Amino acid rapid analysis method

Abstract

The invention belongs to the technical field of amino acid detection and analysis, and provides a rapid analysis method for amino acid, which efficiently completes derivatization of amino acid and fluorescein isothiocyanate in a short time by utilizing a microwave-assisted reaction, and remarkably shortens the traditional time-consuming and tedious pretreatment time. A capillary electrophoresis technology is adopted, derivatized amino acid is separated under constant voltage, and by monitoring voltage and current signals in the electrophoresis process in real time, the migration charge density independent of single migration time is calculated to serve as a qualitative criterion. The separated components are subjected to high-sensitivity detection through a diaphragm coupled laser-induced fluorescence system. Finally, qualitative and quantitative analysis is performed in combination with migration time and migration charge density, and identification of various common amino acids can be realized without a standard substance. According to the invention, full-process automation from sample introduction to result output is realized, the analysis speed is high, the sample consumption is low, and the reproducibility is high.

Description

Technical Field

[0001] This invention relates to the field of amino acid detection and analysis technology, and in particular to a rapid amino acid analysis method. Background Technology

[0002] The existing technology has the following drawbacks: Currently, the mainstream analytical platforms for amino acids are chromatographic, meaning that amino acid analysis generally relies on large analytical instruments such as high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS), which require complex gradient elution programs to achieve amino acid separation. The equipment used is bulky and expensive, requires dedicated personnel for daily maintenance, and involves frequent replacement of consumables and parts, making it unsuitable for the needs of grassroots laboratories or rapid on-site testing.

[0003] Sample pretreatment is cumbersome: Because amino acids themselves do not have strong UV / fluorescence absorption and their detection sensitivity is insufficient, manual derivatization methods (such as OPA / FDAA / AccQ-Tag) are usually required before analysis to react the amino acids in the sample and convert them into a detectable form. This step requires strict control of temperature, time, and pH, and is prone to introducing errors.

[0004] The analysis process is lengthy: Traditional chromatography requires several hours to analyze a single sample. The entire process includes multiple steps such as sample preparation, injection, elution, detection and data processing, which greatly limits the analytical throughput and efficiency.

[0005] Limited ability to identify unknown components: Existing analytical systems have difficulty identifying unknown amino acids or derivatives in the absence of standards, relying on manual interpretation, and have low versatility and automation. Summary of the Invention

[0006] Therefore, one object of the present invention is to provide a rapid amino acid analysis method to solve the problems mentioned in the background art and overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention provides a rapid amino acid analysis method, comprising: Capillary pre-cleaning and activation, and air peak calibration; The sample is automatically injected through a capillary tube, mixed with the derivatization reagent, and subjected to a microwave-assisted derivatization reaction. The derivatized samples were separated by capillary electrophoresis, and the migration charge density and migration time were measured and recorded in real time during the separation process. Simultaneously, real-time optical detection was performed on the separated amino acid derivatives; Based on the detection signal, qualitative and quantitative analysis of amino acids is performed according to migration time and migration charge density.

[0008] Preferably, the capillary pre-cleaning and activation and air peak calibration include: First, the capillary is pre-cleaned by rinsing with alkaline washing solution to remove residue from the inner wall. Then, it is neutralized by rinsing with ultrapure water. Finally, it is activated by rinsing with separation buffer to bring the capillary to a stable separation state. An air pulse is applied at the capillary inlet to achieve air peak calibration.

[0009] Preferably, the alkaline washing solution is a 0.5 mol / L NaOH solution, and the rinsing time is 90 seconds; The ultrapure water rinsing time is 45 seconds; The separation buffer contains 40 mM borate and 15 mM sulfonated cyclodextrin derivative, and the rinsing time is 110 seconds. The air peak calibration is achieved by applying a 0.2-second air pulse.

[0010] Preferably, the derivatizing reagent is a dimethyl sulfoxide solution containing fluorescein isothiocyanate at a concentration of 10 mg / mL; The microwave-assisted derivatization was performed by heating at 150W power for 60 seconds at a reaction temperature of 50°C. The automatic sampler uses a positive pressure injection method with a pressure of 10 Pa and an injection time of 5 seconds.

[0011] Preferably, in the electrophoretic separation, capillary electrophoresis is carried out at a constant temperature of 22°C, and a constant voltage of 26.8kV is applied to both ends of the capillary to drive the derivatized amino acids to migrate along the effective length of the capillary of 83cm.

[0012] Preferably, the migration charge density is calculated using the instantaneous voltage and instantaneous current of the real-time reverse output.

[0013] Preferably, in the real-time optical detection, a laser with a wavelength of 488nm and a power of 25mW is used as the light source. The laser passes through a grating, a collimating lens, a focusing lens, and an aperture in sequence before illuminating the capillary detection area and generating fluorescence.

[0014] Preferably, the fluorescence signal is sampled by a photomultiplier tube at a sampling frequency of 25 Hz to obtain real-time fluorescence intensity data, and the dynamic gain adjustment of the photomultiplier tube is determined based on the electrophoretic current signal.

[0015] Preferably, during the synchronization of capillary electrophoretic separation and real-time optical detection, dynamic gain adjustment is performed, specifically including: Using the initial current at the start of electrophoresis as a reference, the changes in the real-time current during the separation process are monitored in real time. When the real-time current fluctuates relative to the initial current, the operating voltage of the photomultiplier tube is automatically adjusted according to a preset proportional control algorithm to compensate for the fluctuation in fluorescence signal intensity caused by changes in sample migration speed.

[0016] Preferably, the qualitative and quantitative analysis of amino acids based on the detection signal, according to migration time and migration charge density, includes: Baseline correction and filtering are performed on real-time fluorescence intensity data to obtain electrophoresis spectra and peak identification is completed, and the migration time of each target peak is extracted; Based on the comparison of characteristic parameters such as migration time and migration charge density with the built-in database, the identification of amino acid types and the quantification of concentrations can be achieved under the condition of no standard.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention employs an automated capillary activation process involving alkaline washing, neutralization in ultrapure water, and balancing with separation buffer. This ensures that the capillary inner wall is in a stable and consistent initial state. Furthermore, air peak calibration further corrects for drift in the detection window position and time axis. This solves the problem of poor separation reproducibility caused by residues on the capillary inner wall or inconsistent initial states. It ensures the reproducibility of migration time and peak shape during each electrophoretic separation from the source, achieving highly consistent electrophoretic separation and laying a stable foundation for subsequent qualitative and quantitative analysis.

[0018] This invention calculates the migration charge density using real-time feedback of instantaneous voltage and current signals, and combines the migration charge density with the migration time as a qualitative criterion. This overcomes the limitations of traditional capillary electrophoresis, which relies solely on the migration time, which is susceptible to temperature and electric field fluctuations, for identification. Since the migration charge density simultaneously reflects the electric field strength, effective capillary length, and electrophoretic state, it can significantly reduce the influence of factors such as temperature fluctuations and changes in liquid level. This allows the qualitative judgment to offset the interference of common factors, making the qualitative identification more stable and reliable when facing complex or unknown samples.

[0019] This invention employs a grating for beam shaping and uses a collimating lens and a focusing lens to focus the laser beam onto the capillary detection window. Simultaneously, an aperture is used to limit the spot size, effectively suppressing stray light. This optical path structure maintains a signal-to-noise ratio consistently at ≥100:1, significantly superior to traditional single-lens excitation methods, providing higher sensitivity for the detection of weakly fluorescent amino acid derivatives.

[0020] This invention introduces a fluorescein internal standard, and both the qualitative and quantitative processes are calibrated with the internal standard peak as a reference, so that the detection results are not affected by different sample matrices, conductivity differences or slight operational deviations; the combination of internal standard calibration and migration charge density calculation greatly improves the accuracy and repeatability of amino acid identification.

[0021] This invention employs a fixed-power microwave-assisted heating method, enabling the FITC derivatization of amino acids to be completed within 60 seconds, significantly faster than traditional water bath or chemical heating methods (which typically require 15–30 minutes). Combined with automated sample introduction, this invention allows for single-sample pretreatment to be completed within minutes, making it suitable for high-throughput analysis scenarios.

[0022] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0023] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic flowchart of a rapid amino acid analysis method according to the present invention; Figure 2 This is a front view of the analyzer of the present invention; Figure 3 This is a schematic diagram of the analyzer of the present invention with part of the housing hidden. Figure 4 This is an overall assembly diagram of the detection unit and separation unit of the present invention; Figure 5 This is an isometric view of the detection unit of the present invention; Figure 6 This is a front view of the detection unit of the present invention; Figure 7 This is a right view of the detection unit of the present invention; Figure 8 This is an isometric view of the separation unit of the present invention; Figure 9 This is a front view of the separation unit of the present invention; Figure 10 This is a rear view of the separation unit of the present invention; Figure 11 for Figure 10 The AA section view is shown below; Figure 12 for Figure 10 The BB section view shown; Figure 13 This is an isometric view of the sample injection and cleaning control unit of the present invention; Figure 14 This is a rear view of the sample injection and cleaning control unit of the present invention. Figure 15 This is a top view of the two-dimensional mobile platform of the present invention; Figure 16 This is a schematic diagram of the reagent kit of the present invention; Figure 17This is an isometric view of the sample dispensing needle assembly of the present invention; Figure 18 This is a cross-sectional view of the sample dispensing needle assembly of the present invention.

[0024] The components include: 1. Housing; 2. Sample inlet and cleaning control unit; 3. Separation unit; 4. Detection unit; 5. Photomultiplier tube; 6. First connection port; 7. Adjustable grating; 8. Triaxial adjuster for the photomultiplier tube; 9. Capillary slot; 10. Sample inlet; 11. Sample outlet; 12. Fixing component; 13. Fixing block; 14. Constant temperature fan; 15. First through hole; 16. Laser light source; 17. Adjustable platform; 18. Second connection port; 19. Second through hole. 20. Sample carrying platform; 21. Lifter; 22. Two-dimensional moving platform; 23. Sample holder; 24. Reagent table; 25. Reagent kit; 26. Tube slot; 27. Sample vial; 28. Base plate; 29. ​​First guide rail; 30. Second guide rail; 31. First slide; 32. Second slide; 33. Motor; 34. Lead screw; 35. Guide block; 36. Sample dispensing needle assembly; 37. Needle holder; 38. Capillary sample dispensing needle; 39. Capillary guide slot. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0026] On the one hand, such as Figure 2 and Figure 3 As shown, the present invention provides an amino acid analyzer, comprising: 2. Sample introduction and cleaning control unit for automatically introducing nano-sized micro-samples and performing cleaning and activation operations; 3. At least one separation unit for capillary electrophoresis separation of amino acid components in the sample; and 4. At least one detection unit for real-time optical detection of the separated amino acid components. The separation unit 3 and the detection unit 4 are located above the sample injection and cleaning control unit 2. The separation unit 3 is fluidly connected to the sample injection and cleaning control unit 2, and the detection unit 4 is connected to the separation unit 3.

[0027] It is understood that the number of separation unit 3 and detection unit 4 in this invention is at least one; in one embodiment, such as... Figure 3 As shown, there are four separation units 3 and four detection units 4. That is to say, the number of separation units 3 and four detection units 4 can be adaptively reduced or increased according to actual needs.

[0028] The fluorescent capillary amino acid analyzer of this invention successfully overcomes the main defects inherent in traditional amino acid analysis platforms that rely on large-scale chromatography-mass spectrometry instruments by employing miniaturized and integrated capillary electrophoresis-fluorescence detection technology. With its compact structural design and nano-scale micro-injection system, it significantly reduces the purchase, consumables, and maintenance costs of the equipment. Through a highly automated numerical control unit and simplified fluid path, it greatly improves the convenience and stability of operation. It eliminates the dependence on a professional laboratory environment and can achieve efficient and low-cost amino acid analysis in grassroots laboratories or on-site rapid detection scenarios.

[0029] Furthermore, such as Figures 4-7 As shown, the detection unit 4 includes a photomultiplier tube 5 and a connection port. An adjustable grating 7 is provided on the side of the photomultiplier tube 5 near the separation unit 3. A triaxial adjuster 8 is provided above the photomultiplier tube 5 and the adjustable grating 7. One end of the connection port passes through the adjustable grating 7 and is connected to the photomultiplier tube 5. The other end of the connection port is connected to the separation unit 3.

[0030] As one implementation method, such as Figures 5-9 As shown, the connection port includes a first connection port 6 and a second connection port 18. The first connection port 6 is disposed on the detection unit 4, and the second connection port 18 is disposed on the separation unit 3. The first connection port 6 on the detection unit 4 and the second connection port 18 on the separation unit 3 are connected to realize the connection between the separation unit 3 and the detection unit 4.

[0031] The detection unit 4 of the present invention includes a photomultiplier tube 5, a connector, an adjustable grating 7, and a triaxial adjuster 8. The photomultiplier tube 5 is connected to the separation unit 3 through the connector, the adjustable grating 7 is used to adjust the optical path, and the triaxial adjuster allows for fine-tuning of the position to optimize signal acquisition. This design enhances the sensitivity and stability of detection, ensures accurate capture of weak light signals in fluorescence detection mode, improves the accuracy of quantitative analysis, and thus enhances the reliability of the instrument in complex sample analysis.

[0032] Furthermore, the separation unit 3 also includes a high-voltage power supply module, which is used to apply a constant voltage to both ends of the capillary and output instantaneous voltage and current signals in real time.

[0033] Furthermore, such as Figure 8 , Figure 9 and Figure 10As shown, the separation unit 3 includes a capillary slot 9, which has a cavity inside. The capillary slot 9 has an inlet 10 and an outlet 11 below it. The capillary slot 9 is fixed to the front of the fixing block 13 by a fixing member 12. The back of the fixing block 13 is provided with a constant temperature fan 14. The fixing block 13 is connected to the cavity inside the capillary slot 9 through a first through hole 15 at the position where the constant temperature fan 14 is installed.

[0034] It is understandable that a capillary tube is installed and fixed in the cavity inside the capillary slot 9, and the capillary tube is connected to the sample inlet 10.

[0035] The separation unit 3 includes a capillary slot 9, a cavity, an inlet 10, an outlet 11, a fixing block 13, and a thermostatic fan 14. The thermostatic fan 14 is connected to the cavity through a first through hole 15 to achieve precise temperature control of the capillary region. This thermostatic design ensures that the electrophoretic separation process is carried out in a stable environment, reduces the impact of temperature fluctuations on migration time and separation efficiency, and improves the accuracy of amino acid component separation. It is suitable for clinical or scientific research applications with strict requirements.

[0036] Furthermore, such as Figure 10 , Figure 11 and Figure 12 As shown, the separation unit 3 further includes a laser light source 16 and an adjustable platform 17. The laser light source 16 is disposed on the adjustable platform 17. The laser light source 16 and the adjustable platform 17 are disposed on the back side of the fixed block 13. A second through hole 19 is provided on the back side of the fixed block 13 directly opposite the laser light source 16.

[0037] The separation unit 3 also includes a laser source 16 and an adjustable platform 17. The laser source 16 is aligned with the sample through a second through-hole 19, and the adjustable platform 17 allows the position of the light source to be adjusted to optimize the excitation optical path. This design supports flexible fluorescence detection modes, can adapt to different amino acid labeling methods, enhance the selectivity and sensitivity of detection, thereby expanding the application range of the instrument and improving the performance of sample analysis.

[0038] Understandably, the separation unit 3 also integrates a high-voltage DC power supply and an electrode system (i.e., a high-voltage power supply module). The high-voltage DC power supply, electrode system, and capillary are used to form a migration charge density separation path, and the electrophoresis principle is used to efficiently separate various amino acid components.

[0039] Furthermore, such as Figure 13 and Figure 14As shown, the sample loading and cleaning control unit 2 includes a sample loading platform 20, a lifter 21 is connected below the sample loading platform 20, and a two-dimensional moving platform 22 is connected below the lifter 21. The two-dimensional moving platform 22 is used to move the sample loading platform 20 along the XY axis in the horizontal plane, and the lifter 21 is used to move the sample loading platform 20 along the Z axis in the longitudinal direction.

[0040] The sample loading and cleaning control unit 2 includes a sample platform 20, a lifter 21, and a two-dimensional moving platform 22, which enables precise movement and positioning of the sample in the XYZ axis direction. This automated design improves the speed and accuracy of sample processing, reduces human error, supports high-throughput continuous analysis, and enhances the efficiency and reliability of the instrument.

[0041] Furthermore, such as Figure 13 and Figure 16 As shown, the sample carrying platform 20 includes a sample carrier 23, which is provided with an a×b matrix reagent station 24. Each reagent station 24 is provided with a reagent kit 25, and each reagent kit 25 is provided with a c×d matrix tube slot 26. Each tube slot 26 accommodates a sample vial 27.

[0042] The sample loading platform 20 adopts a matrix reagent station 24 and tube slot 26 design, which can efficiently accommodate and organize multiple sample vials 27 and reagent kits 25. This layout optimizes the placement of samples and reagents, and together with the automated moving platform, enables rapid sequence analysis, improves the working efficiency and sample throughput of the instrument, and is particularly suitable for large-scale or batch detection scenarios.

[0043] Furthermore, such as Figure 15 As shown, the two-dimensional mobile platform 22 includes a base plate 28, on which a first guide rail 29 and a second guide rail 30 are provided. A first slide 31 is provided on the first guide rail 29, and a second slide 32 is provided on the second guide rail 30. The lifting device 21 is connected to the first slide 31 and the second slide 32 through a support plate. The first guide rail 29 and the second guide rail 30 are arranged parallel to each other. A motor 33 is provided at one end of the first guide rail 29. The motor 33 is connected to a lead screw 34. The lead screw 34 is perpendicular to the first guide rail 29 and the second guide rail 30. The lead screw 34 is connected to a guide block 35, which is connected to the sample carrying platform 20.

[0044] The two-dimensional moving platform 22 includes a base plate 28, a guide rail, a slide table, a motor 33, a lead screw 34, and a guide block 35. Precise positioning is achieved by driving the motor 33. This high-precision moving mechanism ensures the repeatability and accuracy of sample import, reduces positional errors, and improves the overall performance and stability of the instrument.

[0045] Furthermore, such as Figure 17 and Figure 18 As shown, both the inlet 10 and the outlet 11 are connected to a sample dispensing needle assembly 36. The sample dispensing needle assembly 36 includes a needle holder 37 and a capillary sample dispensing needle 38. The needle holder 37 is provided with a capillary guide groove 39 that penetrates the needle holder 37. One end of the capillary guide groove 39 is connected to the inlet 10 and the outlet 11, and the other end of the capillary guide groove 39 is connected to the capillary sample dispensing needle 38.

[0046] The sample dispensing needle assembly 36 includes a needle holder 37 and a capillary dispensing needle 38. The capillary guide groove 39 ensures smooth sample introduction and outflow. This design minimizes sample dead volume and contamination risk, improves injection accuracy and separation efficiency, and is especially important for the analysis of nanoscale micro-samples, ensuring the reliability of detection results.

[0047] Furthermore, the analyzer also includes a numerical control unit (NC unit), which is connected to the cleaning control unit, the separation unit 3, and the detection unit 4 respectively, and is used to control the operation of each unit. The hardware part of the NC unit includes an industrial computer and an I / O interface with multi-channel input / output function. The I / O interface is connected to the control terminal and signal output terminal of each unit through a cable.

[0048] This invention connects to the signals of each unit via an industrial computer and a multi-channel I / O interface, enabling programmable control and real-time monitoring of the entire analysis process. This integrated control system supports method programming, data acquisition, and result output, improving the automation level of the instrument. It also facilitates integration with external databases such as LIS systems, meeting the needs of laboratory information management.

[0049] Furthermore, such as Figure 2 As shown, the analyzer also includes a housing 1, the cleaning control unit, the separation unit 3, and the detection unit 4 are integrated inside the housing 1, and the industrial computer of the CNC unit is located inside the housing 1 or connected to the outside of the housing through the I / O interface.

[0050] This invention integrates all units into the housing 1, and the CNC unit can be built-in or externally connected. This housing 1 design protects precision components from environmental interference, optimizes the instrument layout, facilitates transportation, installation and maintenance, and supports external connections through standard interfaces, enhancing the instrument's practicality and adaptability to meet the needs of various application scenarios.

[0051] The analyzer of this invention operates on the following principle: Operation begins with the automated operation of the sample loading and cleaning control unit 2: the sample platform 20, precisely driven by the two-dimensional moving platform 22 and the lifter 21, accurately moves the sample vial 27 to below the dispensing needle; subsequently, the dispensing needle assembly 36 absorbs and dispenses a small amount of sample, automatically completing the injection. After the sample enters the separation unit 3, under the action of a high-voltage electric field, based on the principle of capillary electrophoresis, different amino acid components achieve efficient separation within the capillary due to differences in migration charge density and migration time. During the separation process, the constant-temperature fan 14 provides a stable temperature environment for the capillary through a precision air duct, ensuring the reproducibility of the separation. The separated amino acid components sequentially enter the detection area, where they are excited by the laser light source 16 to generate fluorescence. The fluorescence signal is captured and converted into an electrical signal by the photomultiplier tube 5, whose position is optimized by the triaxial adjuster, in the high-sensitivity detection unit 4. Ultimately, these signals are transmitted to the numerical control unit, where the intelligent data processing software built into the industrial computer performs qualitative (e.g., by comparing retention time / mobility) and quantitative (e.g., by calculating peak area / peak height) analysis, automatically generating analysis reports and supporting integration with the LIS system, thus completing a fully automated, high-precision analysis process from micro-sample injection to result output.

[0052] On the other hand, such as Figure 1 As shown, this embodiment of the invention provides a rapid amino acid analysis method using a previously described amino acid analyzer, comprising: S1: Capillary pre-cleaning and activation and air peak calibration; S2: Automatic capillary injection mixes the sample with the derivatization reagent and performs microwave-assisted derivatization reaction; S3: Separate the derivatized sample by capillary electrophoresis, and measure the migration charge density and record the migration time in real time during the separation process; S4: Simultaneously perform real-time optical detection on the separated amino acid derivatives; S5: Based on the detection signal, qualitative and quantitative analysis of amino acids is performed according to migration time and migration charge density.

[0053] Furthermore, the capillary pre-cleaning and activation and air peak calibration described in S1 include: First, the capillary is pre-cleaned by rinsing with alkaline washing solution to remove residue from the inner wall. Then, it is neutralized by rinsing with ultrapure water. Finally, it is activated by rinsing with separation buffer to bring the capillary to a stable separation state. An air pulse is applied at the capillary inlet to achieve air peak calibration.

[0054] The alkaline washing solution is a 0.5 mol / L NaOH solution, and the rinsing time is 90 seconds. The ultrapure water rinsing time is 45 seconds; The separation buffer contains 40 mM borate and 15 mM sulfonated cyclodextrin derivative, and the rinsing time is 110 seconds. The air peak calibration is achieved by applying a 0.2-second air pulse.

[0055] Specifically, after the instrument is started, the sample injection and cleaning control unit first activates the 83cm capillary before analysis. The XYZ three-dimensional motion platform, consisting of the two-dimensional moving platform and the lift, sequentially positions the capillary inlet to the alkaline washing solution bottle, the pure water bottle, and the buffer solution bottle.

[0056] After switching to the corresponding liquid path, the three-stage activation process is completed by pressure drive: rinsing with 0.5 mol / L NaOH solution for 90 seconds to remove residue from the inner wall; then rinsing with ultrapure water for 45 seconds for neutralization; and finally rinsing with separation buffer containing 40 mM borate and 15 mM sulfonated cyclodextrin derivative for 110 seconds to bring the capillary to a stable separation state.

[0057] After activation, an air peak calibration is performed on the capillary. A short 0.2-second air pulse is applied at the capillary inlet, and the position of the air peak in the detection window is recorded for time axis calibration and to confirm the collimation status of the optical path in the detection window.

[0058] Furthermore, the derivatizing reagent mentioned in S2 is a dimethyl sulfoxide solution containing fluorescein isothiocyanate at a concentration of 10 mg / mL; The microwave-assisted derivatization was performed by heating at 150W power for 60 seconds at a reaction temperature of 50°C. The automatic sampler uses a positive pressure injection method with a pressure of 10 Pa and an injection time of 5 seconds.

[0059] Specifically, after capillary activation, the sample processing stage begins. First, the XYZ three-dimensional motion platform is driven to position the sample tray, so that the sealed sample dispensing needle is precisely moved above the target sample tube.

[0060] The sampling needle descends along the Z-axis and inserts below the sample liquid surface. A positive pressure of 10 Pa is applied through the side air inlet structure for 5 seconds, directly pressing the sample to the capillary inlet for initial injection. This positive pressure method avoids disturbances to the early migration time caused by electric injection, which is beneficial to the reproducibility of subsequent derivatization reactions.

[0061] The XYZ 3D motion platform then moves the FITC reagent vial to a position below the dispensing needle, and draws 10 μL of DMSO solution containing fluorescein isothiocyanate (10 mg / mL) under the same positive pressure of 10 Pa. After the FITC reagent enters the dispensing needle, it automatically mixes with the previously drawn sample solution using capillary action, forming a homogeneous derivatization reaction solution within the needle cavity, without the need for an additional mechanical mixing structure.

[0062] After mixing, the sample needle is moved to the inlet of the microwave reaction cell, where a sealed connection is achieved using the self-positioning mechanism between the needle tip and the guide groove of the reaction cell. Then, with microwave assistance, the reaction solution is heated at a fixed power of 150W and subjected to a derivatization reaction for 60 seconds, allowing the free amino acids in the sample to fully react and form fluorescein isothiocyanate-thiourea derivatives.

[0063] Furthermore, in the electrophoretic separation described in S3, capillary electrophoresis is carried out at a constant temperature of 22°C, and a constant voltage of 26.8kV is applied to both ends of the capillary to drive the derivatized amino acids to migrate along the effective length of the capillary of 83cm.

[0064] Furthermore, the migration charge density is calculated using the instantaneous voltage and instantaneous current output in real time.

[0065] Specifically, after the derivatized sample enters the capillary, the separation unit is activated, and the capillary is maintained at 22°C under the action of the temperature control module. A constant voltage of 26.8kV is applied to both ends of the capillary by a high-voltage power supply, driving the derivatized amino acids to migrate along the effective length of the capillary of 83cm.

[0066] The instrument's high-voltage power supply module has the function of real-time reverse output voltage and current signals, and can synchronously output instantaneous voltage during the separation process. Instantaneous current And the migration time recorded by the system The industrial computer in the CNC unit automatically calculates the migration charge density based on this real-time data. This allows peak identification to be independent of a single migration time, ensuring higher reproducibility of the analysis.

[0067] In one embodiment, electromobility The calculation formula is as follows: ; in, The effective length of the capillary. The total length of the capillary tube. The instantaneous voltage applied across the capillary tube. This refers to the migration time.

[0068] migration charge density as follows: ; in, For the initial current, For calibration constant, The specific value can be determined by calibrating the instrument using a standard.

[0069] Furthermore, in the real-time optical detection described in S4, a laser with a wavelength of 488nm and a power of 25mW is used as the light source. The laser passes through a grating, a collimating lens, a focusing lens, and an aperture in sequence before illuminating the capillary detection area and generating fluorescence.

[0070] Furthermore, the fluorescence signal is sampled by the photomultiplier tube at a sampling frequency of 25Hz to obtain real-time fluorescence intensity data, and the dynamic gain adjustment of the photomultiplier tube is determined based on the electrophoretic current signal.

[0071] Furthermore, during the synchronization of capillary electrophoretic separation and real-time optical detection, dynamic gain adjustment is performed, specifically including: Using the initial current at the start of electrophoresis as a reference, the changes in the real-time current during the separation process are monitored in real time. When the real-time current fluctuates relative to the initial current, the operating voltage of the photomultiplier tube is automatically adjusted according to a preset proportional control algorithm to compensate for the fluctuation in fluorescence signal intensity caused by changes in sample migration speed.

[0072] Specifically, the detection unit automatically starts after electrophoresis begins. A laser source with a wavelength of 488nm and a power of 25mW is used. The laser beam is first spectrally shaped by a grating, and then forms a highly uniform focused spot through a combination of collimating and focusing lenses. After optical path shaping, the laser beam is further limited in spot size by a 0.8mm diameter aperture, ultimately precisely focused on the capillary detection area, causing the FITC derivative to fluoresce at that location.

[0073] Fluorescence emitted from the detection window is focused by a collecting lens onto a photomultiplier tube (PMT). After the separated derivatized amino acids enter the detection window, the laser light passes sequentially through a grating, collimating lens, focusing lens, and aperture before illuminating the capillary detection area and generating fluorescence. The PMT continuously acquires the fluorescence signal at a sampling frequency of 25 Hz. Simultaneously, the high-voltage power supply module provides real-time feedback on the current changes during the electrophoresis process to the numerical control unit (NC unit). The industrial computer in the NC unit determines the instantaneous electrophoresis state within the capillary based on this real-time current signal.

[0074] In one embodiment, the high-voltage power supply module feeds back a real-time / instantaneous current signal to the control system in real time. The numerical control unit continuously calculates the relative rate of change of the current or its relationship with the initial current. The ratio. If the system detects... Compared to If the voltage rises steadily and continuously, it indicates that the current system temperature may be increasing, leading to increased buffer conductivity and thus faster migration of all samples. In this case, the PMT gain (operating voltage) should be increased. Conversely, if... Compared to If the rate of decrease is continuous and stable, it is determined that the migration speed has slowed down, and the gain (operating voltage) of the PMT should be reduced.

[0075] In one embodiment, the adjustment amount of the PMT gain (operating voltage) can be based on proportional control of the relative rate of change of current: Determine the relative rate of change of current: ; If the relative rate of change of current A positive value indicates a faster migration speed; If the relative rate of change of current A negative value indicates a slower migration speed; Determine the gain adjustment amount: ; in The proportional gain coefficient is determined experimentally during the instrument development phase and then embedded in the intelligent data processing software of the industrial computer. The adjustment range of the gain is linearly proportional to the fluctuation range of the current. The greater the interference, the greater the compensation force.

[0076] The numerical control unit dynamically adjusts the operating voltage of the PMT using the acquired current signal, ensuring that the PMT's detection gain matches the migration speed of the sample bands during electrophoresis. When current fluctuations cause changes in the migration speed, the PMT's high-voltage compensation signal intensity is increased or decreased to maintain peak shape integrity and detection stability. Through this interconnected adjustment mechanism, real-time optimization of amino acid separation quality can be achieved without altering the electric field strength.

[0077] Furthermore, the qualitative and quantitative analysis of amino acids based on the detection signal, according to migration time and migration charge density, as described in S5 includes: Baseline correction and filtering are performed on real-time fluorescence intensity data to obtain electrophoresis spectra and peak identification is completed, and the migration time of each target peak is extracted; Based on the comparison of characteristic parameters such as migration time and migration charge density with the built-in database, the identification of amino acid types and the quantification of concentrations can be achieved under the condition of no standard.

[0078] Specifically, the fluorescence intensity data generated during the separation and detection process is transmitted to the numerical control unit (NC unit) in real time. The industrial computer in the NC unit first performs baseline correction on the raw signal, uses an adaptive algorithm to eliminate background drift, and suppresses high-frequency noise through digital filtering to obtain an electrophoretic spectrum with a high signal-to-noise ratio. Subsequently, peak identification is automatically completed by combining peak shape changes and fluorescence intensity derivative information, and the migration time of each target peak is extracted.

[0079] Migration time With the voltage output in real time by the high-voltage power supply Current The migration charge density was calculated from the data. Industrial computers with migration time and migration charge density Based on the combined characteristic parameters, the system compares the results with a built-in amino acid feature database to achieve automatic qualitative identification of each amino acid. Subsequently, it calculates the concentration of each amino acid in the sample according to the calibration curve of the corresponding amino acid and automatically generates an analysis report containing electrophoresis spectra, qualitative results, quantitative concentration tables, and anomaly alerts. This invention compares the migration time of the internal standard peak calibration with the database, employing multi-dimensional feature joint identification based on migration time, migration charge density, peak shape parameters, and fluorescence intensity to achieve automatic spectrum interpretation and realize the automatic identification and concentration calculation of 20 amino acids under standard-free conditions.

[0080] Specifically, the data in the built-in database is accumulated through prior system calibration using standards. It includes the characteristic migration time, migration charge density, and corresponding standard calibration curves (i.e., the relationship curve between concentration and detection signal intensity or peak area) for each known amino acid. In actual detection, the measured migration time and migration charge density of each peak in the unknown sample are combined into a characteristic coordinate, which is then matched with the standard characteristic coordinates in the database (e.g., by calculating Euclidean distance or similarity algorithms). When a match is successful, the type of amino acid corresponding to the peak can be determined (qualitative). The calibration curve of that amino acid is then invoked, and its precise concentration is automatically calculated based on the signal intensity of the current peak (quantitative). This enables rapid, fully automated analysis without the need for simultaneous running of standards.

[0081] This invention provides a rapid amino acid analysis method that utilizes microwave-assisted reaction to efficiently derivatize amino acids with fluorescein isothiocyanate in a short time, significantly reducing the traditional time-consuming and cumbersome pretreatment process. Capillary electrophoresis is used to separate the derivatized amino acids under a constant voltage. By real-time monitoring of the voltage and current signals during electrophoresis, the migration charge density, independent of a single migration time, is calculated as a qualitative criterion. The separated components are then detected with high sensitivity using an aperture-coupled laser-induced fluorescence system. Finally, qualitative and quantitative analysis is performed by combining migration time and migration charge density, enabling the identification of 20 common amino acids without the need for standards.

[0082] Furthermore, the analytical method and analyzer of this invention are combined to construct a fully automated sample processing system, which realizes the automated execution of the entire process from sample aspiration and reagent mixing to microwave derivatization and result output. This avoids the systematic errors caused by inaccurate pipetting, inconsistent reaction time, or unstable temperature control in traditional manual operation. It has the advantages of fast analysis speed, low sample consumption, and high reproducibility, which significantly improves the derivatization efficiency and reaction repeatability.

[0083] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0084] It will be readily understood by those skilled in the art that this invention includes any combination of the inventive description and specific embodiments outlined in the foregoing specification, as well as the various parts shown in the accompanying drawings. Due to space limitations and for the sake of brevity, not all of these combinations have been described in detail. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0085] Although embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid amino acid analysis method, characterized in that, include: Capillary pre-cleaning and activation, and air peak calibration; The sample is automatically injected through a capillary tube, mixed with the derivatization reagent, and subjected to a microwave-assisted derivatization reaction. The derivatized samples were separated by capillary electrophoresis, and the migration charge density and migration time were measured and recorded in real time during the separation process. Real-time optical detection of isolated amino acid derivatives; Based on the detection signal, qualitative and quantitative analysis of amino acids is performed according to migration time and migration charge density.

2. The rapid amino acid analysis method as described in claim 1, characterized in that, The capillary pre-cleaning and activation, and air peak calibration include: First, the capillary is pre-cleaned by rinsing with alkaline washing solution to remove residue from the inner wall. Then, it is neutralized by rinsing with ultrapure water. Finally, it is activated by rinsing with separation buffer to bring the capillary to a stable separation state. An air pulse is applied at the capillary inlet to achieve air peak calibration.

3. The rapid amino acid analysis method as described in claim 2, characterized in that, The alkaline washing solution is a 0.5 mol / L NaOH solution, and the rinsing time is 90 seconds; The ultrapure water rinsing time is 45 seconds; The separation buffer contains 40 mM borate and 15 mM sulfonated cyclodextrin derivative, and the rinsing time is 110 seconds. The air peak calibration is achieved by applying a 0.2-second air pulse.

4. The rapid amino acid analysis method as described in claim 1, characterized in that, The derivatization reagent is a dimethyl sulfoxide solution containing fluorescein isothiocyanate at a concentration of 10 mg / mL; The microwave-assisted derivatization was performed by heating at 150W power for 60 seconds at a reaction temperature of 50°C. The automatic sampler uses a positive pressure injection method with a pressure of 10 Pa and an injection time of 5 seconds.

5. The rapid amino acid analysis method as described in claim 1, characterized in that, In the electrophoretic separation, capillary electrophoresis is carried out at a constant temperature of 22°C, and a constant voltage of 26.8kV is applied to both ends of the capillary to drive the derivatized amino acids to migrate along the effective length of the capillary of 83cm.

6. The rapid amino acid analysis method as described in claim 5, characterized in that, The migration charge density is calculated using the instantaneous voltage and instantaneous current output in real time.

7. The rapid amino acid analysis method as described in claim 1, characterized in that, In the real-time optical detection, a laser with a wavelength of 488nm and a power of 25mW is used as the light source. The laser passes through a grating, a collimating lens, a focusing lens, and an aperture in sequence before illuminating the capillary detection area and generating fluorescence.

8. The rapid amino acid analysis method as described in claim 7, characterized in that, The fluorescence signal is sampled by a photomultiplier tube at a sampling frequency of 25 Hz to obtain real-time fluorescence intensity data, and the dynamic gain adjustment of the photomultiplier tube is determined based on the electrophoretic current signal.

9. The rapid amino acid analysis method as described in claim 8, characterized in that, During the synchronization of capillary electrophoretic separation and real-time optical detection, dynamic gain adjustment is performed, specifically including: Using the initial current at the start of electrophoresis as a reference, the changes in the real-time current during the separation process are monitored in real time. When the real-time current fluctuates relative to the initial current, the operating voltage of the photomultiplier tube is automatically adjusted according to a preset proportional control algorithm to compensate for the fluctuation in fluorescence signal intensity caused by changes in sample migration speed.

10. The rapid amino acid analysis method according to any one of claims 1-9, characterized in that, The qualitative and quantitative analysis of amino acids based on the detection signal, according to migration time and migration charge density, includes: Baseline correction and filtering are performed on real-time fluorescence intensity data to obtain electrophoresis spectra and peak identification is completed, and the migration time of each target peak is extracted; Based on the comparison of characteristic parameters such as migration time and migration charge density with the built-in database, the identification of amino acid types and the quantification of concentrations can be achieved under the condition of no standard.

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