Raman spectrum detection device for frozen biological fluid sample

Abstract

The utility model discloses a Raman spectrum detection device for freezing a biological fluid sample, and particularly relates to the technical field of biomedical detection equipment, the Raman spectrum detection device comprises a detection box, a sample table, a refrigeration device, a vacuum pump and a Raman spectrometer, the sample table is fixed in the detection box, the sample table is used for placing a sample test tube, the refrigeration device is connected with the sample table, and the vacuum pump is connected with the Raman spectrometer. The detection box is connected with the vacuum pump through a pipeline, a probe of the Raman spectrometer is fixed on the detection box, and the probe of the Raman spectrometer is used for emitting laser and collecting a Raman spectrum of the sample test tube. According to the utility model, accurate temperature regulation and control can be realized, sample denaturation and inactivation are avoided, and the spectrum quality and the detection stability are remarkably improved.

Description

Technical Field

[0001] This utility model relates to the field of biomedical detection equipment technology, and in particular to a Raman spectroscopy detection device for frozen biological fluid samples. Background Technology

[0002] Raman spectroscopy, based on the principle of inelastic scattering of molecular vibrations and rotations, can provide "fingerprint-level" characteristic information of the chemical composition and molecular structure of a sample.

[0003] However, applying Raman spectroscopy to the detection of frozen blood samples still faces several severe technical bottlenecks: First, during freeze-thaw cycles, irreversible changes in water crystallization, protein conformation, and the spatial arrangement of biomolecules (especially the formation of destructive ice crystals) severely distort the original molecular information of the sample, directly affecting the accuracy and repeatability of Raman spectroscopy. Second, the Raman signal itself is extremely weak, requiring extended integration time to obtain an effective spectrum, which places stringent requirements on the low-temperature stability of the sample. Even small temperature fluctuations can trigger changes in molecular conformation or phase transitions, leading to peak position drift, abnormal peak intensity, or even spurious peaks. Third, mechanical vibrations caused by the low-temperature system, thermal drift caused by the thermal expansion and contraction of materials, and frost formation on the Raman probe window can further deteriorate the spectral quality and even lead to detection failure.

[0004] Currently, most commercially available low-temperature Raman spectroscopy systems or hot / cold stage accessories are general-purpose designs and have not been specifically optimized for biological fluid samples such as frozen blood, which have high water content, are sensitive to ice crystals, and often exist in trace amounts, making it difficult to meet the needs of accurate detection. Utility Model Content

[0005] The purpose of this invention is to provide a Raman spectroscopy detection device for frozen biological fluid samples, in order to solve the problems existing in the prior art, achieve precise temperature control, avoid sample denaturation and inactivation, and significantly improve spectral quality and detection stability.

[0006] To achieve the above objectives, this utility model provides the following solution:

[0007] This invention provides a Raman spectroscopy detection device for frozen biological fluid samples, comprising a detection chamber, a sample stage, a refrigeration device, a vacuum pump, and a Raman spectrometer. The sample stage is fixedly disposed inside the detection chamber and is used to place sample tubes. The refrigeration device is connected to the sample stage. The detection chamber is connected to the vacuum pump through a pipe. The probe of the Raman spectrometer is mounted on the detection chamber and is used to emit laser light and acquire the Raman spectral signal of the sample tubes.

[0008] Preferably, it also includes an inert gas storage bottle, which is connected to the detection box via an inlet pipe.

[0009] Preferably, the sample stage is also included, wherein the temperature sensor is connected to the sample stage and is used to monitor the temperature of the sample stage in real time.

[0010] Preferably, the detection box includes a box body and a door. The box body has an opening, and the door is rotatably connected to the opening side of the box body. A sealing structure is provided between the box body and the door. The probe of the Raman spectrometer is fixedly connected to the door through a mounting flange, and the probe and the door are sealed together by a sealing ring.

[0011] Preferably, the window of the probe is made of quartz, and both ends of the window are coated with an antireflection film.

[0012] Preferably, the sample stage includes a top plate and multiple legs fixedly connected to the top plate. The top plate has fixing holes for fixing sample tubes. The top plate is made of oxygen-free copper and has a gold-plated layer on its surface. Each of the legs is made of plastic. The cooling device is connected to the top plate.

[0013] Preferably, the cooling device includes a TEC module, a heat dissipation component, and a flexible thermal connector. One end of the flexible thermal connector is connected to the cold end of the TEC module, and the other end of the flexible thermal connector passes through the side wall of the testing chamber and is connected to the top plate. The hot end of the TEC module is connected to the heat dissipation component.

[0014] Preferably, the flexible thermal connector is formed by stacking multiple oxygen-free copper sheets with a thickness of 0.1 mm.

[0015] Preferably, the heat dissipation component includes a water-cooled heat sink and a circulating water chiller. The water-cooled heat sink is in close contact with the hot end of the TEC module, and the water-cooled heat sink is connected to the circulating water chiller through a pipeline.

[0016] Preferably, it also includes a controller, wherein the refrigeration device, the vacuum pump and the temperature sensor are all signal-connected to the controller.

[0017] The present invention achieves the following technical advantages over the prior art:

[0018] This invention provides a Raman spectroscopy detection device for frozen biological fluid samples. Connected to the sample stage via a cooling device, it enables precise and continuous low-temperature control of the sample tubes, preventing freeze-thaw cycles in frozen blood samples and avoiding irreversible changes in water crystallization, protein conformation, and the spatial arrangement of biomolecules. This maximizes the preservation of the original molecular information of the sample, improving the accuracy and repeatability of Raman spectroscopy detection. Simultaneously, a vacuum pump is used to evacuate the detection chamber, reducing the water vapor content and solving the problem of frost formation on the Raman probe window. This ensures unobstructed laser emission and spectral acquisition, preventing spectral quality deterioration and detection failure caused by frost. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this utility model. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of a Raman spectroscopy detection device used for frozen biological fluid samples.

[0021] In the diagram: 1. Detection box; 2. Sample stage; 3. Cooling device; 4. Raman spectrometer; 5. Probe; 6. Sample tube; 7. Temperature sensor; 8. Flexible thermal connector. Detailed Implementation

[0022] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0023] The purpose of this invention is to provide a Raman spectroscopy detection device for frozen biological fluid samples, in order to solve the problems existing in the prior art, achieve precise temperature control, avoid sample denaturation and inactivation, and significantly improve spectral quality and detection stability.

[0024] To make the above-mentioned objectives, features and advantages of this utility model more apparent and understandable, the present utility model will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1

[0026] This embodiment provides a Raman spectroscopy detection device for frozen biological fluid samples, such as... Figure 1As shown, the system includes a detection chamber 1, a sample stage 2, a cooling device 3, a vacuum pump, and a Raman spectrometer 4. The sample stage 2 is fixed inside the detection chamber 1 and is used to hold sample tubes 6. The cooling device 3 is connected to the sample stage 2. The detection chamber 1 is connected to the vacuum pump via a pipe. The probe 5 of the Raman spectrometer 4 is fixed on the detection chamber 1 and is used to emit laser light and collect the Raman spectral signal of the sample tubes 6. By coupling the cooling device 3 with the sample stage 2, precise and continuous low-temperature control of the sample tubes 6 is achieved, effectively preventing freeze-thaw cycles in frozen blood samples, avoiding water crystallization, protein conformational changes, and irreversible changes in the spatial arrangement of biomolecules, thereby preserving the original molecular information of the sample to the greatest extent and improving the accuracy and repeatability of Raman spectroscopy detection. At the same time, the vacuum pump evacuates the detection chamber 1, which reduces the heat transferred from the outside to the sample stage 2 through gas convection and also reduces the water vapor content inside the detection chamber 1, solving the problem of frost formation at the window of the Raman probe 5. This not only ensures the continuous smooth operation of the laser excitation and signal acquisition optical path, but also avoids the risk of spectral signal attenuation, quality deterioration, and detection failure caused by frost.

[0027] Preferably, the chamber sidewall of the testing chamber 1 is provided with a KF16 interface, which is connected to a vacuum pump via a metal bellows, enabling the chamber vacuum of the testing chamber 1 to be evacuated to 10. -4 mbar or higher.

[0028] Furthermore, the testing chamber 1 is also equipped with a pressure relief valve. After the test is completed, air is slowly introduced through the pressure relief valve to make the pressure inside and outside the testing chamber 1 consistent, which facilitates safe opening of the chamber and removal of samples.

[0029] In a further preferred embodiment of this invention, the Raman spectroscopy detection device for frozen biological fluid samples also includes an inert gas storage bottle; the side wall of the detection chamber is provided with a 1 / 4-inch VCR interface or a Swagelok interface, which is connected to the inert gas storage bottle via an inlet pipe. During the detection process, high-purity dry nitrogen or helium is backfilled into the evacuated detection chamber 1, creating a slightly positive pressure inert atmosphere inside the chamber. Compared to a vacuum environment, this improves heat exchange, resulting in a more uniform sample temperature distribution, and effectively prevents frost formation on the Raman spectrometer probe window.

[0030] In a further preferred embodiment of this invention, the Raman spectroscopy detection device for frozen biological fluid samples further includes a temperature sensor 7, which is connected to the sample stage 2 and is used to monitor the temperature of the sample stage 2 in real time.

[0031] In a further preferred embodiment of this invention, the detection box 1 includes a box body and a door. The box body has an opening, and the door is rotatably connected to the opening side of the box body. A sealing structure is provided between the box body and the door. The probe 5 of the Raman spectrometer 4 is fixedly connected to the door through a mounting flange, and the probe and the door are sealed together by a sealing ring.

[0032] In a further preferred embodiment of this invention, the window of the probe 5 of the Raman spectrometer 4 is made of quartz, and both ends of the window are coated with an antireflection film. The window of the probe 5 of the Raman spectrometer 4 is the only optical path between the low-temperature detection chamber 1 and the external room-temperature spectrometer; the laser beam enters through this path, and the Raman signal from the sample also exits through it. The performance of the window directly affects the signal quality. The ultraviolet-grade fused silica window undergoes a customized coating process with multi-layer antireflection coating (AR coating) on ​​both sides. This ensures stable optical and mechanical properties at -80°C, preventing cracking or deformation, while also improving wavelength transmittance and significantly enhancing excitation and signal collection efficiency.

[0033] In a further preferred embodiment of this invention, the sample stage 2 includes a top plate and multiple legs fixedly connected to the top plate. The top plate has fixing holes for fixing sample tubes 6. The top plate is made of oxygen-free copper and has a gold-plated layer on its surface. The legs are made of plastic. The cooling device 3 is connected to the top plate. The top plate is made of high-purity oxygen-free copper (OFHC) and has a gold-plated surface. Copper provides excellent thermal conductivity, ensuring that the cooling energy transferred from the flexible thermal connector 8 can be quickly and evenly distributed throughout the top plate. The gold plating not only prevents copper oxidation but also provides a highly reflective surface, reducing the radiative heat load from the chamber walls. The legs are made of plastic with low thermal conductivity and low coefficient of thermal expansion to achieve thermal insulation with the inner bottom surface of the detection chamber 1. To facilitate the placement of blood collection tubes, centrifuge tubes, and other packaging, the fixing holes of the top plate have flexible limiting rings to fix the sample tubes 6 in the center of the optical path.

[0034] In a further preferred embodiment of this invention, the cooling device 3 includes a TEC module, a heat dissipation assembly, and a flexible thermal connector 8. One end of the flexible thermal connector 8 is connected to the cold end of the TEC module, and the other end of the flexible thermal connector 8 passes through the side wall of the detection chamber 1 and connects to the top plate. A sealing structure is provided at the penetration point of the flexible thermal connector 8 into the side wall of the detection chamber 1 to ensure the chamber of the detection chamber 1 is sealed. The hot end of the TEC module is connected to the heat dissipation assembly. The TEC module operates based on the Peltier effect of semiconductor materials. When direct current passes through a thermocouple composed of different semiconductor materials, one end absorbs heat to form a cold end, and the other end releases heat to form a hot end. By connecting multiple TEC modules in series, a larger cooling temperature difference can be achieved. The TEC module adopts a pure solid-state cooling method with no moving parts, and there is no mechanical vibration or noise during operation. High-precision temperature control can be achieved by precisely controlling the current flowing into the TEC module, and the overall system structure is compact and lightweight.

[0035] In a further preferred embodiment of this invention, the flexible thermal connector 8 is composed of multiple stacked oxygen-free copper sheets, each 0.1 mm thick. Copper exhibits excellent thermal conductivity at low temperatures. This layered structure makes the flexible thermal connector 8 highly flexible in the direction perpendicular to the copper sheet plane, capable of absorbing vibrations and compensating for displacements. Simultaneously, due to its large total cross-sectional area, it maintains extremely high thermal conductivity along the length of the copper sheets. The two ends of the stacked oxygen-free copper sheets are fixed to two copper end blocks using pressure welding or diffusion welding techniques. The cold end of the TEC module and one end block are tightly bolted together, and the sample stage 2 is also bolted together with the other end block.

[0036] In a further preferred embodiment of this invention, the heat dissipation component includes a water-cooled heat sink and a circulating water chiller. The water-cooled heat sink is in close contact with the hot end of the TEC module, and the water-cooled heat sink is connected to the circulating water chiller through a pipeline.

[0037] In a further preferred embodiment of this invention, the Raman spectroscopy detection device for freezing biological fluid samples also includes a controller. The cooling device 3, vacuum pump, and temperature sensor 7 are all signal-connected to the controller. The controller is a commercially available high-precision, multi-channel PID temperature controller. It supports four-wire sensor measurement to eliminate errors caused by lead resistance. Adjustable PID parameters (proportional, integral, derivative) are provided to optimize for the system's thermal characteristics (heat capacity, thermal conductivity, etc.), achieving rapid, stable, and overshoot-free temperature control. It can provide a stable, low-noise DC power output to drive the TEC module. For example, the controller continuously reads the temperature value T_actual from the temperature sensor 7 and compares it with the user-set target temperature T_setpoint. Based on the difference (T_setpoint - T_actual), the PID algorithm calculates an accurate output power and adjusts the current applied to the TEC module accordingly. If T_actual is lower than T_setpoint, the current is reduced; if it is higher, the current is increased. Through this closed-loop feedback, the system can automatically resist external thermal disturbances and lock the temperature of the sample stage 2 at the set value.

[0038] Example 2

[0039] This embodiment provides a detection method using the Raman spectroscopy detection device for frozen biological fluid samples according to Embodiment 1, comprising the following steps:

[0040] Step 1: Start the equipment controller and preset the target cooling temperature of the cooling device;

[0041] Step 2: Open the door of the testing chamber, place the sample tubes stably on the sample stage, close the door and ensure that the door and the chamber are sealed tightly.

[0042] Step 3: The controller starts the vacuum pump to evacuate the chamber until the vacuum gauge shows that the pressure inside the chamber is below 10. -3 mbar; Close the vacuum valve and stop pumping, then open the inert gas valve and slowly backfill the chamber with dry nitrogen gas to a pressure slightly above atmospheric pressure. After completion, close the inert gas valve.

[0043] Step 4: The controller activates the cooling device to cool the sample stage. When the temperature sensor detects that the sample stage temperature has reached the preset value, the device automatically enters the constant temperature mode. In this state, it is maintained for 10-15 minutes to ensure that the temperature reading is stable and the internal temperature of the sample is completely consistent with the set temperature.

[0044] Step 5: Set the relevant parameters of the spectrometer according to the detection requirements (such as excitation wavelength, integration time, scanning range, etc.), and emit a laser to the sample tube through the Raman spectroscopy acquisition probe to simultaneously acquire the Raman spectral signal of the sample;

[0045] Step six: After the test is completed, the controller controls the cooling device to heat the sample stage (from -80℃ to about 0℃), and then slowly raises the sample stage to room temperature. After the temperature inside the chamber reaches room temperature, open the chamber door and take out the sample. Then, turn off the spectrometer, controller, vacuum pump and other related equipment in sequence. Finally, clean and tidy the inside of the sample chamber.

[0046] This utility model uses specific examples to illustrate its principles and implementation methods. The above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​this utility model. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the idea of ​​this utility model. In summary, the content of this specification should not be construed as a limitation of this utility model.

Claims

1. A Raman spectroscopy detection device for freezing a biological fluid sample, characterized by: The device includes a detection chamber, a sample stage, a cooling device, a vacuum pump, and a Raman spectrometer. The sample stage is fixedly installed inside the detection chamber and is used to hold sample tubes. The cooling device is connected to the sample stage. The detection chamber is connected to the vacuum pump through a pipe. The probe of the Raman spectrometer is mounted on the detection chamber and is used to emit laser light and collect the Raman spectral signal of the sample tubes. The device also includes an inert gas storage bottle, which is connected to the detection chamber through an inlet pipe.

2. The Raman spectroscopic detection apparatus for frozen biological fluid samples of claim 1, wherein: It also includes a temperature sensor, which is connected to the sample stage and is used to monitor the temperature of the sample stage in real time.

3. The Raman spectroscopic detection apparatus for frozen biological fluid samples of claim 1, wherein: The detection box includes a box body and a box door. The box body has an opening, and the box door is rotatably connected to the opening side of the box body. A sealing structure is provided between the box body and the box door. The probe of the Raman spectrometer is fixedly connected to the box door through a mounting flange, and the probe and the box door are sealed together by a sealing ring.

4. The Raman spectroscopy detection device for frozen biological fluid samples according to claim 1, characterized in that: The sample stage includes a top plate and multiple legs fixedly connected to the top plate. The top plate has fixing holes for fixing sample tubes. The refrigeration device is connected to the top plate.

5. The Raman spectroscopy detection device for frozen biological fluid samples according to claim 4, characterized in that: The cooling device includes a TEC module, a heat dissipation component, and a flexible thermal connector. One end of the flexible thermal connector is connected to the cold end of the TEC module, and the other end of the flexible thermal connector passes through the side wall of the testing chamber and is connected to the top plate. The hot end of the TEC module is connected to the heat dissipation component.

6. The Raman spectroscopy detection device for frozen biological fluid samples according to claim 5, characterized in that: The flexible thermal connector is made of multiple oxygen-free copper sheets stacked together, each 0.1 mm thick.

7. The Raman spectroscopy detection device for frozen biological fluid samples according to claim 5, characterized in that: The heat dissipation component includes a water-cooled heat sink and a circulating water chiller. The water-cooled heat sink is in close contact with the hot end of the TEC module, and the water-cooled heat sink is connected to the circulating water chiller through a pipeline.

8. The Raman spectroscopy detection device for frozen biological fluid samples according to claim 2, characterized in that: It also includes a controller, and the refrigeration device, the vacuum pump and the temperature sensor are all signal-connected to the controller.

Images