Endotoxin quantitative detection method and detection system

Abstract

The invention relates to the technical field of endotoxin detection, in particular to an endotoxin quantitative detection method and a detection system. The invention aims to improve the detection sensitivity and convenience of endotoxin and reduce the use amount of a sample. A first purpose of the present invention is to provide an endotoxin quantitative detection method, which comprises: respectively injecting a standard tachypleus amebocyte lysate solution and endotoxin standard solutions with different concentrations into a microfluidic channel embedded with an optical fiber biochemical sensor according to a specific volume ratio, and inputting signal light into the optical fiber biochemical sensor, acquiring spectral data output when the optical fiber biochemical sensor contains different standard mixed solutions, drawing an endotoxin concentration logarithm-time fitting curve chart, then replacing the endotoxin standard solution with a to-be-detected sample, repeating the steps, extracting corresponding time parameters, and comparing the time parameters with the fitting curve chart, so as to obtain the endotoxin content in the to-be-detected sample solution. The second purpose of the invention is to provide an endotoxin quantitative detection system.

Description

Technical Field

[0001] The present invention relates to the technical field of endotoxin detection, and more specifically, to an endotoxin quantitative detection method and a detection system. Background Art

[0002] Endotoxins, also known as lipopolysaccharides (LPS), are the main components of the outer membrane of Gram-negative bacteria and some cyanobacteria. Bacterial cells can release large amounts of LPS during cell death, or release small amounts during normal metabolism, which can be toxic to the human body. LPS is an important pollutant in the fields of pharmaceuticals, food industry, healthcare, and environmental monitoring. The rabbit fever test (RPT) is considered to be the oldest and simplest endotoxin detection technology. However, the RPT detection method has been criticized due to the need for live testing and large samples, as well as lower sensitivity and accuracy compared to other methods.

[0003] The horseshoe crab reagent LAL detection technology (the reagent is taken from the blood of horseshoe crabs, and the reagent will coagulate into blocks after contact with endotoxins) came into being. It is more accurate than RPT and no longer involves living test subjects. The Chinese Pharmacopoeia, the United States Pharmacopoeia and the United States Food and Drug Administration have approved this test method as an endotoxin testing strategy for oral / injectable drugs, hospital USP-grade water and medical devices, and have used it as an official endotoxin testing method and have been widely recognized. Although the LAL test is of great significance in applications, it often encounters certain chemical or physical interferences, which are usually solved by continuously diluting the test sample until the interference no longer affects the detection level, which not only prolongs the test time but also increases labor costs and sample consumption. Summary of the invention

[0004] The present invention aims to overcome at least one of the defects of the above-mentioned prior art and provide a quantitative detection method and detection system for endotoxin to improve the detection sensitivity and convenience and reduce the sample usage.

[0005] The first aspect of the present invention is to provide a method for quantitative detection of endotoxins, comprising the following steps:

[0006] S1. Prepare at least 4 endotoxin standard solutions of different concentrations;

[0007] S2. The standard horseshoe crab reagent solution and the endotoxin standard solution of different concentrations are respectively injected into the microfluidic channel embedded with the optical fiber biochemical sensor according to a specific volume ratio, wherein the optical fiber biochemical sensor is provided with a sample slot, and the standard mixed solution obtained by mixing the standard horseshoe crab reagent solution and the endotoxin standard solution covers the sample slot;

[0008] S3. Inputting the signal light into the optical fiber biochemical sensor, obtaining the spectral data output by the optical fiber biochemical sensor when containing standard mixed solutions of different concentrations, extracting the time required for the spectral valley value in the spectral data to drift to a specific wavelength, and drawing a logarithmic-time fitting curve of the endotoxin concentration;

[0009] S4. Inject the standard horseshoe crab reagent solution in step S2 and the sample solution to be tested into the microfluidic channel embedded with the fiber optic biochemical sensor according to the specific volume ratio in step 2, and mix the standard horseshoe crab reagent solution and the sample solution to be tested to obtain a sample mixed solution that covers the sample tank, obtain the spectral data output by the fiber optic biochemical sensor when accommodating the sample mixed solution, extract the time required for the spectral valley value to drift to the specific wavelength described in step S3, and compare it with the fitting curve graph to obtain the endotoxin content in the sample solution to be tested.

[0010] After the standard horseshoe crab reagent solution is mixed with the standard solution or sample solution containing endotoxin, it will gradually solidify, thereby causing the refractive index of the mixture to change. When the mixture is filled in the sample slot of the optical fiber biochemical sensor, the signal light emitted by the light source will undergo spectral changes after being output by the optical fiber biochemical sensor. The research of the present invention shows that the time required for the spectral valley value in the spectral data to drift a specific wavelength is related to the logarithm of the endotoxin concentration. Therefore, a fitting curve can be prepared based on endotoxin standard solutions of different concentrations, and the time required for the drift of the specific wavelength of the sample liquid to be tested is detected, thereby conveniently knowing the endotoxin content of the sample liquid to be tested or the sample to be tested. The present invention embeds the optical fiber biochemical sensor in a microfluidic channel, and the injection amount is accurately controlled by the microfluidic channel to ensure the accuracy of the quantitative measurement results, and significantly reduce the sampling amount, and the optical fiber biochemical sensor has high sensitivity, so that the method of the present invention has the advantages of convenience, sensitivity, and low sample dosage.

[0011] Furthermore, the concentration of the endotoxin standard solution is 10 -4 -10 -1 EU / mL.

[0012] Furthermore, the specific volume ratio is: the ratio of the standard horseshoe crab reagent solution to the endotoxin standard solution is 1:1-50:1; or the ratio of the standard horseshoe crab reagent solution to the sample solution to be tested is 1:1-50:1.

[0013] The volume ratio of the present invention can ensure a faster detection speed on the one hand, and can also avoid excessive consumption of horseshoe crab reagent on the other hand. In particular, when the volume of horseshoe crab reagent solution and the volume of the sample to be tested are 1:1, the amount of horseshoe crab reagent can be greatly saved, and the accuracy and high efficiency of botulinum toxin content detection can be guaranteed.

[0014] Furthermore, the specific wavelength is 2-20 nm.

[0015] The wavelength range of the present invention can overcome spectral fluctuations, avoid background signal interference and expand the range of endotoxin detection, especially when the wavelength is 5nm, the effect is best.

[0016] Furthermore, the wavelength range of the signal light is 450-2400nm, and the output power is 5-20dBm.

[0017] Furthermore, by connecting a spectrum analyzer to the output end of the optical fiber biochemical sensor to obtain the spectral data output by the optical fiber biochemical sensor, the spectrum analyzer has a resolution of 10-20pm, a test wavelength range of 600-1700nm, a wavelength resolution of 0.03-1nm, a dynamic range of 40-60dB, and a scanning speed of 20-40nm / second.

[0018] The second aspect of the present invention is to provide an endotoxin quantitative detection system, which is applicable to the above-mentioned endotoxin detection method, and the detection system includes a light source emitting device, an incident light lead, a microfluidic channel, an optical fiber biochemical sensor, an output light lead and a spectrum analyzer;

[0019] The optical fiber biochemical sensor is embedded in the microfluidic channel and a gap is left between the optical fiber biochemical sensor and the inner wall of the microfluidic channel. The optical fiber biochemical sensor is provided with a sample holding area. The microfluidic channel is provided with at least two sample inlets and at least one sample outlet. The microfluidic channel area between any sample inlet and any sample outlet covers the sample holding area.

[0020] One end of the incident light lead is connected to the light source generating device, the other end of the incident light lead passes through the microfluidic channel and is connected to the optical fiber biochemical sensor, one end of the output light lead is connected to the optical fiber biochemical sensor, and the other end of the output light lead passes through the microfluidic channel and is connected to the spectral analysis instrument.

[0021] Furthermore, the incident light lead is fixedly connected to the microfluidic channel, and the outgoing light lead is fixedly connected to the microfluidic channel.

[0022] This solution can stabilize the optical fiber biochemical sensor in the area between the sample inlet and the sample outlet, ensuring that the optical fiber biochemical sensor can reliably receive the mixed solution of the sample solution and the standard horseshoe crab reagent solution.

[0023] Furthermore, the optical fiber biochemical sensor includes a first single-mode optical fiber, a hollow optical fiber and a second single-mode optical fiber connected in sequence, the hollow optical fiber is provided with a sample slot serving as the sample holding area, and the light source generating device inputs signal light from one end of the first single-mode optical fiber during detection, and the signal light passes through the first single-mode optical fiber, the hollow optical fiber filled with the sample liquid to be tested, and the second single-mode optical fiber in sequence, and then is output from one end of the second single-mode optical fiber to the spectrometer.

[0024] This solution uses a sample tank to hold a mixed solution of sample liquid and horseshoe crab reagent. Therefore, when the signal light is input into the first single-mode optical fiber and output to the hollow-core optical fiber through its end face, the mixed solution and the cladding of the hollow-core optical fiber form an anti-resonance reflection optical waveguide structure. This structure has a periodic conduction mode and a leakage mode. The mixed solution filled in the hollow-core optical fiber can limit some wavelengths of light to achieve a more complete interaction with the mixed solution. The output signal light then enters the end face of the second single-mode optical fiber and is coupled back to the core of the second single-mode optical fiber, and then output from its end, forming a fiber refractive index optical fiber sensor device that directly interacts with the mixed solution. Since the sample liquid is allowed to be used as a low-loss transmission waveguide and a slight change in the refractive index will cause a shift in the spectrum, a higher sensitivity can be achieved.

[0025] Furthermore, the length of the hollow core optical fiber is 0.5-2.5 mm.

[0026] The length dimension of this scheme is sufficient to ensure that the sensing principle of the optical fiber biochemical sensor is based on the anti-resonance effect.

[0027] Furthermore, the injection port comprises a first injection port and a second injection port, and the first injection port and the second injection port are relatively arranged on two sides of the flow direction of the microfluidic channel.

[0028] This solution enables the sample liquid and the labeled horseshoe crab reagent respectively input into the first injection port and the second injection port to have relative incident directions, which helps to achieve a rapid mixing effect.

[0029] Furthermore, it also includes a micro-injection pump connected to the injection port.

[0030] Furthermore, a drainage pipeline connected to the injection port is also provided on the outer surface of the microfluidic channel, and the injection port is connected to the micro-injection pump through the drainage pipeline.

[0031] This solution facilitates connection with a microinjection pump through a drainage pipeline, improves connection strength, and helps maintain sample input accuracy.

[0032] Furthermore, the drainage pipeline is arranged to be inclined relative to the microfluidic channel, and the inclined direction is from the injection port toward a direction away from the optical fiber biochemical sensor.

[0033] This solution enables the sample to have an initial velocity toward the optical fiber biochemical sensor when it is input into the microfluidic channel, and can ensure that the sample liquid and the standard horseshoe crab reagent solution are mixed and then directed to the optical fiber biochemical sensor.

[0034] Further, the microfluidic channel is a rectangular parallelepiped, and the length of the microfluidic channel is 5-50 mm, the width is 5-20 mm, and the thickness is 2-10 mm; and / or,

[0035] The aperture of the injection port is 1-2 mm; and / or,

[0036] The aperture of the sample outlet is 1-2 mm.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: through the microfluidic channel in conjunction with a micro-injection pump, the test sample and the standard horseshoe crab reagent solution are micro-quantitated and highly precisely injected into the system, and the hollow optical fiber of the optical fiber biochemical sensor is used to contain the reaction liquid, and a spectrometer is used to record and analyze the spectral data in real time, and the spectral drift rate is used to quantify the endotoxin concentration, providing a new solution for the high-sensitivity, low-sample consumption, and rapid quantitative detection of endotoxins in the fields of medicine, drug research, food safety, and environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure 2 is a structural diagram of the endotoxin detection system.

[0039] Figure 2 This is the structural diagram of the optical fiber biochemical sensor.

[0040] Figure 3 This is a cross-sectional view of the optical fiber biochemical sensor.

[0041] Figure 4 Figure 2 is a diagram of the microfluidic channel structure.

[0042] Figure 5 This is a graph showing the variation of the transmittance spectrum of the optical fiber biochemical sensor input spectrum caused by different test samples measured in Example 1 over time.

[0043] Figure 6 The left side is a summary diagram of the response curves of the trough shift change of the optical fiber biochemical sensor input spectrum caused by different test samples measured in Example 1 over time. Figure 6 The right side is the fitting curve of the relationship between the response time and concentration corresponding to the 5nm trough shift of the optical fiber biochemical sensor input spectrum caused by different test samples measured in Example 1.

[0044] Figure numerals: light source emitting device 100, incident light lead 200, microfluidic channel 300, first sample inlet 310, second sample inlet 320, sample outlet 330, drainage pipeline 340, base 350, output pipeline 360, fiber optic biochemical sensor 400, first single-mode optical fiber 410, first polishing area 411, hollow optical fiber 420, sample slot 421, second single-mode optical fiber 430, second polishing area 431, fiber core 440, output light lead 500, spectrum analyzer 600, micro-injection pump 700. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels by those skilled in the art.

[0047] Example 1

[0048] This embodiment provides a method for quantitative detection of endotoxin, comprising the following steps:

[0049] S1. Prepare endotoxin standard solution. The endotoxin content in the endotoxin standard solution is 10 -4 -10 -1 EU / mL, the four concentrations selected were 10 -4 EU / mL, 10 -3 EU / mL, 10 -2 EU / mL, 10 -1 EU / mL;

[0050] S2. Take 5uL of the standard horseshoe crab reagent solution and the four different concentrations of endotoxin standard solutions in S1 each time, and inject them into the microfluidic channel embedded with the fiber optic biochemical sensor, wherein the fiber optic biochemical sensor is provided with a sample holding area. After each injection of the endotoxin standard solution and the standard horseshoe crab reagent solution, the standard mixed solution obtained by mixing the standard horseshoe crab reagent solution and the endotoxin standard solution can completely cover the sample holding area through the rapid mixing effect of the microfluidic channel. In order to improve the accuracy and reliability of the test, two groups of negative control groups are also prepared. Among them, the first negative control group uses 5uL of standard horseshoe crab reagent solution + 5uL of horseshoe crab reagent test water injected into the microfluidic channel embedded with the fiber optic biochemical sensor, and the second negative control group uses 5uL of horseshoe crab reagent test water + 5uL of horseshoe crab reagent test water injected into the microfluidic channel embedded with the fiber optic biochemical sensor.

[0051] Regarding the standard horseshoe crab reagent solution: the optional detection limit range of the horseshoe crab reagent solution includes 0.005-0.1EU / mL. The detection limit of the horseshoe crab reagent solution specifically used in this embodiment is 0.06EU / mL. The greater the concentration of the horseshoe crab reagent solution used, the faster the detection speed is usually, but it will also lead to an increase in the amount of horseshoe crab reagent used and increase the detection cost. The preparation method of horseshoe crab reagent is to dissolve the standard horseshoe crab reagent solid product in the corresponding 100μL standard horseshoe crab reagent test water to prepare a solution with the corresponding detection limit. The horseshoe crab reagent test water is a commercially available horseshoe crab reagent accessory, which is water after the pyrogen is removed.

[0052] In the specific implementation, in order to improve the detection sensitivity, the optical fiber biochemical sensor is prepared by referring to the embodiments 1-5 of the patent document CN114965359A (a refractive index optical fiber sensor and its manufacturing method), which is equivalent to the refractive index optical fiber sensor. Specifically, Figure 2-3 The optical fiber biochemical sensor includes a first single-mode optical fiber 410, a hollow-core optical fiber 420, and a second single-mode optical fiber 430 connected in sequence. The hollow-core optical fiber 420 is provided with a sample slot 421, and the sample slot 421 is used as a sample holding area for holding the sample to be tested. The first single-mode optical fiber 410 and the second single-mode optical fiber 430 are both polished by side edges to form a first polishing area 411 and a second polishing area 431, respectively. Thus, the hollow-core optical fiber 420 is fused between the first single-mode optical fiber 410 and the second single-mode optical fiber 430. The first polishing area 411, the hollow-core optical fiber 420, and the second polishing area 431 together form a D-type slot. The D-type slot facilitates the convergence of the mixed solution in the microfluidic channel, so that the mixed solution is quickly and fully filled in the sample slot 421. At the same time, it can also ensure the transmission power of the first single-mode optical fiber 410 and the second single-mode optical fiber 430, improve the efficiency of exciting multi-order modes in the mixed solution waveguide, and thus improve the detection speed and accuracy. Reference Figure 3 The inner diameter of the sample groove 421 is larger than the diameter of the core 440 covering the first single-mode optical fiber 410 and the second single-mode optical fiber 430. In this way, when the mixed solution completely covers the sample groove 421, the mixed solution can completely cover the core 440 of the first single-mode optical fiber 410 and the second single-mode optical fiber 430, avoiding light leakage at the core 440 that affects the detection results.

[0053] It should be pointed out that the sensitivity of the detection system is independent of the volume of the filling solution, so it is only necessary to ensure that the mixed solution completely fills the sample tank to achieve the detection purpose, and the volume of other external solutions has no effect on the sensitivity of the detection system. In specific implementation, the length of the hollow fiber 420 is 0.5-2.5mm, for example, 1.6mm, the remaining thickness of the hollow fiber 420 is between 62.5-75um, and the inner diameter (equivalent to the inner diameter of the sample tank 421) is 10-100um, preferably 30um, thereby ensuring that the sensing principle of the optical fiber biochemical sensor is based on the anti-resonance effect and has good sensitivity. In addition, the diameter of the first single-mode optical fiber 410 and the second single-mode optical fiber 430 can be selected from 8-9um, for example, 8.2um, and the outer diameter of the cladding is 125-150um, preferably 125um. As for the length of the first single-mode optical fiber 410 and the second single-mode optical fiber 430, it can be adaptively set according to the actual length of the microfluidic channel used to ensure that the microfluidic channel is sufficient to stably assemble the optical fiber biochemical sensor. Among them, the light source is a supercontinuum laser light source or a tunable laser light source, the wavelength range of the light source emitting device is 450-2400nm, and the output power is 5-20dBm, thus providing the endotoxin detection system with high reliability, wide spectral range and high output power, which is sufficient to meet the requirements of optical fiber biochemical sensors of different specifications for light sources.

[0054] S3. After injecting the endotoxin standard solution and the standard horseshoe crab reagent solution (or injecting the first negative control group reagent and the second negative control group reagent in S2), the signal light is input into the optical fiber biochemical sensor to obtain the spectral data output by the optical fiber biochemical sensor when containing standard mixed solutions of different concentrations, such as Figure 5 , which shows the corresponding curve of the trough drift change over time obtained by the spectrum analyzer when the optical fiber biochemical sensor contains different concentrations of endotoxin standard solution or negative control group reagent, combined with Figure 6 As can be seen from the left, as the endotoxin content increases, the slope of the curve increases, that is, there is a positive correlation between the trough offset per unit time and the endotoxin concentration. Figure 6 As shown on the right, by extracting the time required for the spectrum valley value in the spectrum data to drift by 5nm, a logarithmic-time fitting curve of endotoxin concentration is drawn. Figure 6 Right, in the present invention, as the concentration of endotoxin in the test sample gradually increases, the response time y required for the trough to drift 5nm is linearly correlated with the logarithm of the test endotoxin concentration lgC. -4 -10 -1 The linear relationship of EU / mL endotoxin concentration is y=-84.9*lgC-3.8, and the linear correlation is 0.9935.

[0055] The principle of the change of the trough position in the experiment is that when the standard horseshoe crab reagent reacts with the endotoxin in the test sample, the mixed solution will gradually show a coagulation effect over time. Specifically, the solution state changes from a liquid to a colloidal state, and the equivalent refractive index will increase. The optical fiber biochemical sensor used in the present invention is a device for sensing refractive index changes dominated by the anti-resonance principle. In this structure, as the equivalent refractive index of the mixed solution gradually increases, the trough value of the spectrum will gradually decrease. Based on this principle, the present invention analyzes the relationship between the trough value and the endotoxin content, thereby realizing quantitative monitoring of the sample to be tested with an unknown endotoxin content.

[0056] During the specific operation, in order to simplify the environmental requirements for the reaction of the endotoxin standard solution and the standard horseshoe crab reagent, the experimental environment temperature is maintained at room temperature at 25°C. The test process is to record the transmission spectrum of the optical fiber biochemical sensor once every 3 seconds through a spectrometer connected to the output end of the optical fiber biochemical sensor, and continuously monitor the changes in the resonant wavelength within the selected range. To ensure the reusability of the device, the maximum recording time of the spectrum does not exceed 6 minutes. When the spectrum changes rapidly, the recording time can be appropriately reduced. The specific spectrometer used has a resolution of 10-20pm, a test wavelength range of 600-1700nm, a wavelength resolution of 0.03-1nm, a dynamic range of 40-60dB, and a scanning speed of 20-40nm / second. Provide fast, high-resolution, and high-dynamic range equipment support for obtaining detection spectra.

[0057] S4. Inject the standard horseshoe crab reagent solution in step S2 and the sample solution to be tested into the microfluidic channel embedded with the fiber optic biochemical sensor according to the specific volume ratio (1:1) in step 2, and mix the standard horseshoe crab reagent solution and the sample solution to be tested to obtain a sample mixed solution that covers the sample holding area, obtain the spectral data output by the fiber optic biochemical sensor when accommodating the sample mixed solution, extract the time required for the spectral valley value to drift to the specific wavelength (5nm) described in step S3, and compare it with the fitting curve graph obtained in step S3. Specifically, the concentration of endotoxin in the test sample is obtained by the concentration formula C=10y+3.8 / -84.9, so as to achieve quantitative detection of endotoxin.

[0058] In addition, after each test, the fiber optic biochemical sensor was taken out, rinsed three times with alcohol to remove impurities on the fiber surface, dried on a hot plate, and then soaked in the prepared piranha solution for 30 minutes to clean residual endotoxins and organic impurities; after cleaning, the waste liquid was absorbed with a glass pipette, and then rinsed repeatedly three times with a large amount of deionized water, and finally placed in a 100°C drying oven for 1 hour, and the fiber optic biochemical sensor can be reused.

[0059] Example 2

[0060] like Figure 1-4As shown, this embodiment provides an endotoxin quantitative detection system, which is applicable to the endotoxin quantitative method in Example 1, including a light source emitting device 100, an incident light lead 200, a microfluidic channel 300, an optical fiber biochemical sensor 400, an output light lead 500 and a spectrum analyzer 600;

[0061] The optical fiber biochemical sensor 400 is embedded in the microfluidic channel 300 and has a gap with the inner wall of the microfluidic channel 300. The optical fiber biochemical sensor 400 is provided with a sample receiving area. The microfluidic channel 300 is provided with at least two sample inlets and at least one sample outlet 330. The area of ​​the microfluidic channel 300 between any sample inlet and any sample outlet 330 covers the sample receiving area.

[0062] One end of the incident light lead 200 is connected to the light source generating device, the other end of the incident light lead 200 penetrates into the microfluidic channel 300 and is connected to the optical fiber biochemical sensor 400, one end of the outgoing light lead 500 is connected to the optical fiber biochemical sensor 400, and the other end of the outgoing light lead 500 penetrates out of the microfluidic channel 300 and is connected to the spectrum analyzer 600. The optical fiber biochemical sensor 400 is arranged in the manner described in Example 1.

[0063] In a specific implementation, the incident light lead 200 is introduced from the front opening of the microfluidic channel 300, and the outgoing light lead 500 is led out from the rear opening of the microfluidic channel 300. By straightening the incident light lead 200 and the outgoing light lead 500 inside the microfluidic channel 300, and encapsulating the incident light lead 200 in the front opening of the microfluidic channel 300, and encapsulating the outgoing light lead 500 in the rear opening of the microfluidic channel 300, the incident light lead 200, the optical fiber biochemical sensor 400, and the outgoing light lead 500 can be well fixed. Therefore, the optical fiber biochemical sensor 400 is stabilized in the area between the sample inlet and the sample outlet 330, ensuring that the optical fiber biochemical sensor 400 can reliably receive the mixed solution of the sample solution and the standard horseshoe crab reagent solution. Furthermore, since the optical fiber biochemical sensor 400 needs to be cleaned after each sample test, in a preferred embodiment, the incident light lead 200 and the outgoing light lead 500 are fixed to the microfluidic channel 300 in a detachable manner, thereby facilitating the loading and unloading of the optical fiber biochemical sensor 400. In addition, in order to improve the stability of signal light transmission, the incident light lead 200 and the outgoing light lead 500 can be implemented in the form of optical fiber jumpers.

[0064] During detection, the sample liquid to be tested and the standard horseshoe crab reagent solution are respectively injected into the microfluidic channel 300 through two injection ports, and then the two solutions are quickly and evenly mixed through the microfluidic channel 300. Since the sample slot of the optical fiber biochemical sensor 400 is arranged between the injection port and the outlet port 330, the mixed solution can fully cover the sample slot before flowing out of the outlet port 330. The mixed solution gradually solidifies, resulting in a change in the refractive index, thereby causing the signal light output from the optical fiber biochemical sensor 400 to undergo a spectral change. Since the spectral change law is related to the endotoxin content, therefore, referring to the detection method of Example 1, the spectral data of the emitted signal light is analyzed by the spectrometer 600 to obtain the endotoxin content in the sample liquid.

[0065] refer to Figure 4 In some embodiments, the microfluidic channel 300 is a rectangular parallelepiped, and the length L1 of the microfluidic channel 300 is 5-50 mm, the width L2 is 5-20 mm, and the thickness L3 is 2-10 mm, so that the optical fiber biochemical sensor 400 with a length dimension of usually 1-50 mm can be assembled. In addition, the aperture of the sample inlet and the sample outlet 330 is 1-2 mm, which helps to reduce the liquid tension at the sample inlet and the sample outlet 330, making it easier to inject the solution into the microfluidic channel 300. In the specific implementation, refer to Figure 1 , 4 In order to improve the structural stability, the microfluidic channel 300 is embedded in the base 350, and the microfluidic channel 300 and the base 350 can be made of the same material, such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), silicate glass, borosilicate glass or metal material.

[0066] refer to Figure 1 , 4 In this embodiment, a first injection port 310 and a second injection port 320 are specifically provided. The first injection port 310 and the second injection port 320 are relatively arranged on both sides of the flow direction of the microfluidic channel 300. Therefore, the sample liquid and the labeled horseshoe crab reagent respectively input by the first injection port 310 and the second injection port 320 have relative incident directions, which helps to achieve a rapid mixing effect.

[0067] like Figure 1 As shown, in order to achieve accurate injection, a microinjection pump 700 connected to the injection port is also provided. When implemented specifically, the number of microinjection pumps 700 can be a dual-channel mode, thereby extending two connecting pipes to be connected to the first injection port 310 and the second injection port 320, respectively, so as to facilitate accurate control of the injection volume of the two injection ports to remain consistent. In some embodiments, the specification of the microinjection pump 700 is 0.5-60mL, the injection rate is 0.008nL / h-1246L / min, the injection accuracy is ±0.5%, and the reproducibility is ±0.2%.

[0068] In addition, reference Figure 1 In order to improve the connection strength between the microinjection pump 700 and the injection port, the outer surface of the microfluidic channel 300 is also provided with a drainage pipeline 340 connected to the injection port. In some embodiments, the drainage pipeline 340 is integrally formed on the surface of the microfluidic channel 300 and is made of the same material as the microfluidic channel 300, so that the drainage pipeline 340 and the microfluidic channel 300 are highly connected, which can play a stable drainage role for the solution input by the microinjection pump 700. Similarly, the output pipeline 360 ​​can be connected to the sample outlet 330 to facilitate the discharge of the sample liquid in the microfluidic pipeline.

[0069] Furthermore, if Figure 1 As shown, the drainage pipeline 340 is inclined relative to the microfluidic channel 300, and the inclination direction is from the injection port toward the direction away from the optical fiber biochemical sensor 400. In this way, the sample liquid and the standard horseshoe crab reagent solution have an initial velocity toward the optical fiber biochemical sensor 400 when input into the microfluidic channel 300, which can ensure that the sample liquid and the standard horseshoe crab reagent solution are mixed and then guided to the optical fiber biochemical sensor 400.

[0070] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for quantitative detection of endotoxin, characterized in that: The following steps are involved: S1. Prepare at least 4 endotoxin standard solutions of different concentrations; S2. The standard horseshoe crab reagent solution and the endotoxin standard solution of different concentrations are respectively injected into the microfluidic channel embedded with the optical fiber biochemical sensor according to a specific volume ratio, wherein the optical fiber biochemical sensor is provided with a sample receiving area, and the standard mixed solution obtained by mixing the standard horseshoe crab reagent solution and the endotoxin standard solution covers the sample receiving area; S3. Inputting the signal light into the optical fiber biochemical sensor, obtaining the spectral data output by the optical fiber biochemical sensor when containing standard mixed solutions of different concentrations, extracting the time required for the spectral valley value in the spectral data to drift to a specific wavelength, and drawing a logarithmic-time fitting curve of the endotoxin concentration; S4. Inject the standard horseshoe crab reagent solution in step S2 and the sample solution to be tested into the microfluidic channel embedded with the fiber optic biochemical sensor according to the specific volume ratio in step 2, and mix the standard horseshoe crab reagent solution and the sample solution to be tested to obtain a sample mixed solution that covers the sample holding area, obtain the spectral data output by the fiber optic biochemical sensor when accommodating the sample mixed solution, extract the time required for the spectral valley value to drift to the specific wavelength as described in step S3, and compare it with the fitting curve graph to obtain the endotoxin content in the sample solution to be tested.

2. The method for quantitative detection of endotoxin according to claim 1, characterized in that: The endotoxin content of the endotoxin standard solution is 10 -4 -10 -1 EU / mL.

3. The method for quantitative detection of endotoxin according to claim 1, characterized in that: The specific volume ratio is: the ratio of the standard horseshoe crab reagent solution to the endotoxin standard solution is 1:1-50:1; or the ratio of the standard horseshoe crab reagent solution to the sample solution to be tested is 1:1-50:

1.

4. The method for quantitative detection of endotoxin according to claim 1, characterized in that: The specific wavelength is 2-20 nm.

5. The method for quantitative detection of endotoxin according to any one of claims 1 to 4, characterized in that: The wavelength range of the signal light is 450-2400nm, and the output power is 5-20dBm.

6. The method for quantitative detection of endotoxin according to any one of claims 1 to 4, characterized in that: The spectrum data output by the optical fiber biochemical sensor is obtained by connecting a spectrum analyzer to the output end of the optical fiber biochemical sensor. The resolution of the spectrum analyzer is 10-20pm, the test wavelength range is 600-1700nm, the wavelength resolution is 0.03-1nm, the dynamic range is 40-60dB, and the scanning speed is 20-40nm / second.

7. An endotoxin quantitative detection system, characterized in that: Applicable to the detection method according to any one of claims 1 to 6, the detection system comprises a light source emitting device, an incident light lead, a microfluidic channel, an optical fiber biochemical sensor, an output light lead and a spectrum analyzer; The optical fiber biochemical sensor is embedded in the microfluidic channel and a gap is left between the optical fiber biochemical sensor and the inner wall of the microfluidic channel. The optical fiber biochemical sensor is provided with a sample holding area. The microfluidic channel is provided with at least two sample inlets and at least one sample outlet. The microfluidic channel area between any sample inlet and any sample outlet covers the sample holding area. One end of the incident light lead is connected to the light source generating device, the other end of the incident light lead passes through the microfluidic channel and is connected to the optical fiber biochemical sensor, one end of the output light lead is connected to the optical fiber biochemical sensor, and the other end of the output light lead passes through the microfluidic channel and is connected to the spectral analysis instrument.

8. The endotoxin quantitative detection system according to claim 7, characterized in that: The optical fiber biochemical sensor includes a first single-mode optical fiber, a hollow optical fiber, and a second single-mode optical fiber connected in sequence. The hollow optical fiber is provided with a sample slot used as the sample holding area. During detection, a light source generating device inputs signal light from one end of the first single-mode optical fiber. The signal light passes through the first single-mode optical fiber, the hollow optical fiber filled with a sample liquid to be tested, and the second single-mode optical fiber in sequence, and then is output from one end of the second single-mode optical fiber to a spectrometer.

9. The endotoxin quantitative detection system according to claim 8, characterized in that: The length of the hollow core optical fiber is 0.5-2.5 mm.

10. The endotoxin quantitative detection system according to claim 7, characterized in that: Also included is a microinjection pump connected to the injection port.

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