Micro thermophoretic molecule analysis system and method based on multi-laser cooperative control

Abstract

The invention belongs to the technical field of thermophoresis analysis, and particularly relates to a microscale thermophoresis molecule analysis system and method based on multi-laser cooperative control. The system is characterized in that a laser module outputs infrared laser and at least two preset excitation light sources through an optical beam combining unit; generating a local temperature gradient and fluorescence excitation in the microfluidic sample cavity based on the output light path; the fluorescence analysis module is used for detecting the intrinsic fluorescence change of the protein sample based on the sample temperature change so as to determine the thermal stability of the protein sample, and judging to give up the trace thermophoresis detection, re-processing the sample or carrying out the interaction detection of the trace thermophoresis based on the thermal stability analysis result; detecting fluorescence change caused by trace thermophoresis due to local temperature gradient generated based on infrared laser heating; and the characteristic determination module determines the molecular characteristics of the protein sample based on the trace thermophoresis detection result and the thermal stability detection result. According to the invention, the system applicability is improved.

Description

Technical Field

[0001] The present disclosure belongs to the technical field of thermophoresis analysis, and in particular to a micro-thermophoresis molecular analysis system and method based on multi-laser coordinated control. Background Art

[0002] Microscale Thermophoresis (MST) technology is an optical detection method that introduces a temperature gradient at the microscale to induce thermal surge migration of fluorescently labeled molecules and studies the interaction between molecules through changes in fluorescence intensity.

[0003] Traditional MST systems typically use a single-wavelength infrared laser (e.g., 1480 nm) to generate a local temperature gradient, followed by a single excitation light source (e.g., a blue or red LED) to excite the fluorescent probe. However, this single-wavelength laser and excitation light source system has application limitations. Summary of the Invention

[0004] To solve the above problems, the present disclosure provides a micro-thermophoresis molecular analysis system and method based on multi-laser coordinated control, which can solve the application limitations caused by the single wavelength.

[0005] In a first aspect, the present disclosure provides a micro-thermophoresis molecular analysis system based on multi-laser coordinated control, The system comprises, A laser module is used to output infrared laser and at least two preset excitation light sources through an optical beam combining unit, and generate a local temperature gradient and fluorescence excitation in the microfluidic sample cavity based on the output light path; The laser module includes: a first excitation light unit for providing a first excitation light source for thermal stability detection; a second excitation light unit for providing at least one visible light laser as a second excitation light source to excite a fluorescent marker or a FRET probe for micro-thermophoresis detection; a heating unit for heating a protein sample based on an infrared laser to obtain a temperature change of the sample; and an optical beam combining unit for combining any of the infrared lasers, the first excitation light source, and the second excitation light source to obtain the output light path, and focusing the output light path to a focal point in the microfluidic sample chamber to generate a local temperature gradient in the microfluidic sample chamber. A fluorescence analysis module is used to detect changes in intrinsic fluorescence of a protein sample based on changes in sample temperature, thereby determining the thermal stability of the protein sample based on the changes in intrinsic fluorescence. Based on the results of the thermal stability analysis, it is determined whether to abandon the microthermophoresis test and reprocess the sample, or to perform a microthermophoresis interaction test; The fluorescence analysis module further includes detecting fluorescence changes caused by microthermophoresis based on the local temperature gradient generated by the infrared laser heating; The property determination module is used to determine the molecular properties of the protein sample based on the microthermophoresis detection results and the thermal stability detection results.

[0006] Further, The heating unit also includes heating the sample by using a resistance heating technology to obtain a temperature change of the sample.

[0007] Further, The optical beam combining unit comprises: A control subunit, configured to achieve synchronization or step-by-step control of the infrared laser and the first excitation light source and the second excitation light source through a unified timing controller; The superposition subunit is used to superimpose the wavelengths of the regulated infrared laser, the first excitation light source and the second excitation light source on a coaxial path based on a multi-stage dichroic mirror or an acousto-optic tunable filter to obtain the output light path.

[0008] Further, The fluorescence analysis module includes: A thermal stability analysis unit is used to collect intrinsic fluorescence signals through a dual-channel fluorescence detector, calculate the curve of the fluorescence ratio of the intrinsic fluorescence signal changing with temperature, and fit the curve to obtain the actual Tm value and aggregation trend; a judgment unit connected to the thermal stability analysis unit, configured to compare the actual Tm value with a preset Tm threshold value to obtain a Tm comparison result, determine the aggregation trend of the change curve, obtain a trend judgment result, and determine whether to abandon the micro-thermophoresis detection and re-process the sample based on the Tm comparison result and the trend judgment result, or to perform a micro-thermophoresis interaction detection; The thermophoresis detection unit is used to collect FRET donor, acceptor or single-label fluorescence signals under the local temperature gradient, and analyze the interaction strength of the target molecules in the thermophoresis state through the change value of thermophoretic mobility.

[0009] Further, The thermal stability analysis unit comprises: A Tm value calculation unit is used to fit the change curve, determine the temperature point at which the slope of the curve corresponding to the change curve is the largest, and use this point as the actual Tm value; A trend judgment unit, configured to judge that the trend is gathering when the change curve shows abnormal fluctuation after the Tm value, or shows a plateau in advance before reaching the Tm value; If the curve is smooth and only a single inflection point occurs near the Tm value, it is judged that the trend is non-aggregated.

[0010] Further, The judging unit includes: Abandoning subunit, for abandoning the microthermophoresis detection and re-processing the sample when the actual Tm value is less than the preset Tm threshold, or the trend of the detection change curve is aggregation; The thermophoresis detection subunit is used to perform micro-thermophoresis interaction detection when the actual Tm value is greater than or equal to the preset Tm threshold and the trend of the detection change curve is non-aggregation.

[0011] Further, The thermophoresis detection unit comprises: An acquisition subunit, used to acquire FRET donor, acceptor or single-label fluorescence signals based on a multi-channel sCMOS or PMT sensor; A separation subunit for synchronously separating FRET donor, acceptor or single-label fluorescence signals based on a multi-bandwidth filter wheel; The fitting unit is used to obtain the fluorescence migration curve, calculate the thermophoretic mobility change value based on the fluorescence migration curve, and calculate the binding constant Kd based on the thermophoretic mobility change value fitted by the automatic fitting tool to determine the interaction strength.

[0012] Further, The microfluidic sample chamber is a quartz capillary structure with a volume of ≤10μL. A thermocouple temperature sensor is integrated at the bottom to provide real-time feedback of the solution temperature to the control subunit to achieve closed-loop power regulation.

[0013] Furthermore, an algorithm module is included to recommend an optimal buffer or optimal measurement conditions based on the actual Tm value and the aggregation onset temperature.

[0014] In the second aspect, based on the same inventive concept, the present disclosure provides a micro-thermophoresis molecular analysis method based on multi-laser coordinated control, The method comprises, The infrared laser and at least two preset excitation light sources are output through an optical beam combining unit, and a local temperature gradient and fluorescence excitation are generated in the microfluidic sample cavity based on the output light path; Among them, at least two excitation light sources are preset, including a first excitation light source for thermal stability detection, and at least one visible light laser as a second excitation light source for exciting fluorescent markers or FRET probes for micro-thermophoresis detection; and, heating the protein sample based on infrared laser to obtain the temperature change of the sample; and combining any of the infrared lasers, the first excitation light source, and the second excitation light source through an optical beam combining unit to obtain the output light path, and focusing the output light path onto a focal point in the microfluidic sample chamber to generate a local temperature gradient in the microfluidic sample chamber; Detecting intrinsic fluorescence changes of the protein sample based on changes in sample temperature to determine the thermal stability of the protein sample based on the intrinsic fluorescence changes, and deciding to abandon the microthermophoresis test and reprocess the sample based on the results of the thermal stability analysis, or to perform a microthermophoresis interaction test; and detecting fluorescence changes caused by microthermophoresis based on the local temperature gradient generated by the infrared laser heating; The molecular characteristics of the protein sample were determined based on the results of microthermophoresis and thermal stability tests.

[0015] Compared with the prior art, the present disclosure has the following advantages: 1. This invention achieves fully coaxial, unified output of laser thermal control and fluorescence excitation. By combining infrared laser light with multi-wavelength excitation beams through a multi-stage beam splitter or AOTF, it eliminates the misalignment of the laser hot zone and excitation region in conventional systems, improving spatial resolution and signal reproducibility.

[0016] 2. The system described in this invention supports multiple excitation wavelengths, enabling compatibility with multiple fluorescent probes, including FRET. The system is configured with at least two visible light lasers, such as 488nm, 561nm, and 635nm. The same platform supports simultaneous detection of multiple probes, including FITC, Cy5, and Alexa series. FRET donor-acceptor pairs are supported for analyzing molecular conformational changes or affinity states, significantly enhancing the platform's versatility and analytical depth.

[0017] 3. The system integrates protein thermal stability analysis (nanoDSF) to enhance data reliability. Prior to MST testing, the protein's intrinsic fluorescence thermal curve is used to determine its Tm value, conformational consistency, and aggregation propensity. This system automatically determines whether a sample is suitable for the MST stage, effectively eliminating sources of error and forming a closed-loop "quality control + testing" process.

[0018] The system and method described in this invention can automate the detection process, enabling linked judgment and parameter optimization. A single optical system enables nanoDSF evaluation and MST detection, driving the system to select a buffer solution or terminate the experiment, reducing manual intervention and improving experimental success rate and throughput.

[0019] Other features and advantages of the present disclosure will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present disclosure. The purposes and other advantages of the present disclosure can be realized and obtained by the structures indicated in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 The structure block diagram of the micro-thermophoresis molecular analysis system based on multi-laser coordinated control according to an embodiment of the present disclosure is shown; Figure 2 A structural block diagram of a specific example of a micro-thermophoresis molecular analysis system based on multi-laser coordinated control according to an embodiment of the present disclosure is shown; Figure 3 A structural block diagram showing another specific example of a micro-thermophoresis molecular analysis system based on multi-laser coordinated control according to an embodiment of the present disclosure is shown; Figure 4 A schematic diagram of the detection process of molecular interactions according to an embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present disclosure without making any creative efforts shall fall within the scope of protection of the present disclosure.

[0023] Figure 1 FIG. 4 shows a structural block diagram of a micro-thermophoresis molecular analysis system based on multi-laser coordinated control according to an embodiment of the present disclosure. Figure 1 As shown, the micro-thermophoresis molecular analysis system based on multi-laser coordinated control of the embodiment of the present disclosure includes: The laser module 10 is used to output infrared laser and at least two preset excitation light sources through an optical beam combining unit, and generate a local temperature gradient and fluorescence excitation in the microfluidic sample chamber based on the output light path; In the embodiment of the present disclosure, the laser module 10 includes: A first excitation light unit, for providing a first excitation light source with a wavelength of 280–295 nm for thermal stability detection; A second excitation light unit, for providing at least one visible light laser as a second excitation light source to excite fluorescent markers or FRET probes for microthermophoresis detection; A heating unit, used to heat the protein sample based on infrared laser to obtain the temperature change of the sample; An optical beam combining unit is used to combine any of the infrared lasers, the first excitation light source, and the second excitation light source to obtain the output light path, and focus the output light path to a focal point in the microfluidic sample chamber to generate a local temperature gradient in the microfluidic sample chamber.

[0024] In the disclosed embodiments, visible light lasers include 488 nm, 561 nm, and 635 nm. These wavelengths can be independently controlled in power, enabling selective excitation of different fluorescent markers (e.g., FITC, Cy5, etc.) or FRET (fluorescence resonance energy transfer) donor and acceptor pairs, thereby enabling specific excitation and acquisition of target molecule fluorescence signals in microthermophoresis assays.

[0025] In the embodiment of the present disclosure, the heating unit further comprises heating the sample by means of a resistance heating technology to obtain a temperature change of the sample.

[0026] In the embodiment of the present disclosure, the optical beam combining unit includes: A control subunit, configured to achieve synchronization or step-by-step control of the infrared laser and the first excitation light source and the second excitation light source through a unified timing controller; The superposition subunit is used to superimpose the wavelengths of the regulated infrared laser, the first excitation light source and the second excitation light source on a coaxial path based on a multi-stage dichroic mirror or an acousto-optic tunable filter to obtain the output light path.

[0027] In the disclosed embodiment, synchronization control requires simultaneous activation of the infrared laser (heating) and the dual-wavelength visible laser (excitation of the donor / acceptor) during FRET detection, with a time deviation of <1ms to avoid signal asynchrony. The step-by-step control for switching from nanoDSF to MST requires turning off the UV laser first and then turning on the visible laser, with a 10ms delay inserted in between to prevent crosstalk.

[0028] In the embodiment of the present disclosure, the response time of the control subunit is less than 10ms.

[0029] Fluorescence analysis module 20, used to detect changes in intrinsic fluorescence of the protein sample based on changes in sample temperature, to determine the thermal stability of the protein sample based on the changes in intrinsic fluorescence, and to decide whether to abandon the microthermophoresis test and re-process the sample based on the results of the thermal stability analysis, or to perform microthermophoresis interaction detection; The fluorescence analysis module 20 further includes detecting fluorescence changes caused by microthermophoresis based on the local temperature gradient generated by the infrared laser heating; In the embodiment of the present disclosure, the fluorescence analysis module 20 includes: Thermal stability analysis unit, used to collect protein intrinsic fluorescence signals through a dual-channel fluorescence detector (such as a 330nm and 350nm peak detector) and calculate the fluorescence ratio (F330 / F350) curve as a function of temperature; a judgment unit connected to the thermal stability analysis unit, configured to compare the actual Tm value with a preset Tm threshold value to obtain a Tm comparison result, determine the aggregation trend of the change curve, obtain a trend judgment result, and determine whether to abandon the micro-thermophoresis detection and re-process the sample based on the Tm comparison result and the trend judgment result, or to perform a micro-thermophoresis interaction detection; The thermophoresis detection unit is used to collect FRET donor, acceptor or single-label fluorescence signals under the local temperature gradient, and analyze the interaction strength of the target molecules in the thermophoresis state through the change value of thermophoretic mobility.

[0030] In the embodiment of the present disclosure, the thermal stability analysis unit includes: A Tm value calculation unit is used to fit the change curve using a sigmoid function fitting curve, determine the temperature point at which the slope of the curve corresponding to the change curve is the largest, and use this point as the actual Tm value (melting temperature); A trend judgment unit, configured to judge that the trend is gathering when the change curve shows abnormal fluctuation after the Tm value, or shows a plateau in advance before reaching the Tm value; If the curve is smooth and only a single inflection point occurs near the Tm value, it is judged that the trend is non-aggregated.

[0031] In the embodiment of the present disclosure, the abnormal fluctuation is a sudden increase or decrease in the fluorescence ratio after the Tm value.

[0032] In the embodiment of the present disclosure, the judgment unit includes: Abandoning subunit, for abandoning the microthermophoresis detection and re-processing the sample when the actual Tm value is less than the preset Tm threshold, or the trend of the detection change curve is aggregation; The thermophoresis detection subunit is used to perform micro-thermophoresis interaction detection when the actual Tm value is greater than or equal to the preset Tm threshold and the trend of the detection change curve is non-aggregation.

[0033] In the disclosed embodiments, the preset Tm threshold is set according to actual conditions, such as ≥40°C for enzymes and ≥60°C for antibodies.

[0034] In the embodiment of the present disclosure, the thermophoresis detection unit includes: An acquisition subunit, used to acquire FRET donor, acceptor or single-label fluorescence signals based on a multi-channel sCMOS or PMT sensor; A separation subunit for synchronously separating FRET donor, acceptor or single-label fluorescence signals based on a multi-bandwidth filter wheel; The fitting unit is used to obtain the fluorescence migration curve, calculate the thermophoretic mobility change value based on the fluorescence migration curve, and calculate the binding constant Kd based on the thermophoretic mobility change value fitted by the automatic fitting tool to determine the interaction strength.

[0035] In the disclosed embodiments, θ = L / (Kd + L), where θ is the binding fraction (0-1), L is the free ligand concentration, and Kd is the dissociation constant; △S=△S max ×θ, △S is the mobility change value, △S max is the maximum mobility change value at saturation binding (obtained by fitting), △S= (S bound -S free ) / S free , S free is the fluorescence gradient slope of the free molecule (ligand concentration = 0), S bound is the fluorescence gradient slope of the bound molecules.

[0036] In the disclosed embodiment, the microfluidic sample chamber is a quartz capillary structure with a volume of ≤10 μL. A thermocouple temperature sensor is integrated at the bottom to provide real-time feedback of the solution temperature to the control subunit to achieve closed-loop power regulation.

[0037] In the disclosed embodiment, the temperature change is monitored in real time by a sensor, and the controller automatically adjusts the control of the heating power according to the deviation.

[0038] In the embodiment of the present disclosure, an algorithm module is further included to recommend an optimal buffer solution or measurement conditions based on the actual Tm value and the aggregation onset temperature.

[0039] In the disclosed embodiments, an example of an algorithm module is that the selection of a buffer should be aimed at reducing protein aggregation and improving thermal stability (optimizing the Tm value). Specific recommendation rules include: if the actual Tm value is lower than a preset Tm threshold (e.g., 45°C), indicating that the protein has poor thermal stability, the algorithm module will prioritize recommending a buffer that can improve protein stability (e.g., Tris-HCl or HEPES, which have a more stable pH buffer range and are more likely to maintain protein conformation than PBS); if the Tm value is higher (e.g., ≥60°C), the more versatile PBS may be recommended; If the aggregation initiation temperature is low (i.e., the protein begins to aggregate at a low temperature), the algorithm module will exclude buffers that are prone to inducing aggregation (such as systems containing high salt or detergents) and recommend buffers with low ionic strength and no UV absorption (such as 10-50mM Tris-HCl, pH 7.0-7.5) to delay aggregation; The algorithm module will call the historical detection data of similar proteins. If a certain type of buffer shows a lower aggregation rate in samples with similar Tm values ​​and aggregation onset temperatures, this buffer will be recommended first.

[0040] Measurement conditions, including infrared laser power, heating rate, and excitation light parameters, must be adapted to the thermal stability and aggregation characteristics of the protein. The specific rules are as follows: If the protein aggregation onset temperature is low (prone to aggregation), the algorithm module recommends reducing the infrared laser power (e.g., 20-30 mW) to reduce local thermal stress; If the Tm value is high (good thermal stability), the power can be appropriately increased (e.g., 40-50 mW) to enhance the temperature gradient signal; For proteins with low Tm values ​​or early aggregation onset temperatures, it is recommended to reduce the heating rate (e.g., 1-2°C / min) to avoid rapid heating that may cause sudden denaturation and aggregation of the protein. For proteins with good stability, a rate of 3-5°C / min can be used to shorten the detection time.

[0041] For proteins that are prone to aggregation, it is recommended to use a homogenized spot size of ≥1 mm to ensure uniform heating of the solution and reduce aggregation caused by local overheating.

[0042] The property determination module 30 is used to determine the molecular properties of the protein sample based on the microthermophoresis detection results and the thermal stability detection results.

[0043] Figure 2 This is a structural block diagram of a specific example of the embodiment of the present disclosure, see Figure 2 As shown, it includes an infrared laser heating unit 101, excitation light 102, excitation light 103, an optical beam combining module 1001, a microfluidic sample chamber 13, a differential scanning fluorescence (nanoDSF) module 1002, and a fluorescence detection unit 203. Excitation light 102, serving as the excitation light for nanoDSF detection, has a wavelength range of 280 nm to 295 nm, preferably 280 nm. The core signal of nanoDSF comes from tryptophan within the protein, with a maximum absorption peak at 280 nm. Excitation light 103 serves as the excitation light source for the fluorescence signal detected by microsurge calorimetry (MST).

[0044] The optical beam combining module 1001 is composed of a primary dichroic mirror 111, a secondary dichroic mirror 112, a perforated reflector 11, and an objective lens 12. This ensures that the infrared laser heating unit 101, the excitation light 102, and the excitation light 103 are combined and focused onto a focal point within the microfluidic sample chamber 13. The fluorescence signal generated by the sample in the microfluidic sample chamber 13 is collected by the objective lens 12 and reflected by the perforated reflector 11. It is then split by the spectrometer modules 121, 122, and 123 before entering detectors 201, 202, and 203, respectively. The spectrometer modules are preferably dichroic mirrors. Detectors 201 and 202 are 330nm and 350nm peak detectors, respectively. Detector 203 is the fluorescence signal detector for MST detection and can be any of a CMOS, CCD, PMT, PD, or APD, with a PMT being preferred.

[0045] Figure 3 A structural block diagram showing another specific example of a micro-thermophoresis molecular analysis system based on multi-laser coordinated control according to an embodiment of the present disclosure is shown. Figure 3 As shown, the system Figure 2 The system also includes an excitation light source 104, a spectrometer module 113 attached to the optical beam combiner 1001, a detector 204, and a spectrometer module 124. It is used to dynamically monitor FRET-labeled conformational switching aptamers. It is used to analyze the conformational switching behavior of DNA aptamers when they bind to small molecules. The system synthesizes G-quadruplex aptamers labeled with Cy3 at the 5' end (the 5' end refers to the end with a phosphate group attached to the 5th carbon atom of the deoxyribose sugar) and Cy5 at the 3' end (the 3' end refers to the end with a hydroxyl group attached to the 3rd carbon atom). Its Tm value and G structure stability are analyzed using nanoDSF. The infrared laser unit 101 is activated to increase the temperature to form a temperature gradient. The FRET donor and acceptor are excited using excitation light 103 at a wavelength of 561 nm and excitation light 104 at a wavelength of 635 nm, respectively. Detectors 203 and 204 collect the FRET donor and acceptor signals, and the change in FRET efficiency is calculated to determine whether the aptamer undergoes conformational change due to target molecule binding.

[0046] Based on the above system, the present disclosure also provides a micro-thermophoresis molecular analysis method based on multi-laser coordinated control corresponding to the above system, the method comprising: The infrared laser and the preset excitation light source are output through an optical beam combining unit to obtain an output light path, and a local temperature gradient is generated in the microfluidic sample cavity based on the output light path; Detecting intrinsic fluorescence changes of the protein sample based on the local temperature gradient to determine the thermal stability of the protein sample according to the intrinsic fluorescence changes, and deciding to abandon the microthermophoresis test and re-process the sample based on the thermal stability analysis results, or to perform microthermophoresis interaction detection; The molecular characteristics of the protein sample were determined based on the results of microthermophoresis and thermal stability tests.

[0047] Figure 4 A schematic diagram of the detection process of the embodiment of the present disclosure is shown. Figure 4 Shown, including: Sample preparation: Mix the purified target protein with the ligand, use a buffer without high UV absorption (such as PBS), control the protein concentration to 0.05–1 mg / ml, and load 10 μL.

[0048] Thermal stability test: Start the first excitation light source (280 nm) and infrared laser (1450 nm, power 20–50 mW), increase the temperature at a rate of 1–5 °C / min, and record the fluorescence signal every 0.5–1 s using a dual-channel detector.

[0049] The thermal stability analysis unit calculates the F330 / F350 curve and fits it to obtain the actual Tm value and aggregation trend.

[0050] Quality control judgment: The judgment unit compares the actual Tm value with the preset threshold value and combines the aggregation trend: If the standards are not met, prompt to reprocess the sample (e.g., replace the buffer); If the criteria are met, proceed to the next step.

[0051] Thermophoresis detection: The infrared laser forms a stable temperature gradient and synchronously starts the second excitation light source (such as 488nm and 561nm) to excite the fluorescent marker.

[0052] The thermophoresis detection unit collects the fluorescence signal and calculates the mobility change value and Kd.

[0053] Result output: The characteristic determination module 30 generates a comprehensive report, and the algorithm module recommends an optimization solution.

[0054] Example 1: The binding affinity between a class of tyrosine kinase inhibitors and their target protein, EGFR, was analyzed. The nanoDSF module was used to screen the conformational consistency of the expressed and purified EGFR, ensuring that the Tm value was above 45°C and that there were no multimodal structures. The EGFR sample was loaded into the microfluidic sample chamber 13, and a temperature gradient was established using a 1450nm infrared laser heating unit 101. Simultaneously, 488nm excitation light 103 was activated to excite the FITC label, and the thermal surge response curve was recorded. Different concentrations of small molecule drug candidates were added, and the MST fluorescence changes were measured and fitted to the binding constant, Kd. This integrated sample quality control and affinity testing improves data accuracy.

[0055] Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A micro-thermophoresis molecular analysis system based on multi-laser coordinated control, characterized in that: The system comprises, A laser module is used to output infrared laser and at least two preset excitation light sources through an optical beam combining unit, and generate a local temperature gradient and fluorescence excitation in the microfluidic sample cavity based on the output light path; The laser module includes: a first excitation light unit for providing a first excitation light source for thermal stability detection; a second excitation light unit for providing at least one visible light laser as a second excitation light source to excite a fluorescent marker or a FRET probe for micro-thermophoresis detection; a heating unit for heating a protein sample based on an infrared laser to obtain a temperature change of the sample; and an optical beam combining unit for combining any of the infrared lasers, the first excitation light source, and the second excitation light source to obtain the output light path, and focusing the output light path to a focal point in the microfluidic sample chamber to generate a local temperature gradient in the microfluidic sample chamber. A fluorescence analysis module is used to detect changes in intrinsic fluorescence of a protein sample based on changes in sample temperature, thereby determining the thermal stability of the protein sample based on the changes in intrinsic fluorescence. Based on the results of the thermal stability analysis, it is determined whether to abandon the microthermophoresis test and reprocess the sample, or to perform a microthermophoresis interaction test; The fluorescence analysis module further includes detecting fluorescence changes caused by microthermophoresis based on the local temperature gradient generated by the infrared laser heating; The property determination module is used to determine the molecular properties of the protein sample based on the microthermophoresis detection results and the thermal stability detection results.

2. The system according to claim 1, wherein: The heating unit also includes heating the sample by using a resistance heating technology to obtain a temperature change of the sample.

3. The system according to claim 2, characterized in that The optical beam combining unit comprises: A control subunit, configured to achieve synchronization or step-by-step control of the infrared laser and the first excitation light source and the second excitation light source through a unified timing controller; The superposition subunit is used to superimpose the wavelengths of the regulated infrared laser, the first excitation light source and the second excitation light source on a coaxial path based on a multi-stage dichroic mirror or an acousto-optic tunable filter to obtain the output light path.

4. The system according to claim 3, characterized in that The fluorescence analysis module includes: A thermal stability analysis unit is used to collect intrinsic fluorescence signals through a dual-channel fluorescence detector, calculate the curve of the fluorescence ratio of the intrinsic fluorescence signal changing with temperature, and fit the curve to obtain the actual Tm value and aggregation trend; a judgment unit connected to the thermal stability analysis unit, configured to compare the actual Tm value with a preset Tm threshold value to obtain a Tm comparison result, determine the aggregation trend of the change curve, obtain a trend judgment result, and determine whether to abandon the micro-thermophoresis detection and re-process the sample based on the Tm comparison result and the trend judgment result, or to perform a micro-thermophoresis interaction detection; The thermophoresis detection unit is used to collect FRET donor, acceptor or single-label fluorescence signals under the local temperature gradient, and analyze the interaction strength of the target molecules in the thermophoresis state through the change value of thermophoretic mobility.

5. The system according to claim 4, characterized in that The thermal stability analysis unit comprises: A Tm value calculation unit is used to fit the change curve, determine the temperature point at which the slope of the curve corresponding to the change curve is the largest, and use this point as the actual Tm value; A trend judgment unit, configured to judge that the trend is gathering when the change curve shows abnormal fluctuation after the Tm value, or shows a plateau in advance before reaching the Tm value; If the curve is smooth and only a single inflection point occurs near the Tm value, it is judged that the trend is non-aggregated.

6. The system according to claim 5, characterized in that The judging unit includes: Abandoning subunit, for abandoning the microthermophoresis detection and re-processing the sample when the actual Tm value is less than the preset Tm threshold, or the trend of the detection change curve is aggregation; The thermophoresis detection subunit is used to perform micro-thermophoresis interaction detection when the actual Tm value is greater than or equal to the preset Tm threshold and the trend of the detection change curve is non-aggregation.

7. The system according to claim 6, characterized in that The thermophoresis detection unit comprises: An acquisition subunit, used to acquire FRET donor, acceptor or single-label fluorescence signals based on a multi-channel sCMOS or PMT sensor; A separation subunit for synchronously separating FRET donor, acceptor or single-label fluorescence signals based on a multi-bandwidth filter wheel; The fitting unit is used to obtain the fluorescence migration curve, calculate the thermophoretic mobility change value based on the fluorescence migration curve, and calculate the binding constant Kd based on the thermophoretic mobility change value fitted by the automatic fitting tool to determine the interaction strength.

8. The system according to claim 7, characterized in that The microfluidic sample chamber is a quartz capillary structure with a volume of ≤10μL. A thermocouple temperature sensor is integrated at the bottom to provide real-time feedback of the solution temperature to the control subunit to achieve closed-loop power regulation.

9. The system according to claim 8, characterized in that The method further comprises an algorithm module for recommending an optimal buffer or optimal measurement conditions according to the actual Tm value and the aggregation onset temperature.

10. A microthermophoresis molecular analysis method based on multi-laser coordinated control, using the system according to claims 1-9, characterized in that: The method comprises, The infrared laser and at least two preset excitation light sources are output through an optical beam combining unit, and a local temperature gradient and fluorescence excitation are generated in the microfluidic sample cavity based on the output light path; Among them, at least two excitation light sources are preset, including a first excitation light source for thermal stability detection, and at least one visible light laser as a second excitation light source for exciting fluorescent markers or FRET probes for micro-thermophoresis detection; and, heating the protein sample based on infrared laser to obtain the temperature change of the sample; and combining any of the infrared lasers, the first excitation light source, and the second excitation light source through an optical beam combining unit to obtain the output light path, and focusing the output light path onto a focal point in the microfluidic sample chamber to generate a local temperature gradient in the microfluidic sample chamber; Detecting intrinsic fluorescence changes of the protein sample based on changes in sample temperature to determine the thermal stability of the protein sample based on the intrinsic fluorescence changes, and deciding to abandon the microthermophoresis test and reprocess the sample based on the results of the thermal stability analysis, or to perform a microthermophoresis interaction test; and detecting fluorescence changes caused by microthermophoresis based on the local temperature gradient generated by the infrared laser heating; The molecular characteristics of the protein sample were determined based on the results of microthermophoresis and thermal stability tests.

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