 level signal, which can provide real-time feedback on the rotation speed and pumping volume. By generating negative pressure, the sample is drawn in through the sample loading capillary and transported to the capillary sample cell through a closed pipeline.

[0025] Preferably, the microfluidic sample loading structure is also equipped with a photoelectric sensor. The control algorithm of the system control module can detect the position of the liquid column through the photoelectric sensor to ensure that the sample fills the irradiation area of ​​the capillary sample cell without any air bubbles. The peristaltic pump can realize continuous cyclic injection or long-distance pumping of the sample, which is suitable for in-situ dynamic monitoring requirements.

[0026] Preferably, the high-throughput X-ray microstructure characterization module is an original design module, which also includes a multi-pore array reaction cell automated sample introduction robotic arm, an X-ray source, a sample measurement cell, a detector, and a data acquisition system; the sample measurement cell is connected to the in-situ solution condition screening module or the automated sample introduction and motion control module to realize the automatic introduction of samples in a closed state; this module can realize single-sample testing with a time resolution of milliseconds to seconds, and complete the acquisition of structural information of biomacromolecules and soft matter systems in the nano to sub-nanometer scale range; the X-ray source is equipped with a shutter, and the detector can convert the captured photon signal into a two-dimensional image to complete the acquisition and conversion of scattered signals.

[0027] Preferably, the automatic liquid pretreatment module and the high-throughput X-ray microstructure characterization module are linked via pulse signals. After the sample stabilizes in the capillary sample cell, the pretreatment device sends a "ready" pulse signal to the X-ray detection system, the X-ray source shutter opens, the X-ray beam passes through the sample cell, and the SAXS detector and WAXS detector are synchronously triggered and record the scattering signal. The system control module can control the device to cycle through the steps of liquid preparation, positioning, sample loading, and detection by changing the ratio of the pretreatment system or environmental variables, thereby achieving in-situ scattering spectrum acquisition at different concentrations or under different conditions.

[0028] Preferably, the system control module includes an automatic sample preparation and injection system mechanical control system and an X-ray beamline control system, which interact with each other via a communication interface. The X-ray beamline control system adopts the EPICS experimental physics and industrial control system architecture as the underlying control framework. By defining cross-module state synchronization, triggering mechanisms, and experimental process arrangement, it achieves deep integration of various functional modules and completes the seamless connection between sample preparation, transportation, and X-ray testing. The device adopts a miniaturized liquid pretreatment unit and a flow-through sample measurement structure, which significantly reduces the sample volume consumption per experiment while ensuring the quality of scattered signal acquisition.

[0029] According to the present invention, the device is applicable to soft matter and biomacromolecule systems such as proteins, nucleic acids, liposomes, and nanomedicine carriers. It can be applied to the fields of new drug development, precision medicine and personalized formulation optimization, biomanufacturing and synthetic biology, and artificial intelligence-assisted drug and material development, enabling high-throughput, automated, and highly consistent in-situ characterization of the structural stability, conformational changes, and self-assembly behavior of such systems under different solution conditions.

[0030] According to a preferred embodiment of the present invention, the device can perform experimental operations such as concentration gradient dilution and in-situ SAXS testing of biological macromolecules such as lysozyme, and synthesis and in-situ SAXS testing of nanodelivery systems such as mRNA-LNP.

[0031] This invention provides a small-angle scattering experimental device for fully automated liquid pretreatment and nanoscale microstructure characterization based on X-rays. The device consists of an automated liquid pretreatment module, an in-situ solution condition screening module, a high-throughput X-ray microstructure characterization module, an automated sample introduction and motion control module, and a system control module. All modules work collaboratively under unified control, forming an integrated technical solution of "sample preparation – condition control – structural characterization – data acquisition". The core design and innovations of each module are as follows:

[0032] (a) Automatic Liquid Pretreatment Module

[0033] This module includes a multi-channel liquid workstation, which is equipped with at least a robotic arm, a high-precision liquid delivery unit, a sample tray, a reagent tray, and a temperature control and mixing unit. The high-precision liquid delivery unit preferably uses a syringe pump, plunger pump, or droplet dispenser to achieve quantitative liquid dispensing from micro- to nano-scale. The robotic arm automatically picks up, places, positions, and transfers samples and reagents. The sample and reagent trays respectively hold the test samples and various formulation components. The temperature control and mixing unit maintains the sample preparation temperature and ensures uniform mixing. This module can complete sample preparation, gradient dilution, and component mixing without manual intervention, reducing operational errors and improving batch-to-batch sample consistency.

[0034] While the mechanical principle of this module is not original, its integration with this device offers unique innovations: First, it systematically addresses the key issue affecting the quality of scattering data by ensuring the consistency of sample pretreatment, overcoming the technical bias that pretreatment is merely an auxiliary step. Second, it constructs a dedicated pretreatment scheme adapted to soft matter and biomacromolecule systems through the collaborative combination of multiple units, rather than directly applying a general-purpose liquid workstation. Third, it achieves standardization, repeatability, and traceability of the pretreatment process, improving the comparability of scattering experimental data and providing a reliable data foundation for subsequent analysis and AI model training.

[0035] (II) In-situ solution condition screening module

[0036] This module includes a porous array reaction cell, a condition control unit, and a status monitoring unit. The porous array reaction cell consists of multiple independent and isolated reaction wells, which can hold samples with different solution conditions in parallel. The condition control unit is connected to the automatic liquid pretreatment module and can automatically adjust parameters such as pH, ionic strength, and additive concentration of the sample to generate multivariate combination conditions to meet the structural screening requirements of multi-condition space.

[0037] Existing technologies for solution condition screening often involve manually changing sample cells to adjust conditions, or using general-purpose multi-well plates for offline screening followed by transfer testing. This results in a disconnect between the screening and testing processes, making in-situ parallel screening difficult to achieve. Furthermore, there is a widespread technical bias in the industry that multi-condition screening is incompatible with in-situ scattering. While the mechanical principle of this module is not original, its integration achieves innovative breakthroughs: First, it integrates multi-condition screening and small-angle scattering in-situ measurement within the same reaction cell, breaking the technical path dependence that condition screening must be completed offline. Second, through the equivalent optical path and structural isolation design of the multi-well array reaction cell, it solves the scattering interference problem introduced by the porous structure, overcoming the bias that multi-condition parallelism and high-precision scattering measurement are incompatible. Third, through collaboration with the pretreatment module, it achieves automatic generation and traceable screening of multivariable solution conditions, forming a dedicated in-situ screening scheme for soft matter and biomacromolecule systems.

[0038] (III) Automatic Sample Injection and Motion Control Module

[0039] This module serves as the core hub for sample transfer and precise loading between various functional modules, comprising three core units: a three-axis cantilever motion mechanism, a high-precision lifting mechanism, and a microfluidic loading structure. The three-axis cantilever motion mechanism employs a double-layer, three-axis linkage design. The main structure's X / Y / Z-axis motion components achieve overall spatial coarse positioning, while the dedicated X / Y / Z-axis motion components of the cantilever motion module complete fine position adjustment, enabling automated and precise sample transfer between modules. The high-precision lifting mechanism is a Z-axis vertical motion mechanism driven by a precision lead screw stepper motor, paired with an origin sensor to achieve high-precision liquid level positioning during container lifting, avoiding vibration and air bubble introduction issues caused by capillary movement. The microfluidic loading structure consists of a peristaltic pump, a quartz glass loading capillary, a capillary sample cell, and a photoelectric sensor. Real-time feedback and control of pumping parameters are achieved via RS485 level signals, ensuring sealed, bubble-free, and precise sample loading, and supporting continuous cyclic injection mode.

[0040] While the mechanical principles of each unit in this module are not original to this invention, their integration with the device forms a specialized sample loading and movement scheme adapted to soft matter and biomacromolecule systems, possessing unique innovations: First, it solves the problem of accuracy and stability in sample transfer between multiple modules, adapting to the stringent sample positioning requirements of X-ray scattering testing; second, it innovatively adopts a container lifting liquid level positioning design, replacing the traditional capillary moving positioning, structurally avoiding problems such as vibration and bubble introduction that affect test results, thus improving sample loading consistency; third, it achieves the adaptation of closed microfluidic sample loading and in-situ dynamic monitoring, through a combination design of photoelectric sensor liquid column detection and peristaltic pump continuous pumping, ensuring that the sample fills the X-ray irradiation area without bubble retention, while meeting the continuous testing requirements for the dynamic structural evolution study of soft matter systems; fourth, it completes the action linkage and signal interaction between the sample loading and pretreatment, condition screening, and characterization modules, realizing seamless transfer of samples from solution preparation and screening to sample loading and testing, avoiding sample structure distortion, contamination, and loss caused by manual transfer.

[0041] (iv) High-throughput X-ray microstructure characterization module

[0042] This module is integrated into the X-ray scattering experimental environment and includes a multi-pore array reaction cell automated sample introduction robotic arm, an X-ray source, a sample measurement cell, a detector, and a data acquisition system. The sample measurement cell is connected to the in-situ solution condition screening module or the automated sample introduction system to achieve automatic sample introduction in a closed state. The module is equipped with at least a small-angle X-ray scattering (SAXS) detection unit and can also integrate a wide-angle X-ray scattering (WAXS) detection unit as needed to achieve the acquisition of structural information at the nanometer to sub-nanometer scale. Through automated sample introduction, exposure, and temperature control, it can achieve single-sample testing with millisecond to second-level time resolution, improving experimental throughput and efficiency.

[0043] The design challenge of this module lies in achieving automated sample introduction and continuous measurement of porous array samples while maintaining the high requirements of X-ray scattering for sample positioning, optical path, and background stability. It also overcomes the industry's bias that high-throughput automation is incompatible with precision scattering measurements. Its core innovations are: first, the high-throughput coupling and integration of the porous array reaction cell and the X-ray scattering detection system at the structural level, breaking through the technical mode of single-sample measurement in scattering experiments; second, the high-precision matching design of the automated sample introduction robotic arm and the scattering optical path solves the technical challenge of compatibility between high-throughput sample introduction and high-precision scattering measurement, overcoming the bias that high throughput inevitably sacrifices data quality; and third, it forms a complete technical chain with the two preceding modules, realizing a continuous automated process from sample preparation to structural characterization, representing system-level integrated innovation rather than a simple combination.

[0044] (v) System Control Module

[0045] This module consists of an automated sample preparation and injection system mechanical control system and an X-ray beamline control system. The two systems interact with each other via a communication interface. The X-ray beamline control system preferably uses the EPICS architecture to uniformly manage the X-ray source, detector acquisition parameters, and scattering test process.

[0046] The innovation of this module does not lie in the adoption of the EPICS architecture itself, but in the design of a system-level control scheme based on this architecture for multi-module collaborative operation: for the first time, the three major functional modules are deeply integrated under a unified control logic. By defining cross-module state synchronization, triggering mechanisms, and experimental process orchestration, continuous automated operation of sample preparation, condition control, and structural measurement is achieved. At the same time, relying on the EPICS architecture, cross-module collaboration and closed-loop process control are realized, which solves the problems of poor experimental consistency and process fragmentation caused by independent operation of multiple systems. It can also achieve seamless connection between sample preparation, transportation, and X-ray testing, reduce the risk of sample loss, contamination, or structural changes, and improve the stability and repeatability of scattering data.

[0047] Compared with existing technologies, the fully automated in-situ X-ray microstructure characterization device provided by this invention achieves breakthroughs in multiple aspects through its integrated technical solution of automated liquid pretreatment, in-situ condition screening, and high-throughput X-ray scattering characterization. The core beneficial effects are as follows:

[0048] 1) The device achieves full automation and high-throughput operation of the experimental process, significantly improving experimental efficiency. It integrates sample liquid pretreatment, solution condition control, automatic sample injection, and X-ray scattering data acquisition into a continuous automated process, realizing a closed, unattended operation from sample injection to scattering data acquisition. This reduces the time loss caused by manual intervention, and a single device can complete the testing of more samples and conditions per unit time, adapting to the application requirements of multi-condition screening and high-throughput structural characterization.

[0049] 2) Effectively improves the quality of scattering data and batch consistency. The entire sample pretreatment process is completed by a high-precision liquid delivery and automatic control system. The solution ratio, concentration, and processing conditions can be precisely set and stably repeated, fundamentally avoiding errors, cross-contamination, and batch deviations caused by manual operation. This ensures the consistency of sample state and makes the acquired small-angle X-ray scattering data more repeatable and comparable, laying a reliable data foundation for subsequent structural analysis and model building.

[0050] 3) Integrating multiple pretreatment functions enhances the flexibility and adaptability of experimental procedures. By integrating multiple liquid pretreatment units and a controllable flow path switching structure, the device can perform various pretreatment operations such as sample buffer replacement, desalting, concentration adjustment, and formulation reconstruction according to experimental needs. This eliminates the need to switch between different devices, reduces sample transfer steps, and makes the experimental process more compact, thus adapting to the diverse testing needs of different types of biomacromolecules and soft matter systems.

[0051] 4) Significantly reduces sample consumption and expands the application scope of small-angle X-ray scattering (SAXS). The device employs a miniaturized liquid pretreatment unit and a flow-through sample measurement structure, which greatly reduces the sample volume required for a single experiment while ensuring the quality of scattering signal acquisition. It is particularly suitable for the structural characterization of biomolecules or drug candidates that are costly to prepare and have limited availability, effectively expanding the practical application scenarios of SAXS in the field of soft matter research.

[0052] In summary, this invention provides a fully automated in-situ X-ray microstructure characterization device. This device overcomes the technical difficulties of existing technologies, such as reliance on manual sample pretreatment, fragmented experimental procedures, low test throughput, and poor data consistency. Through an integrated technical solution of automated liquid pretreatment, in-situ condition screening, and high-throughput X-ray scattering characterization, it achieves fully automated closed-loop operation of the entire experimental process. This not only significantly improves experimental efficiency, enhances the quality and batch consistency of scattering data, but also integrates multiple pretreatment functions to improve experimental adaptability, while reducing sample consumption and expanding the application range of small-angle X-ray scattering technology. It can meet the needs of multiple fields for high-throughput, automated in-situ characterization of soft matter and biomacromolecule systems. Attached Figure Description

[0053] Figure 1 It is a three-axis cantilever motion mechanism in the automatic sample introduction and motion control module;

[0054] Figure 2 It is the core operating area of ​​the automatic liquid pretreatment module and the in-situ solution condition screening module;

[0055] Figure 3 It is a microfluidic sample loading structure in the automatic sample introduction and motion control module;

[0056] Figure 4 These are SAXS curves of different concentrations of lysozyme;

[0057] Figure 5 This is the SAXS curve of mRNA-LNP.

[0058] The meanings of the reference numerals in the attached figures are as follows:

[0059] 1. Main structure X-axis motion assembly; 2. Main structure Y-axis motion assembly; 3. Main structure Z-axis motion assembly; 4. Cantilever motion module; 5. Pipette assembly; 6. Base plate; 7. Carrier plate; 8. Waste container; 9. Functional module mounting holes; 10. Cantilever motion module X-axis motion assembly; 11. Cantilever motion module Y-axis motion assembly; 12. Cantilever motion module Z-axis motion assembly; 13. Water-cooled refrigeration module; 14. Waste liquid discharge tank. Detailed Implementation

[0060] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the instrument manufacturer. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0061] According to a preferred embodiment of the present invention, a fully automated in-situ X-ray microstructure characterization device is provided. The device includes an automated liquid pretreatment module, an in-situ solution condition screening module, a high-throughput X-ray microstructure characterization module, an automated sample introduction and motion control module, and a system control module.

[0062] The automated liquid pretreatment module includes a multi-channel liquid workstation, which includes a robotic arm, a high-precision liquid delivery unit, a sample tray and a reagent tray. It is also equipped with a liquid preparation structure consisting of a reagent bottle, polytetrafluoroethylene tubing, a mixing chamber and a high-precision flow meter, for completing the automatic sampling, preparation, dilution and transfer of liquid samples.

[0063] The in-situ solution condition screening module includes a porous array reaction cell for parallel processing of samples under different solution conditions to form a multivariate solution condition combination.

[0064] The high-throughput X-ray microstructure characterization module integrates a small-angle X-ray scattering (SAXS) detection unit and / or a wide-angle X-ray scattering (WAXS) detection unit for in-situ microstructure characterization of samples after pretreatment and condition screening.

[0065] The automatic sample introduction and motion control module includes at least one three-axis cantilever motion mechanism, a high-precision lifting mechanism and a microfluidic sample loading structure, which are used to realize the automatic transfer, precise positioning and accurate loading of samples between various functional modules.

[0066] The system control module is electrically connected to the automatic liquid pretreatment module, the in-situ solution condition screening module, the high-throughput X-ray microstructure characterization module, and the automatic sample introduction and motion control module. This enables coordinated program control of liquid sample preparation and X-ray path sample loading robotic arm, as well as signal interaction and linkage control between modules, completing the fully automated operation of the device from sample pretreatment to structural characterization.

[0067] like Figure 1 As shown, the three-axis cantilever motion mechanism in the automatic sample introduction and motion control module serves as the basic motion mechanism of the device of this invention, providing three-dimensional spatial positioning and motion support for the automated operation of all functional modules. The core components of this three-axis cantilever motion mechanism include the main structure X-axis motion component 1, the main structure Y-axis motion component 2, and the main structure Z-axis motion component 3, which work together to form the overall motion foundation of the device. The main structure X-axis motion component 1, the main structure Y-axis motion component 2, and the main structure Z-axis motion component 3 respectively realize the precise movement and positioning of the sample / functional module in the horizontal primary direction, the horizontal secondary direction (perpendicular to the X-axis), and the vertical direction, completing the basic motion in three-dimensional space.

[0068] like Figure 2 As shown, this is the core operating area of ​​the automatic liquid pretreatment module and the in-situ solution condition screening module. The core components include: pipette assembly 5, base plate 6, carrier plate 7, waste box 8, and functional module mounting holes 9. All components are supported by the base plate 6 to ensure the overall operational stability of the device.

[0069] The pipette assembly 5 is the core actuator of the automated liquid pretreatment module. It is an integrated structure of a robotic arm and a high-precision liquid delivery unit. It is fixedly installed at the lower end of the Z-axis motion assembly of the main structure through a flange connection seat. It can achieve precise three-dimensional positioning under the drive of the three-axis motion assembly to complete the automatic aspiration, dispensing and transfer of samples and reagents. The core components of the liquid preparation structure of the automated liquid pretreatment module (mixing chamber and high-precision flow meter) are integrated in this core operating area and arranged adjacent to the carrier plate 7 and the pipette assembly 5. The reagent bottle is externally connected to the side of the operating area and connected to the mixing chamber through a polytetrafluoroethylene pipeline.

[0070] The support plate 7 is located in the operating area below the pipette assembly. It is used to place the sample tray, reagent tray, or multi-pore array reaction cell of the in-situ solution condition screening module. It is the core supporting component for sample pretreatment and multi-condition screening. The liquid preparation structure completes the liquid metering through a high-precision flow meter, and after homogenization through the mixing chamber, the pipette assembly 5 completes the precise aspiration, distribution and transfer of the sample, realizing seamless linkage between liquid preparation and pipetting operations.

[0071] Waste box 8 is located on one side of the support plate and is used to collect waste liquid and discarded pipette tips during the pipetting process to avoid cross-contamination of samples;

[0072] The functional module mounting holes 9 are located in the predetermined area of ​​the base plate 6 of the whole machine. They are detachable and can be used to install cooling, heating and oscillation mixing modules according to experimental needs. They can be functionally adapted to the temperature control and mixing units to meet the pretreatment conditions of different samples.

[0073] As shown in Figure 3, the microfluidic sample loading structure in the automatic sample loading and motion control module serves as a transitional unit connecting the in-situ solution condition screening module and the high-throughput X-ray microstructure characterization module. Its core components are used for precise microfluidic sample loading, pre-test temperature control, and waste liquid collection. The core components include: cantilever motion module 4, water-cooled cooling module 13, and waste liquid discharge tank 14.

[0074] The cantilever motion module 4 is located on one side of the main structure and is fixedly connected to the moving end of the X-axis motion component 1. It can move as a whole with the X-axis motion component 1. At the same time, the cantilever part is independently equipped with a dedicated X / Y / Z axis motion component, which can realize the precise movement of the load-bearing components along the extension direction of the cantilever.

[0075] The cantilever motion module 4 is a three-axis cantilever motion mechanism, which includes an independent cantilever motion module X-axis motion component 10, a cantilever motion module Y-axis motion component 11, and a cantilever motion module Z-axis motion component 12. It can realize the precise positioning and automatic transmission of the sample in three-dimensional space, and provide precise motion support for the delivery of the sample to the X-ray detection unit.

[0076] The water-cooled refrigeration module 13 is located below the cantilever motion module and adjacent to the sample transport flow path. It is used to control the temperature of biological macromolecular liquid samples at constant or low temperatures to ensure the structural stability of the samples during sample loading and testing.

[0077] Waste liquid discharge tank 14 is located below the sample loading structure operation area and is used to collect waste liquid during sample pretreatment, condition screening and sample loading testing, so as to realize centralized treatment of waste liquid.

[0078] The fully automated in-situ X-ray microstructure characterization device provided by the present invention has its overall workflow completed by a unified and coordinated automated control system. This system, with a main control unit (industrial computer) at its core, integrates mechanical action execution and electrical signal interaction logic, sequentially completing four core processes: multi-component fully automated liquid pretreatment and preparation, high-precision liquid level positioning by a lifting mechanism, peristaltic pump-driven microfluidic precise sample loading, and in-situ detection using a SAXS / WAXS linkage system. Each process is seamlessly connected and automatically triggered, achieving fully automated closed-loop operation from sample preparation to scattering data acquisition. The mechanical actions, electrical control logic, and operational details of each process are described in detail below:

[0079] 1) Multi-component fully automated liquid pretreatment and preparation

[0080] This process is completed by an automated liquid pretreatment module, with the main control unit (industrial computer) as the core control terminal. A sample mixing scheme is preset in the system, driving the operation of multiple precision syringe pumps or solenoid valve assemblies. This device innovatively incorporates collaborative control logic between liquid sample preparation and the X-ray path loading robotic arm at the software control level, achieving program-linked operation between liquid preparation and subsequent loading. Structurally, reagent bottles are connected to the mixing chamber via polytetrafluoroethylene (PTFE) tubing, with each PTFE tubing equipped with a high-precision flow meter for accurate liquid delivery. The electrical system sends pulse signals to control the drive motor, which draws the solvent and solute into the mixing chamber according to the preset mixing ratio. The mixing chamber contains a built-in magnetic stir bar or ultrasonic oscillation module to homogenize the drawn-in liquid. Compared to manual liquid preparation, this process effectively avoids the introduction of air bubbles and concentration errors, achieving a precise mixing ratio with a component error of less than 0.1%, providing a highly consistent sample environment for subsequent SAXS experiments.

[0081] 2) Liquid level positioning of the high-precision lifting mechanism

[0082] After the multi-component liquid homogenization and preparation are completed, the mixing container is automatically moved to the sampling position via the device's transport track, triggering the intervention of the high-precision lifting mechanism. This lifting mechanism is driven by a precision lead screw stepper motor, which is connected to the loading platform. The motor drives the platform to move vertically (Z-axis) upwards and downwards. Upon receiving a signal from the origin sensor, the motor drives the loading platform to rise at a constant speed, simultaneously moving the liquid container to be tested upwards until the tip of the quartz glass sample capillary needle is completely submerged to a preset depth below the liquid surface, completing the liquid level positioning operation. This "container lifting" liquid level positioning design avoids vibration problems caused by long-distance movement of the sample capillary, effectively ensuring the structural stability of the injection port, while preventing air from entering the liquid flow path system, thus providing a guarantee for subsequent sample loading processes.

[0083] 3) Precise microfluidic sample loading driven by a peristaltic pump

[0084] After liquid level positioning is completed, the microfluidic sample loading process is initiated. The core component of this process, the peristaltic pump, is connected to the main control unit via an RS485 level signal, allowing real-time feedback to the main control unit on pump speed and liquid volume, enabling precise control of the sample loading volume. During operation, the peristaltic pump generates negative pressure, drawing the sample from the mixing container through the suction capillary. The sample is then transported through a closed tubing and finally enters the capillary sample cell located at the center of the X-ray path. Throughout the pumping process, the device's control algorithm uses photoelectric sensors to monitor the liquid column position in real time, ensuring that the sample completely fills the X-ray irradiation area of ​​the capillary sample cell without air bubbles remaining in the flow path. This device uses a peristaltic pump for sample loading, which, compared to traditional syringe pumps, allows for continuous cyclic sample injection or long-distance pumping, making it easier to achieve in-situ dynamic monitoring of soft matter and biomolecular systems.

[0085] 4) In-situ testing of SAXS / WAXS linkage system

[0086] Once the sample has stabilized within the capillary sample cell, the automated liquid pretreatment module sends a "ready" pulse signal to the X-ray detection system, triggering the in-situ detection process. Upon receiving the pulse signal, the shutter of the X-ray source automatically opens, allowing the X-ray beam to pass through the capillary sample cell. The integrated SAXS (small-angle X-ray scattering) detector and WAXS (wide-angle X-ray scattering) detector are synchronously triggered, recording the scattering signal of the sample in real time. The detector converts the captured photon signal into a two-dimensional image, completing the initial acquisition of scattering data. If structural characterization of the sample at different concentrations or in different states is required, the liquid ratio or environmental variables of the pretreatment system can be changed. The device will automatically cycle through the four major processes of liquid preparation, liquid level positioning, microfluidic sample loading, and in-situ detection, completing the automated acquisition of in-situ scattering spectra of samples under multiple conditions.

[0087] Example 1: Gradual dilution of lysozyme concentration and in situ SAXS test

[0088] The pipetting station used in this embodiment is the core multi-channel liquid workstation of the aforementioned automatic liquid pretreatment module. It is equipped with a high-precision liquid delivery unit of injection pump type, a robotic arm, a temperature control and mixing unit, a mixing chamber connected by polytetrafluoroethylene pipeline and a high-precision flow meter. The mixing chamber has a built-in magnetic stir bar, which can achieve precise liquid preparation with a component error of less than 0.1%.

[0089] This embodiment describes the serial dilution of lysozyme and the completion of an in situ SAXS test. The specific steps are as follows:

[0090] 1) Take a 10.0 mg / mL lysozyme solution (Merck, catalog number L6876-1G) and PBS buffer, and place them in the corresponding positions of the pipetting station as the experimental stock solution. The stock solution is connected to the mixing chamber of the workstation through a polytetrafluoroethylene tube.

[0091] 2) Open the device software interface, select the corresponding plate positions for lysozyme stock solution and PBS buffer on the operation page, and set the specific scheme for sample gradient dilution in the system. The system will measure the liquid volume through a high-precision flow meter, and the stock solution will be pumped into the mixing chamber according to the ratio by the injection pump type high-precision liquid delivery unit. At the same time, the temperature control and mixing unit will be activated, and the liquid will be homogenized by the built-in magnetic stir bar in the mixing chamber to ensure that the error of the solution components is less than 0.1%.

[0092] 3) This serial dilution was set up with four sample protocols: Sample 1 was 60 μL of lysozyme stock solution (concentration 10.0 mg / mL); Sample 2 was 40 μL of lysozyme stock solution mixed with 20 μL of PBS buffer (concentration 6.7 mg / mL); Sample 3 was 30 μL of lysozyme stock solution mixed with 30 μL of PBS buffer (concentration 5.0 mg / mL); Sample 4 was 20 μL of lysozyme stock solution mixed with 40 μL of PBS buffer (concentration 3.3 mg / mL).

[0093] 4) Complete the stage position calibration operation in the software interface: push the pipette station to the designated operating position and fix it with the screw. Adjust the values ​​on the control panel to accurately align the loading needle with the A01 well. After completion, click the "Save Parameters" button on the system interface.

[0094] 5) Start the automated process of the device, and sequentially send the four sets of configured samples into the detection position for SAXS testing; perform one-dimensional data transformation, scattering intensity normalization, scattering absolute intensity correction, and scattering background subtraction on the acquired scattering images in sequence to finally obtain the SAXS scattering curves of different concentrations of lysozyme, such as... Figure 4 As shown.

[0095] Example 2: Synthesis and in-situ SAXS assay of lipid nanoparticles (mRNA-LNP) for mRNA delivery system

[0096] The pipetting station used in this embodiment is the core multi-channel liquid workstation of the aforementioned automatic liquid pretreatment module. It is equipped with a high-precision liquid delivery unit of injection pump type, a robotic arm, a temperature control and mixing unit, a mixing chamber connected by polytetrafluoroethylene pipeline and a high-precision flow meter. The mixing chamber has a built-in magnetic stir bar, which can achieve precise liquid preparation with a component error of less than 0.1%.

[0097] This embodiment describes the synthesis and in-situ SAXS assay of lipid nanoparticles (mRNA-LNP) for an mRNA delivery system. The specific steps are as follows:

[0098] 1) Preparation of lipid and nucleic acid stock solutions. Weigh 15 mg of DLin-MC3-DMA (MC3) (Shanghai Saikerui Biotechnology Co., Ltd., catalog number SCFC-343150), dissolve it thoroughly in 200 μL of anhydrous ethanol, and prepare a stock solution with a concentration of 75 mg / mL. Weigh 10 mg each of DSPC (Avanti® Polar Lipids, catalog number 850365P), cholesterol, and DMG-PEG (Avanti® Polar Lipids, catalog number 880151P), dissolve each in 1.0 mL of anhydrous ethanol, and prepare stock solutions with a concentration of 10 mg / mL. Measure 32.37 μL of MC3 stock solution, 94.82 μL of DSPC stock solution, 178.63 μL of cholesterol stock solution, and 45.16 μL of DMG-PEG stock solution, mix them, and bring the volume to 1 mL with anhydrous ethanol. Mix thoroughly to obtain the lipid stock solution.

[0099] Alternatively, take 23.76 μg of mRNA (see reference: Ling D, Li N, Pan X, et al. A new type of lipid nanoparticles with sphingomyelin-induced active lipid raft structures for improved mRNA cellular uptake[J]. Journal of Controlled Release, 2026, 391(c):114640.DOI:10.1016 / j.jconrel.2026.114640.), dissolve it in 10 mM citrate buffer (pH 4.0) and bring the volume to 1 mL to obtain the nucleic acid stock solution.

[0100] 2) Place the prepared lipid stock solution and nucleic acid stock solution into the corresponding plate positions of the pipetting station for later use.

[0101] 3) Open the device software interface, select the corresponding plate positions for the lipid mother liquor and nucleic acid mother liquor, and set the specific parameter scheme for sample pipetting and mixing in the system.

[0102] 4) Complete the stage position calibration operation in the software interface: push the pipette station to the designated operating position and fix it with the screw. Adjust the values ​​on the control panel to accurately align the loading needle with the A01 well. After completion, click the "Save Parameters" button on the system interface.

[0103] 5) After in-situ synthesis of mRNA-LNP is completed by starting the automated process of the device, the sample is sent to the detection site for SAXS testing; the acquired scattering images are preprocessed sequentially, including one-dimensional data transformation, scattering intensity normalization, absolute scattering intensity correction, and scattering background subtraction, to finally obtain the SAXS scattering curve of mRNA-LNP, as shown below. Figure 5 As shown.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A fully automated in-situ X-ray microstructure characterization apparatus, characterized in that, The device includes an automated liquid pretreatment module, an in-situ solution condition screening module, a high-throughput X-ray microstructure characterization module, an automated sample introduction and motion control module, and a system control module. The automated liquid pretreatment module includes a multi-channel liquid workstation, which includes a robotic arm, a high-precision liquid delivery unit, a sample tray and a reagent tray. It is also equipped with a liquid preparation structure consisting of a reagent bottle, polytetrafluoroethylene tubing, a mixing chamber and a high-precision flow meter, for completing the automatic sampling, preparation, dilution and transfer of liquid samples. The in-situ solution condition screening module includes a porous array reaction cell for parallel processing of samples under different solution conditions to form a multivariate solution condition combination. The high-throughput X-ray microstructure characterization module integrates a SAXS detection unit and / or a WAXS detection unit, and is used to perform in-situ microstructure characterization on samples after pretreatment and condition screening. The automatic sample introduction and motion control module includes at least one three-axis cantilever motion mechanism, a high-precision lifting mechanism and a microfluidic sample loading structure, which are used to realize the automatic transfer, precise positioning and accurate loading of samples between various functional modules. The system control module is electrically connected to the automatic liquid pretreatment module, the in-situ solution condition screening module, the high-throughput X-ray microstructure characterization module, and the automatic sample introduction and motion control module.

2. The apparatus of claim 1, wherein, The multi-channel liquid workstation of the automatic liquid pretreatment module is also equipped with a temperature control and mixing unit, which works in conjunction with the robotic arm and the high-precision liquid delivery unit. The high-precision liquid delivery unit is an injection pump, a plunger pump, or a droplet dispenser. The mixing chamber is equipped with a magnetic stir bar or an ultrasonic oscillation module. The system control module can drive multiple precision injection pumps or solenoid valve groups through a preset ratio scheme by the main control unit to draw the solvent and solute into the mixing chamber in proportion and complete the homogenization process.

3. The apparatus of claim 1, wherein, The in-situ solution condition screening module also includes a condition control unit and a status monitoring unit. The condition control unit is connected to the porous array reaction cell and the automatic liquid pretreatment module. The porous array reaction cell adopts an equivalent optical path and structural isolation design and is composed of multiple independently isolated reaction holes. Each reaction hole is used to independently hold samples with different solution conditions. The status monitoring unit is a dynamic light scattering detection component.

4. The apparatus of claim 1, wherein, The high-precision lifting mechanism of the automatic sample feeding and motion control module is a Z-axis vertical motion mechanism driven by a precision lead screw stepper motor, equipped with a loading platform and an origin sensor.

5. The apparatus of claim 1, wherein, The microfluidic sample loading structure of the automatic sample loading and motion control module includes a peristaltic pump, a sample loading capillary, and a capillary sample cell. The capillary sample cell is located at the center of the X-ray optical path, and the sample loading capillary is made of quartz glass. The peristaltic pump is connected to the main control unit via an RS485 level signal.

6. The apparatus according to claim 5, characterized in that, The microfluidic sample loading structure is also equipped with a photoelectric sensor, and the control algorithm of the system control module can detect the position of the liquid column through the photoelectric sensor.

7. The apparatus according to claim 1, characterized in that, The high-throughput X-ray microstructure characterization module is an original design module, which also includes a multi-pore array reaction cell automated sample introduction robotic arm, an X-ray source, a sample measurement cell, a detector, and a data acquisition system; the sample measurement cell is connected to the in-situ solution condition screening module or the automated sample introduction and motion control module; the X-ray source is equipped with a shutter, and the detector can convert the captured photon signal into a two-dimensional image to complete the acquisition and conversion of the scattered signal.

8. The apparatus according to claim 7, characterized in that, The automatic liquid pretreatment module and the high-throughput X-ray microstructure characterization module are linked by pulse signals; the system control module can control the device to cycle through the steps of liquid preparation, positioning, sample loading and detection by changing the ratio of the pretreatment system or environmental variables, so as to realize the in-situ scattering spectrum acquisition of different concentrations or states.

9. The apparatus according to claim 1, characterized in that, The system control module includes an automated sample preparation and injection system mechanical control system and an X-ray beamline control system, which interact with each other via a communication interface. The X-ray beamline control system uses the EPICS experimental physics and industrial control system architecture as its underlying control framework. The device employs a miniaturized liquid pretreatment unit and a flow-through sample measurement structure.

10. The apparatus according to claim 1, characterized in that, The device is suitable for soft matter and biomacromolecule systems.

Images