A micro-solution sample cell and its preparation method and application

Abstract

The application discloses a trace solution sample cell and a preparation method and application thereof, belongs to the technical field of biomolecule detection, and uses TPX sheet as a transparent material for detection and sets a light transmission film, so that the reflection loss of terahertz waves at the solution interface can be significantly reduced, noise interference caused by the base material is avoided, the problems that the frequency resolution and sensitivity of the existing terahertz time-domain spectroscopy technology are low and the sample spectrum with a microgram concentration gradient cannot be accurately detected are solved, and in addition, the thickness of the solution layer to be detected can be accurately limited by adjusting the thickness or number of the ring-shaped gasket, the biological marking is not needed in the detection process, and the problems that the marking process of the biochemical sensor detection method is complex and the sample is damaged are solved.

Description

Technical Field

[0001] This invention relates to the field of biomolecular detection technology, and in particular to a micro-solution sample cell, its preparation method, and its application. Background Technology

[0002] Biomolecules are the basic building blocks of living organisms, playing crucial physiological and metabolic functions in the human body. Biomolecules exist in cells, tissues, and body fluids, and their diverse types and wide concentration ranges make accurate identification and concentration detection quite complex.

[0003] In existing technologies, biochemical sensors are mainly used to detect and measure specific parameters of biomolecules or biological systems. Before sensing and detection, the sample to be detected needs to be pretreated, such as by fluorescent labeling or chemical modification. This labeling method is complex and can cause some damage to the sample. Traditional terahertz time-domain spectroscopy (THz-TDS) has limited frequency resolution and low sensitivity, and cannot accurately detect differences in the spectra of samples with microgram concentration gradients. Summary of the Invention

[0004] The main objective of this invention is to provide a micro-solution sample cell, its preparation method, and its application, aiming to solve the problems of complex labeling processes and sample damage caused by existing biochemical sensor detection methods, as well as the low frequency resolution and sensitivity of traditional terahertz time-domain spectroscopy, which cannot accurately detect differences in the spectra of samples with microgram concentration gradients.

[0005] To achieve the above objectives, the present invention provides a method for preparing a micro-volume solution sample cell, the method comprising: An annular gasket is disposed on a first TPX sheet, and the first TPX sheet and the annular gasket form a solution cavity; The solution chamber is used to hold the solution to be tested, and the thickness of the solution to be tested can be adjusted by changing the number or thickness of the annular gaskets; A second TPX sheet is placed on the annular gasket to obtain the micro-solution sample cell.

[0006] Optionally, the solute in the test solution is L-valine, L-phenylalanine, L-proline, or glucose.

[0007] Optionally, the diameter of the first TPX sheet is 13mm~15mm and the thickness is 2.0mm~2.2mm, and the diameter of the second TPX sheet is 13mm~15mm and the thickness is 1.95mm~2.05mm.

[0008] Optionally, both the first TPX sheet and the second TPX sheet include an antireflection film, the thickness of which is 0.5 μm to 2 μm.

[0009] Optionally, the annular gasket is a TPX annular gasket or a ceramic annular gasket.

[0010] Optionally, the thickness of the annular gasket is 5μm to 200μm, the inner diameter is 8mm to 10mm, and the outer diameter is 12mm to 14mm.

[0011] Optionally, the first TPX sheet, the second TPX sheet, and the annular gasket are fixed by compression.

[0012] Optionally, both the upper and lower surfaces of the annular gasket include an adhesive coating; the first TPX sheet and the second TPX sheet are fixedly connected to the annular gasket by adhesive bonding, and the adhesive coating has an adhesion strength ≥10 N / cm at 25℃~30℃. 2 .

[0013] To achieve the above objectives, the present invention also provides a micro-solution sample cell, which is prepared by the above-described preparation method.

[0014] To achieve the above objectives, the present invention also provides an application of a micro-solution sample cell, which is used in the detection of terahertz spectra, wherein the solvent used for detection is glycerol, DMSO, DMF or a mixture of water and glycerol.

[0015] Compared with the prior art, the beneficial effects that the present invention can achieve are as follows: 1. In the technical solution of the present invention, the transparent material of the micro-solution sample cell is selected as TPX sheet (4-methylpentene polymer), which has no obvious characteristic absorption peak in the 0.1THz~2.6THz band of terahertz waves, and contains the side of the test solution facing the antireflection membrane. This can not only significantly reduce the reflection loss of terahertz waves at the TPX-solution interface, but also detect the signal directly from the test solution itself, avoiding noise interference introduced by the substrate. This solves the problem that the frequency resolution of the existing terahertz time-domain spectroscopy (THz-TDS) technology is limited and the sensitivity is low, making it unable to accurately detect the difference in the spectrum of samples with microgram concentration gradients.

[0016] 2. In the micro-volume solution sample cell disclosed in this invention, the thickness of the solution layer to be tested can be precisely limited by adjusting the thickness or number of annular gaskets, so that the thickness error of the solution layer to be tested is ≤±1μm, which solves the problems of uneven thickness and unstable optical path in traditional liquid film methods. During detection, the sample cell has a closed structure, and the solution can be directly detected without labeling. Moreover, the terahertz photon energy is extremely low and will not damage biological samples, solving the problems of complex labeling process and sample damage in biochemical sensor detection methods. At the same time, the micro-volume solution sample cell has a disposable cartridge structure. Solution injection can be quickly completed through the micro-volume inlet set on the side of the cartridge structure, and the precise docking with the terahertz spectrometer sample stage through the positioning buckle enables automated batch sample detection. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the micro-solution sample cell structure disclosed in this invention; Figure 2 This is a terahertz spectrum of the micro-solution sample cell in Example 1; Figure 3 The images show the terahertz spectral characteristics of the micro-solution sample cell in Example 1 when the solvent used for detection is glycerol, DMSO or DMF, and the terahertz spectral characteristics of Comparative Example 1 with distilled water as solvent and Comparative Example 2 with acetonitrile as solvent. Figure 4 The terahertz spectral characteristics of the detection solvent in the micro solution sample cell of Example 1 when a mixed solution of water and glycerol in different volume ratios is used as the solvent. Figure 5 The terahertz spectral characteristics of the L-valine solution and the powder tablet sample are shown in the figure. Figure 6 Terahertz spectral characteristics of L-phenylalanine solution and powder tablet samples are shown. Figure 7 Terahertz spectral characteristics of L-proline solution and powder tablet samples are shown. Figure 8 The figures show the terahertz spectral characteristics of the glucose solution and the powder tablet sample. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] To address the limitations of existing terahertz time-domain spectroscopy (THz-TDS) techniques, such as limited frequency resolution and low sensitivity, which prevent accurate detection of spectral differences in samples with microgram concentration gradients, and the complex labeling processes and sample-damaging effects of biochemical sensor detection methods, this invention provides a method for preparing a micro-volume solution sample cell. The preparation method includes: An annular gasket is disposed on a first TPX sheet, and the first TPX sheet and the annular gasket form a solution cavity; The solution to be tested is injected into the solution chamber, wherein the thickness of the solution to be tested can be adjusted by changing the number or thickness of the annular gaskets; A second TPX sheet is placed on the annular gasket to obtain the micro-solution sample cell.

[0020] It should be understood that the side of the first pretreated TPX sheet including the antireflection membrane faces the test solution layer, and the side of the second pretreated TPX sheet including the antireflection membrane faces the test solution layer. Thus, the side of the first pretreated TPX sheet including the antireflection membrane and the side of the second pretreated TPX sheet including the antireflection membrane are arranged opposite to each other.

[0021] Optionally, in the above method of limiting the thickness of the test solution layer by adjusting the thickness or number of annular gaskets, the method of adjusting the thickness of the annular gaskets can be achieved by replacing annular gaskets of different thicknesses or stacking multiple annular gaskets; when adjusting the thickness of the test solution by increasing the number of annular gaskets, multiple annular gaskets can be bonded together.

[0022] It should be noted that by replacing annular gaskets of different thicknesses or stacking multiple annular gaskets, the thickness of the solution layer can be precisely limited to 5μm~200μm, and the thickness error can be controlled within ≤±1μm, thus solving the problems of uneven thickness and unstable optical path in the traditional liquid film method.

[0023] Optionally, to further improve the sealing performance of the sample cell, a sealing ring can be added to the sample cell structure. The material of the sealing ring should be a low-loss material in the terahertz band.

[0024] Optionally, the sealing ring can be made of silicone rubber or fluororubber.

[0025] In one possible implementation, the solute in the test solution is L-valine, L-phenylalanine, L-proline, or glucose.

[0026] Optionally, when the solute in the test solution is a test solution containing protein impurities, an ultra-thin hydrophilic modified layer can be pre-coated on the inner wall of the solution chamber before detection, thereby avoiding detection errors caused by solute adsorption.

[0027] In one possible implementation, the diameter of the TPX sheet is 13mm to 15mm, the thickness of the first TPX sheet is 2.0mm to 2.2mm, and the thickness of the second TPX sheet is 1.95mm to 2.05mm.

[0028] In one possible implementation, both the first TPX sheet and the second TPX sheet include an antireflection film with a thickness of 0.5 μm to 2 μm.

[0029] Optionally, the above-mentioned antireflection membrane can be a MgF2 antireflection membrane.

[0030] Optionally, the antireflective membrane can also be a composite membrane, which is a structure of alternating stacks of SiO2 and TiO2, and the total thickness can be 0.8μm~1.5μm.

[0031] Optionally, the thickness ratio of the SiO2 layer to the TiO2 layer can be optimized according to the target wavelength of terahertz detection. For example, for the 1THz~3THz wavelength band, the SiO2 / TiO2 thickness ratio can be 3:1, so that the reflectivity of the composite film layer in the target detection wavelength band is ≤1%; and the surface hardness of the composite film layer is ≥4H, possessing good wear resistance and corrosion resistance. It should be noted that when the diameter of the TPX sheet is 13mm~15mm, it can match the conventional detection spot size of the terahertz spectrometer sample stage, ensuring that the terahertz pulse completely covers the solution cavity area without signal loss or edge loss. The detection spot diameter of the terahertz time-domain spectrometer is usually 8mm~12mm. In the above scheme, the inner diameter of the annular gasket is 8mm~10mm, which corresponds to the diameter of the solution cavity, and the diameter of the TPX sheet is limited to 13mm~15mm, which ensures that the solution cavity is completely within the spot range, while reserving an edge margin of 3mm~5mm for the fixation and sealing of the cartridge structure. This avoids the problem of the light spot illuminating the sealing ring or card holder frame due to the TPX sheet being too small, or the problem of clamping difficulties and optical path alignment deviation due to the TPX sheet being too large.

[0032] Furthermore, the thickness of the first TPX sheet is 2.0mm~2.2mm, and the thickness of the second TPX sheet is 1.95mm~2.05mm. This avoids coherent superposition of terahertz pulses at the interface of two TPX sheets of equal thickness, preventing coherent interference noise that could cause periodic interference fringes in the frequency domain signal and thus mask the characteristic absorption peaks of the sample under test. Simultaneously, a thickness of approximately 2mm balances mechanical strength and light transmission efficiency. If the thickness is <1.9mm, the TPX sheet is prone to deformation during lamination and assembly, leading to uneven thickness of the solution layer under test; if the thickness is >2.2mm, it increases the propagation loss of terahertz waves in the substrate, reducing the strength of the detection signal. Furthermore, controlling the maximum thickness difference between the first and second pre-treated TPX sheets to 0.25mm avoids interference without altering the propagation path of the terahertz waves, ensuring the stability of the optical path.

[0033] Furthermore, in the above technical solution, the side of the TPX sheet coated with the antireflection film faces the test solution layer. This antireflection film can be a MgF2 antireflection film. This not only significantly reduces the reflection loss of terahertz waves at the TPX-solution interface and improves the transmittance of the sample signal, but also eliminates interface reflection noise and improves detection sensitivity. This is because the dielectric constant of MgF2 is between that of TPX and the test solution layer, so interface reflection can be canceled through a gradual change in refractive index. When the thickness of the MgF2 antireflection film is 0.5μm~2μm, it can match the terahertz band of 0.1THz~2.6THz, corresponding to a quarter wavelength of 100μm~2600μm, achieving the best antireflection effect within the target frequency band and significantly reducing interface reflectivity. At the same time, with one side of the antireflection film facing the test solution layer, it can directly act on the TPX-solution interface, ensuring that the sample information carried by the terahertz pulse after passing through the solution can be efficiently transmitted to the detector, avoiding signal loss at the interface. In the specific implementation process, the thickness of the single-layer MgF2 antireflection membrane can be adjusted according to the refractive index of different solutions. For example, when glycerol is selected as the solution, the thickness of the MgF2 antireflection membrane can be controlled between 0.5 μm and 1 μm. When DMSO is selected as the solution, the thickness of the antireflection membrane can be controlled between 1 μm and 2 μm, thereby ensuring the consistency of the antireflection effect across the entire detection range.

[0034] Optionally, the above-mentioned antireflection film can be deposited using a vacuum evaporation process or a magnetron sputtering process. When a vacuum evaporation process is used, the vacuum level during deposition can be 1×10⁻⁶. -3 Pa~5×10 -3 Pa.

[0035] It should be understood that TPX is a polyolefin polymer with limited solvent resistance. The aforementioned vacuum evaporation and magnetron sputtering are both dry coating processes, requiring no water or organic solvents as a medium. This avoids the swelling or corrosion of the TPX sheet caused by liquid-phase coating, ensuring that the terahertz transmittance of the substrate is not damaged. In specific implementation, the coating process can be selected according to the application scenario. For example, vacuum evaporation can be chosen for the large-scale preparation of conventional precision sample cells; while magnetron sputtering can be chosen for the preparation of high-end sample cells for microgram-level concentration gradient detection.

[0036] It should be noted that the MgF2 antireflective film or composite film obtained by the above-mentioned vacuum evaporation and magnetron sputtering has a purity >99.9% and does not introduce additional terahertz absorption peaks, thus solving the signal noise problem caused by residual impurities in wet deposition. When the vacuum degree is 1×10⁻⁶... -3 Pa~5×10 -3 At a pressure of Pa, the resulting MgF2 antireflective film or composite film exhibits high transmittance and stability, and strong adhesion to the TPX sheet substrate, making it less prone to detachment or cracking. If the vacuum degree is >1×10⁻⁶, -3 If the vacuum level is less than 5 × 10⁻⁶ Pa, there are very few residual gas molecules in the vacuum chamber, resulting in an excessively long mean free path for the metal particles. During evaporation, the particles will collide with the TPX sheet surface at extremely high speeds, leading to excessive internal stress in the film layer and making it prone to cracking, warping, and other problems. -3 If the concentration of residual gas molecules in the cavity is low, the metal particles will frequently collide with the gas molecules during flight, resulting in insufficient kinetic energy for particle deposition. The film layer is loose and porous, which not only has poor adhesion but also reduces the anti-reflection effect due to the scattering of terahertz waves by the pores. In addition, the residual water vapor and oxygen will react with metal ions to form impurity phases, thus introducing additional absorption losses.

[0037] The core premise of terahertz spectroscopy detection is ensuring that the detection signal can effectively penetrate the sample cell and the test solution. However, in existing technologies, TPX substrates are generally uncoated, and the refractive index difference between TPX and air / solution is significant, leading to strong reflection in the terahertz band, resulting in attenuation of the effective transmission signal and a reduced signal-to-noise ratio. Therefore, in the technical solution of this invention, when the antireflection film is a composite film composed of alternating SiO2 and TiO2 stacked structures, interface reflection loss can be significantly reduced. In the terahertz band of 0.1THz to 2.6THz, the reflectivity can be reduced to 0.5%, thereby significantly improving the transmission efficiency of the terahertz signal and ensuring sufficient detection signal strength. Furthermore, the composite film composed of alternating SiO2 and TiO2 stacked structures has no characteristic absorption peaks in the terahertz band, avoiding interference from the film itself on the detection signal.

[0038] In one possible implementation, the aforementioned annular gasket is a TPX annular gasket or a ceramic annular gasket.

[0039] In one possible implementation, the thickness of the aforementioned annular gasket is 5μm to 200μm, the inner diameter is 8mm to 10mm, and the outer diameter is 12mm to 14mm.

[0040] Optionally, the aforementioned ceramic annular gasket can be an aluminum nitride ceramic annular gasket.

[0041] Optionally, the coating area of ​​the above-mentioned adhesive coating can be the entire area of ​​the upper and lower surfaces of the annular gasket, or it can be more than half of the surface area.

[0042] Optionally, the adhesive coating can be FEP (fluorinated ethylene propylene copolymer) or acrylic adhesive.

[0043] Optionally, the aforementioned annular gasket can be composed of a core gasket and an outer ring, wherein the outer ring can be an intelligent control ring, and thus the initial volume of the aforementioned solution cavity is determined by the thickness and inner diameter of the core gasket.

[0044] Optionally, the shape memory alloy of the outer ring can be made of Ni-Ti alloy, with a phase transformation temperature range of 25℃ to 60℃ and a thermal deformation rate of 0.5% to 2%, ensuring the reliability of fine-tuning of the solution thickness.

[0045] When the outer ring is an intelligent control ring, the temperature feedback of the outer intelligent control ring during the injection process monitors the deformation state of the solution cavity in real time to ensure no leakage of the solution. By controlling the temperature of the shape memory alloy of the outer ring, the thermal deformation characteristics are used to drive the core gasket to produce a small deformation, thereby achieving precise fine adjustment of the solution thickness.

[0046] Optionally, when the aforementioned annular gasket can be composed of a core gasket and an outer ring, the upper and lower surfaces of the core gasket can both be coated with a temperature-sensitive adhesive coating.

[0047] Optionally, the aforementioned temperature-sensitive adhesive coating can be a polyurethane-acrylate copolymer system with 5% to 8% by mass of a temperature-sensitive crosslinking agent added, and is in a semi-cured state at 25°C. Therefore, during the sample cell assembly process, the adhesive coating is in a semi-cured state, possessing a certain degree of adhesion to achieve initial positioning of the core gasket and the first and second TPX substrates, while facilitating position adjustment and avoiding sealing failure caused by assembly deviations; during the pressing and fixing stage, when the temperature is 30°C to 35°C and the pressure is 0.5MPa to 1MPa, the adhesive coating is fully cured, with an adhesive strength ≥15N / cm², forming a continuous, gapless sealing interface between the core gasket and the TPX substrate. It should be noted that when the aforementioned annular gasket is a TPX annular gasket, it is made of the same material as the upper and lower substrates of the sample cell, resulting in a high dielectric constant matching degree. This avoids interface reflection caused by material differences. Furthermore, TPX has no characteristic absorption peaks in the 0.1THz~2.6THz band, thus not introducing additional absorption loss. It also possesses good flexibility, making it less likely to damage the antireflection film on the TPX sheet surface during pressing. When the aforementioned annular gasket is a ceramic annular gasket, the dielectric loss is extremely low, and the mechanical strength is much higher than that of TPX. This prevents the gasket from deforming during pressing, making it suitable for preparing ultrathin gaskets with a thickness ≤10μm, which can meet the detection requirements of trace solutions (volume <1μL). At the same time, the strong chemical inertness of ceramic materials can withstand the erosion of strongly polar solvents, expanding the applicability of the sample cell.

[0048] It should be understood that the thickness of the aforementioned annular gasket is the thickness of the solution layer to be tested.

[0049] It should be noted that when the thickness of the aforementioned annular gasket is 5μm~200μm, it can cover the detection of solutions ranging from high to low concentrations. For example, when the thickness of the annular gasket is 5μm, it can be used to detect samples with concentration gradients down to the microgram level; when the thickness of the annular gasket is 200μm, it can be used to detect low-concentration macromolecular solutions, such as protein solutions. Furthermore, the inner diameter of the annular gasket is 8mm~10mm, matching the conventional spot diameter of terahertz spectrometers (8mm~12mm), ensuring complete coverage of the solution cavity and preventing signal loss. The outer diameter is 12mm~14mm, which matches the diameter of the TPX sheet, and allows for a 1mm margin at the edge for adhesive coating bonding, preventing the gasket from exceeding the TPX sheet's range and interfering with the cartridge frame. Simultaneously, the 4mm difference between the inner and outer diameters of the annular gasket ensures sufficient width, preventing deformation or breakage during pressing due to excessive width.

[0050] In one possible implementation, the first TPX sheet, the second TPX sheet, and the annular gasket are fixed by compression.

[0051] Optionally, the first TPX sheet, the second TPX sheet, and the annular gasket can also be fixed by a cartridge structure, such as... Figure 1 As shown, the card holder structure may include an upper structure, a lower structure, and a card housing structure, wherein the upper structure, the lower structure, and the card housing structure are fixedly connected by through pin holes provided thereon.

[0052] Optionally, when the sample cell is fixed by the cartridge structure, an inlet can be provided on the side of the cartridge structure at a position corresponding to the sample cell. The inlet is connected to the solution chamber and is used for injecting the solution to be tested.

[0053] Optionally, when the above-mentioned cartridge structure is provided with an inlet, the inlet can be sealed by means of a disposable capillary tube or a sealing plug.

[0054] Optionally, when the sample cell is fixed by the card holder structure, a positioning buckle can be provided on the card holder structure. The positioning buckle matches the size of the slot on the terahertz spectrometer sample stage, so that the sample cell can be fixed on the sample stage by the card holder structure for easy detection.

[0055] In one possible implementation, both the upper and lower surfaces of the aforementioned annular gasket include an adhesive coating; the first TPX sheet and the second TPX sheet are fixedly connected to the annular gasket by adhesive bonding, and the adhesive coating has an adhesion strength ≥10 N / cm² at 25℃~30℃. 2 .

[0056] It should be noted that the adhesive strength of the above-mentioned adhesive coating at 25℃~30℃ is ≥10N / cm. 2 This ensures that the gasket does not shift or detach during the entire process of sample cell assembly, injection, and testing, preventing wedge-shaped deviations in the solution layer, maintaining the accuracy of the optical path, and preserving structural integrity even under the external force of automated clamping. When both the upper and lower surfaces of the annular gasket include an adhesive coating, it can form a seamless fit with the first and second TPX sheets, blocking the leakage channels of the test solution.

[0057] To achieve the above objectives, the present invention also provides a micro-solution sample cell, which is prepared by the above method.

[0058] It should be understood that when the aforementioned micro-solution sample cell is fixed by the aforementioned cartridge structure, the micro-solution sample cell is used in terahertz spectroscopy detection. It can be precisely fixed to the sample stage of the spectrometer by the positioning buckle of the cartridge structure. Then, a micropipette is used to draw up the solution to be tested, and the needle of the pipette is inserted into the injection port on the side of the sample cell. The solution is slowly injected until the solution fills the entire solution chamber. The injection process should not be too fast to prevent the generation of air bubbles. After the injection is completed, the injection port can be sealed with a sealing plug to prevent the evaporation of volatile solvents from causing changes in solution concentration. Then, the spectrometer can be started to acquire the time-domain signal of the sample.

[0059] Optionally, the aforementioned micro-solution sample cell can be a disposable sample cell or a reusable sample cell. When it is a reusable sample cell, after use, the inlet and solution chamber can be rinsed with anhydrous ethanol or the corresponding solvent and dried with nitrogen; after retesting the empty cell transmittance and confirming that there is no performance degradation, it can be sealed and stored for future use.

[0060] To achieve the above objectives, the present invention also provides an application of a micro-solution sample cell, which is used in the detection of terahertz spectra.

[0061] In one possible implementation, the detection solvent in the above-mentioned terahertz spectroscopy is glycerol, DMSO, DMF, or a mixture of water and glycerol.

[0062] It should be understood that the above DMSO is dimethyl sulfoxide and DMF is N,N-dimethylformamide.

[0063] It should be noted that when glycerol, DMSO, or DMF is used as the solvent for terahertz spectroscopy detection, it exhibits no obvious characteristic absorption peaks within the target detection frequency band of 0.1 THz to 3 THz, and has low dielectric loss, thus avoiding strong background noise. This ensures that the terahertz fingerprint spectrum peaks of the sample are clearly distinguishable, accurately differentiating sample differences at microgram-level concentration gradients. Glycerol, DMSO, or DMF possess strong polarity or hydrogen bonding properties, dissolving most biomolecules, such as proteins, peptides, small organic molecule drugs, and polymers, allowing for the preparation of homogeneous solution samples and avoiding light scattering interference caused by sample aggregation. In practical applications, glycerol can prepare high-concentration sample solutions and is not easily volatile, maintaining concentration stability for long-term detection; DMSO can be used for terahertz spectral analysis of drug molecules; and DMF can be used for solution detection of polymeric materials and poorly soluble compounds.

[0064] The micro-volume solution sample cell disclosed in this invention, applied to terahertz spectroscopy detection, possesses advantages such as low interference, high precision, and high sensitivity. Furthermore, the thickness of the sample solution layer can be precisely controlled by adjusting the thickness of the annular gasket. Through the synergistic effect of the TPX sheet and the MgF2 antireflection membrane, interfacial reflection loss is reduced to below 95%, and the signal-to-noise ratio of the sample signal is improved to above 37 dB, making it suitable for detecting low-concentration samples. A multi-layer seal is formed through the annular gasket, adhesive coating, and sealing ring, ensuring not only uniform thickness of the sample solution layer but also preventing leakage. The precise matching of the positioning buckle of the cartridge structure with the terahertz spectrometer sample stage enables batch sample detection, improving detection efficiency.

[0065] Example 1 A method for preparing a micro-volume solution sample cell, comprising: Take two TPX sheets with a diameter of 14 mm, and label them as the first TPX sheet and the second TPX sheet, respectively. The thickness of the first TPX sheet is 2.1 mm and the thickness of the second TPX sheet is 2.0 mm. Clean them with anhydrous ethanol by ultrasonication for 10 min and then dry them with nitrogen. The first and second TPX sheets were placed into a vacuum coating machine, and the vacuum level was set to 3×10. -3 Pa, evaporation temperature 200℃, evaporation time 30min, a 1.2μm thick MgF2 antireflection film was deposited on one side of the TPX sheet to obtain the first pretreated TPX sheet and the second pretreated TPX sheet respectively; Take a TPX annular gasket with a thickness of 10μm, an inner diameter of 9mm, and an outer diameter of 13mm. Its upper and lower surfaces are coated with an FEP adhesive coating, and the adhesive coating has a protective film. When the sample cell is fixed by pressing, the pressing method can be as follows: peel off the FEP adhesive coating protective film of the TPX annular gasket, accurately align it with the center of the first pretreated TPX sheet, and press it lightly to make it firmly bonded to the first pretreated TPX sheet; then inject the test solution into the solution cavity surrounded by the annular gasket to form the test solution layer; then take the second pretreated TPX sheet, with the side including the MgF2 antireflection membrane facing the test solution layer, cover the upper surface of the TPX annular gasket, and press it lightly until it is bonded, thereby obtaining a micro solution sample cell; When fixing the sample cell using a cartridge structure, the method is as follows: Take a silicone rubber sealing ring and embed it into the groove at the bottom of the inner frame of the cartridge structure. Then, take the first pretreated TPX sheet, place it above the sealing ring, and press it lightly to adhere it, with the side of the first pretreated TPX sheet including the MgF2 antireflection membrane facing the test solution layer. Take a TPX annular gasket, peel off the FEP adhesive coating protective film of the TPX annular gasket, accurately align it with the center of the first pretreated TPX sheet, and press it lightly to firmly adhere it to the first pretreated TPX sheet. Then, take a second pretreated TPX sheet, with the side including the MgF2 antireflection membrane facing the test solution layer, and cover the upper surface of the TPX annular gasket, pressing it lightly until it adheres, forming a 10μm thick solution cavity. Finally, close the upper frame of the cartridge structure and fix it with the positioning buckle to complete the assembly of the sample cell. The test solution can be injected into the solution cavity through the designated inlet.

[0066] Terahertz spectroscopy was performed on the trace solution sample cell obtained in Example 1. Specifically, the time-domain spectra of the empty sample cell and the sample cell without an empty sample cell were measured respectively. The results are as follows: Figure 2 As shown.

[0067] like Figure 2 Neutron diagram a shows the terahertz time-domain waveform. The black curve represents the time-domain waveform without an empty sample cell, i.e., the terahertz pulse in air; the orange curve represents the time-domain waveform after the terahertz pulse passes through the empty sample cell. Observation reveals that the main pulse peak of the orange curve is significantly delayed compared to the black curve. This is because the terahertz wave propagates slower in TPX material than in air, and its amplitude is slightly attenuated. Furthermore, it can be observed that, apart from the main pulse, the orange curve shows no obvious additional pulses or strong noise, indicating that the TPX sheet, gasket, and antireflection membrane in the obtained trace solution sample cell did not introduce any additional reflection or scattering interference.

[0068] After Fourier transform, the transmission spectrum of TPX was observed as follows: Figure 2 As shown in sub-figure b, observation reveals that the transmittance of the empty sample cell remains stable between 0.88 and 1.0 in the 0.2 THz to 2.4 THz frequency band, indicating that the transmittance of the obtained trace solution sample cell is >88%. This demonstrates that the synergistic effect of the antireflection membrane and TPX material effectively reduces interfacial reflection loss. Furthermore, the transmittance curve shows no obvious sharp drop, indicating that the TPX, gasket, and antireflection membrane components in the sample cell do not experience additional absorption interference in this frequency band, resulting in extremely low background noise in the empty sample cell. This ensures that the spectral signal of the subsequent sample will not be masked by the signal from the sample cell itself.

[0069] The micro-solution sample cell obtained in Example 1 was used in terahertz spectroscopy detection, and the detection solvent was glycerol, DMSO or DMF.

[0070] Comparative Example 1 The solvent used for detection in Example 1 was replaced with distilled water.

[0071] Comparative Example 2 The detection solvent in Example 1 was replaced with acetonitrile.

[0072] The detection results of terahertz spectra in Examples 1 and 2 are as follows: Figure 3 As shown.

[0073] according to Figure 3 It can be seen that the transmittance of the blue curve of DMF remains above 0.8 in the 0~2.2THz frequency range, the purple curve of DMSO remains between 0.6 and 0.8, and that of glycerol is around 0.7. In contrast, the transmittance of the yellow curve of distilled water drops rapidly to below 0.2 after 0.4THz, and approaches 0.1 at 2.0THz. This is because water is a strongly polar molecule with multiple strong absorption peaks in the terahertz frequency range, which severely absorbs terahertz waves, completely masking the signal of the sample being tested. The transmittance of acetonitrile (blue triangular curve) drops rapidly from 0.6 to 0.2 in the 0~2.0THz range, indicating that acetonitrile has strong terahertz absorption, interfering with the spectral signal of the sample being tested. Therefore, in the technical solution of this invention, using glycerol, DMSO, or DMF as the detection solvent can avoid the solvent's own absorption masking the spectral characteristics of the sample being tested, thus ensuring the sensitivity of the detection.

[0074] To verify the compatibility of glycerol, DMSO, or DMF as detection solvents with biomacromolecules, 1 mL of glycerol, DMSO, DMF, distilled water, and acetonitrile solution were taken respectively and mixed with 20 mg of glucose and 10 mg of valine. The solubility was observed and recorded, and the results are shown in Table 1.

[0075] Table 1

[0076] In the table, "√" represents dissolved, "-" represents not dissolved, and "×" represents not smooth.

[0077] The data in the table shows that glycerol, DMSO, and DMF have good solubility for glucose but cannot dissolve amino acids. Therefore, they are suitable for detecting non-amino acid biomolecules such as proteins, polysaccharides, and organic drugs. Analysis of THz transmittance and spectral smoothness reveals that glycerol, DMSO, and DMF have high THz transmittance and smooth spectra, indicating that they have no strong absorption peaks and low noise in the terahertz band, ensuring clear and distinguishable spectral signals of the samples being tested.

[0078] As a preferred embodiment of the present invention, the present invention studies a mixed solution of water and glycerol as a mixed solvent for terahertz spectroscopy detection. This mixed solvent can dissolve amino acid biomolecules, thereby enabling the detection of amino acid biomolecules by terahertz spectroscopy.

[0079] A mixed solution of water and glycerol was prepared with volume ratios of 8:2, 3:2, 2:3, and 2:8. The terahertz spectral characteristics of the mixed solutions with different volume ratios were measured, and the results are as follows: Figure 4 As shown in the figure, 80% glycerol, 60% glycerol, 40% glycerol and 20% glycerol represent the volume ratios of water and glycerol as 8:2, 3:2, 2:3 and 2:8, respectively.

[0080] like Figure 4 Sub-figure a shows the Hertz amplitude spectrum. As can be seen, glycerol has the highest amplitude, which gradually decreases with increasing water content in the mixed solvent; pure water has the lowest amplitude. Sub-figure b shows the terahertz transmittance spectrum. As can be seen, pure glycerol has the highest transmittance, which gradually decreases with increasing water content. Sub-figure c shows the terahertz refractive index spectrum. As can be seen, pure glycerol has the lowest and most stable refractive index; with increasing water content, the refractive index gradually increases, and the fluctuations intensify. Sub-figure d shows the terahertz absorption coefficient spectrum. As can be seen, pure glycerol has the lowest absorption coefficient; with increasing water content, the absorption coefficient increases significantly.

[0081] comprehensive Figure 4 The test results of the intermediate amplitude spectrum, transmission spectrum, refractive index spectrum and absorption spectrum were compared in terms of spectral line smoothness, transmittance and solubility. It can be seen that when the volume ratio of water to glycerol is 2:8, the mixed solution of water and glycerol is the best for use as a solvent for terahertz spectroscopy detection.

[0082] The terahertz spectral characteristics of biomolecules in human body fluids were determined using the micro-solution sample cell prepared in Example 1. A mixed solution of water and glycerol at a volume ratio of 2:8 was used as the detection solution. L-valine, L-phenylalanine, L-proline, and glucose were selected as the detection targets. The specific detection method is as follows: (1) Solution preparation: Solutions with a concentration greater than 10 mM can be prepared by weighing using an analytical balance. The specific method is as follows: if the relative molecular mass of a certain substance is known to be Mr, weigh a mass of the substance in mmg and dissolve it in a μL of solvent, then the concentration C (mmol / L) of this solution can be calculated using the following formula:

[0083] For preparing solutions with a concentration less than 10 mM, such as a 1 mM solution, you can first prepare 1000 μL of the 10 mM solution. After it is dissolved evenly, take 100 μL of the solution and mix it evenly with 900 μL of solvent.

[0084] Using the above preparation method, solutions of L-valine, L-phenylalanine, L-proline, and glucose with concentrations of 5 mM, 10 mM, 15 mM, and 20 mM were prepared sequentially.

[0085] As a comparison, powder tablets of L-valine, L-phenylalanine, L-proline, and glucose were prepared and subjected to terahertz spectroscopy for testing.

[0086] (2) Detection of terahertz spectra Data processing methods: A. Processing of data for solution samples: During the detection process, the transmittance of the pure mixed solvent is first measured, followed by the transmittance of the L-valine solution (solvent + L-valine). The transmittance of the solvent is then compared with that of the solution to subtract the solvent's own absorption, retaining only the transmittance of L-valine. Because the original transmittance values ​​of different batches may vary, the solution signal after solvent subtraction needs to be normalized to make the obtained data more objective and easier to compare. B. Processing of solid sample measurement data: Because the molecular environments of solid and solution are different, the transmittance baseline trends of the two will differ. Therefore, it is necessary to fit the transmittance baseline of the solid sample by a linear function, and substitute this baseline pattern into the normalized solution signal to correct the baseline deviation of the solution state, and finally obtain a data processing curve that matches the baseline trend of the solid sample.

[0087] According to this data processing method, the solution measurement results and mixed solvent measurement results of L-valine, L-phenylalanine, L-proline, and glucose were compared separately. Solvent absorption was subtracted, and then normalization was performed to obtain the transmittance of L-valine, L-phenylalanine, L-proline, and glucose, respectively. These results were then compared with the measurement results of powder tablets of L-valine, L-phenylalanine, L-proline, and glucose. The results are as follows: Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown in the figure, "solid" represents the powder tablet sample to be tested, and "solid" represents the solution sample to be tested.

[0088] like Figure 5As shown in the figure, the terahertz spectral characteristic curves of L-valine solution and powder tablet samples are presented. The figure shows that the transmittance curve of the powder dot curve (L-valine solution sample) exhibits a clear transmittance change in the frequency range of 0 THz to 2.4 THz, with a significant decrease in transmittance near 1.70 THz and 2.19 THz. This is consistent with the characteristic absorption peak frequency of the blue dot curve (L-valine powder tablet sample). This demonstrates that after dissolving L-valine in a water-glycerol mixed solvent, the terahertz wave can effectively detect the molecular vibration or rotation mode of L-valine. This indicates that the micro-solution sample cell prepared according to this invention, using a water-glycerol mixed solvent as the detection solvent, can effectively detect the terahertz spectral characteristics of L-valine, thereby effectively determining human biomolecules. Furthermore, the characteristic absorption peak frequencies of L-valine in solution and solid form are perfectly matched, indicating that the dissolution process did not damage the molecular structure of L-valine. Terahertz spectroscopy can accurately capture its inherent "fingerprint" characteristics, solving the problem that traditional solvents cannot dissolve amino acids or that the structure is destroyed after dissolution.

[0089] like Figure 6 The figure shows the terahertz spectral characteristics of the L-phenylalanine solution and powder tablet samples. As can be seen, the L-phenylalanine solution sample exhibits a significant decrease in transmittance around 1.53 THz and 1.98 THz, which is completely consistent with the characteristic absorption peak frequencies of its powder tablet sample. This demonstrates that the combination of the water-glycerol mixed solvent and the micro-sample cell can not only dissolve L-valine but also accommodate the dissolution and detection of L-phenylalanine, reflecting the universality of this technique for different amino acids. Furthermore, the characteristic absorption peaks of L-phenylalanine differ from those of L-valine, indicating that terahertz spectroscopy can distinguish the molecular structures of different amino acids. Simultaneously, the consistent absorption peak frequencies in both the solution and solid states prove that the dissolution process did not destroy the molecular structure of L-phenylalanine.

[0090] L-proline is a special amino acid with a cyclic side chain, and its molecular structure differs from the straight-chain side chain structure of L-valine and L-phenylalanine. For example... Figure 7 The figure shows the terahertz spectral characteristic curves of the L-proline solution sample and the powder tablet sample. As can be seen from the figure, the transmittance of the solution curve decreases at 1.99 THz, which perfectly matches the characteristic absorption peak of the solid curve. This proves that the combination of the micro-solid sample cell disclosed in this invention with water and glycerol solvents can effectively detect amino acids of different structural types through terahertz spectroscopy, and has a wide range of applications.

[0091] like Figure 8The figure shows the terahertz spectral characteristic curves of glucose solution and powder tablet samples. As can be seen, the glucose solution sample exhibits a significant decrease in transmittance at 1.43 THz, which is completely consistent with the characteristic absorption peak frequency of its powder tablet sample. This proves that the technical solution disclosed in this invention can not only detect amino acids but also adapt to the terahertz detection of carbohydrates such as glucose in solution. Furthermore, the figure also shows that the characteristic absorption peak of glucose differs significantly in position from those of L-valine, L-phenylalanine, and L-proline. This difference is due to the vibrational mode differences caused by the polyhydroxy cyclic structure of glucose, which reflects the molecular specificity of terahertz fingerprint spectroscopy and can therefore be used to distinguish different types of biomolecules.

[0092] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing a micro-solution sample cell, characterized in that, The preparation method includes: An annular gasket is disposed on a first TPX sheet, and the first TPX sheet and the annular gasket form a solution cavity; The solution chamber is used to hold the solution to be tested, and the thickness of the solution to be tested can be adjusted by changing the number or thickness of the annular gaskets; A second TPX sheet is placed on the annular gasket to obtain the micro-solution sample cell.

2. The preparation method according to claim 1, characterized in that, The solute in the test solution is L-valine, L-phenylalanine, L-proline, or glucose.

3. The preparation method according to claim 1, characterized in that, The first TPX sheet has a diameter of 13mm~15mm and a thickness of 2.0mm~2.2mm, and the second TPX sheet has a diameter of 13mm~15mm and a thickness of 1.95mm~2.05mm.

4. The preparation method according to claim 1, characterized in that, Both the first TPX sheet and the second TPX sheet include an antireflection film, the thickness of which is 0.5μm~2μm.

5. The preparation method according to claim 1, characterized in that, The annular gasket is a TPX annular gasket or a ceramic annular gasket.

6. The preparation method according to claim 5, characterized in that, The thickness of the annular gasket is 5μm~200μm, the inner diameter is 8mm~10mm, and the outer diameter is 12mm~14mm.

7. The preparation method according to claim 1, characterized in that, The first TPX sheet, the second TPX sheet, and the annular gasket are fixed by compression.

8. The preparation method according to claim 1, characterized in that, The upper and lower surfaces of the annular gasket both include an adhesive coating; the first TPX sheet and the second TPX sheet are fixedly connected to the annular gasket by adhesive bonding, and the adhesive coating has an adhesion strength ≥10 N / cm at 25℃~30℃. 2 .

9. A micro-volume solution sample cell, characterized in that, The micro-solution sample cell is prepared by the preparation method according to any one of claims 1 to 8.

10. An application of a micro-solution sample cell, characterized in that, The micro-solution sample cell of claim 9 is applied to the detection of terahertz spectra, wherein the solvent used for detection is glycerol, DMSO, DMF or a mixture of water and glycerol.

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