Endotoxin quantitative detection method and detection system

Abstract

The present application relates to the technical field of endotoxin detection, and more particularly to a method and system for quantitatively detecting endotoxin. The present application aims to improve the sensitivity, convenience and reduce the sample amount of endotoxin detection. The first object of the present application is to provide a method for quantitatively detecting endotoxin, which comprises the following steps: injecting standard limulus reagent solution and endotoxin standard solution with different concentrations into a microfluidic channel embedded with an optical fiber biochemical sensor according to a specific volume ratio; inputting signal light into the optical fiber biochemical sensor; obtaining the spectral data output by the optical fiber biochemical sensor when containing different standard mixed solutions; drawing a logarithmic endotoxin concentration-time fitting curve; then, replacing the endotoxin standard solution with a sample to be tested and repeating the above steps; extracting the corresponding time parameters; comparing the time parameters with the fitting curve; and obtaining the endotoxin content in the sample to be tested. The second object of the present application is to provide a system for quantitatively detecting endotoxin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of endotoxin detection, and more particularly to an endotoxin quantitative detection method and system. BACKGROUND

[0002] Endotoxin, also known as lipopolysaccharide (LPS), is the main component of the outer membrane of gram-negative bacteria and some blue-green algae. Bacterial cells can release a large amount of lipopolysaccharide during cell death, or a small amount of lipopolysaccharide during normal metabolism, thereby being toxic to the human body. LPS is an important pollutant in the fields of pharmaceutical, food industry, health care and environmental monitoring. Rabbit pyrogen test (RPT) is considered to be the oldest and simplest endotoxin detection technology, however, due to the need for live detection and a large number of samples, as well as lower sensitivity and accuracy compared with other methods, RPT detection method is criticized.

[0003] LAL detection technology (reagent taken from the blood of Limulus, which will coagulate into a block after contacting with endotoxin) emerged as the times require, which is more accurate than RPT and no longer involves live detection objects. The Chinese Pharmacopoeia, the United States Pharmacopoeia and the United States Food and Drug Administration approve this test method as the endotoxin test strategy for oral / injection drugs, hospital USP grade water and medical devices, and as the official endotoxin test method, which is widely recognized. Although LAL detection has important significance in application, it often encounters certain chemical or physical interference, which is usually solved by continuous dilution of the detection sample until the interference no longer affects the detection level, which not only prolongs the test time and increases the labor cost and sample consumption. SUMMARY

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

[0005] The first aspect of the present application is to provide an endotoxin quantitative detection method, comprising the following steps:

[0006] S1. Preparing at least four endotoxin standard solutions with different concentrations;

[0007] S2. Injecting the standard Limulus reagent solution and the endotoxin standard solution with different concentrations into the microfluidic channel embedded with the optical fiber biochemical sensor in a specific volume ratio, wherein the optical fiber biochemical sensor is provided with a sample groove, and the standard mixed solution obtained by mixing the standard Limulus reagent solution and the endotoxin standard solution covers the sample groove;

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

[0009] S4. Inject the standard limulus reagent solution in step S2 and the sample liquid to be measured into the microfluidic channel embedded with the optical fiber biochemical sensor according to the specific volume ratio in step 2, mix the standard limulus reagent solution and the sample liquid to be measured to obtain a sample mixed solution, cover the sample groove with the sample mixed solution, obtain the spectral data output by the optical fiber biochemical sensor when containing the sample mixed solution, extract the time required for the spectral valley value to drift to a specific wavelength as described in step S3, and compare with the fitting curve graph, so as to obtain the endotoxin content in the sample liquid to be measured.

[0010] The standard limulus reagent solution is mixed with the standard solution containing endotoxin or the sample liquid, and then gradually solidifies, thereby causing the refractive index of the mixture to change. When the mixture fills the sample groove of the optical fiber biochemical sensor, the signal light emitted by the light source will change in spectrum after being output by the optical fiber biochemical sensor. The research of the present application shows that the time required for the spectral valley value in the spectral data to drift to a specific wavelength is related to the logarithm of the endotoxin concentration. Therefore, a fitting curve can be obtained based on different concentrations of endotoxin standard solutions, and the endotoxin content of the sample liquid to be measured or the sample to be measured can be conveniently obtained by detecting the time required for the sample liquid to be measured to drift to a specific wavelength. The present application embeds the optical fiber biochemical sensor in the microfluidic channel, accurately controls the sample amount through the microfluidic channel, ensures the accuracy of the quantitative determination result, significantly reduces the sample amount, and the optical fiber biochemical sensor has high sensitivity, so that the method of the present application has the advantages of convenience, sensitivity and low sample amount.

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

[0012] Further, the specific volume ratio is that the ratio of the standard limulus reagent solution to the endotoxin standard solution is 1:1-50:1, or the ratio of the standard limulus reagent solution to the sample liquid to be measured is 1:1-50:1.

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

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

[0015] The wavelength range of the application can overcome spectrum fluctuation, avoid background signal interference, and expand the detection range of endotoxin, especially when the wavelength is 5nm, the effect is best.

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

[0017] Further, the output end of the optical fiber biochemical sensor is connected with a spectrum analyzer to obtain the spectrum data output by 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 / s.

[0018] The second aspect of the application provides an endotoxin quantitative detection system suitable for the endotoxin detection method, and the detection system comprises a light source emitting device, an incident light lead, a microfluidic channel, an optical fiber biochemical sensor, an outgoing light lead and a spectrum analyzer.

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

[0020] One end of the incident light lead is connected with the light source generating device, the other end of the incident light lead penetrates into the microfluidic channel and is connected with the optical fiber biochemical sensor, one end of the outgoing light lead is connected with the optical fiber biochemical sensor, and the other end of the outgoing light lead penetrates out of the microfluidic channel and is connected with the spectrum analyzer.

[0021] Further, the incident light lead and the microfluidic channel are fixedly connected, and the outgoing light lead and the microfluidic channel are fixedly connected.

[0022] The scheme can make the optical fiber biochemical sensor stable in the area between the sample inlet and the sample outlet, and ensure that the optical fiber biochemical sensor can reliably receive the mixed solution after the sample liquid and the standard limulus reagent solution are mixed.

[0023] Further, the optical fiber biochemical sensor comprises 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 groove serving as the sample containing area, the light source generating device inputs signal light from one end of the first single-mode optical fiber during detection, the signal light sequentially passes through the first single-mode optical fiber, the hollow optical fiber filled with the sample liquid to be detected, and the second single-mode optical fiber, and then is output from one end of the second single-mode optical fiber to the spectrum analyzer.

[0024] The present scheme is filled with the mixed solution of sample liquid and limulus reagent through the sample tank, so that when the signal light is input into the first single-mode optical fiber and output to the hollow optical fiber through the end face, the mixed solution and the cladding of the hollow optical fiber constitute the anti-resonance reflection optical waveguide structure. The structure has periodic transmission mode and leakage mode, and the mixed solution filled in the hollow optical fiber can limit part of the wavelength of light, so as to realize more sufficient interaction with the mixed solution. Then the output signal light 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 is output from the end thereof, thereby constituting the optical fiber refractive index fiber sensor device which directly interacts with the mixed solution. Since the sample liquid allows as a low-loss transmission waveguide, and slight refractive index change will cause spectral shift, higher sensitivity can be realized.

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

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

[0027] Further, the sample inlet includes a first sample inlet and a second sample inlet, and the first sample inlet and the second sample inlet are oppositely arranged on both sides of the microfluidic channel in the flow direction.

[0028] The present scheme makes the sample liquid and the labeled limulus reagent input by the first sample inlet and the second sample inlet have opposite incident directions, which helps to achieve rapid mixing effect.

[0029] Further, the micro-amount injection pump in communication with the sample inlet is further included.

[0030] Further, the micro-amount injection pump in communication with the sample inlet is further included.

[0031] The present scheme is connected with the micro-amount injection pump through the drainage pipeline, which improves the connection strength and helps to maintain the sample input accuracy.

[0032] Further, the drainage pipeline is inclined relative to the microfluidic channel, and the inclination direction is from the sample inlet to the direction away from the optical fiber biochemical sensor.

[0033] The present scheme makes the sample input into the microfluidic channel have an initial velocity towards the optical fiber biochemical sensor, which can ensure that the sample liquid and the standard limulus reagent solution are mixed and guided to the optical fiber biochemical sensor.

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

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

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

[0037] Compared with existing technologies, the beneficial effects of this invention are as follows: by using a microfluidic channel in conjunction with a micro-injection pump, the test sample and standard horseshoe crab reagent solution are injected into the system in miniaturized and high-precision quantities. The reaction solution is contained in the hollow optical fiber of the fiber optic biochemical sensor, and the spectral data is recorded and analyzed in real time using a spectrometer. The endotoxin concentration is quantified by using the spectral drift rate. This provides 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. Attached Figure Description

[0038] Figure 1 This is a structural diagram of an endotoxin detection system.

[0039] Figure 2 This is a structural diagram of a fiber optic biochemical sensor.

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

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

[0042] Figure 5 The graph shows the change of the transmission spectrum of the fiber optic biochemical sensor input spectrum over time caused by different test samples measured in Example 1.

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

[0044] Reference 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 tube 340, base 350, output tube 360, fiber optic biochemical sensor 400, first single-mode fiber 410, first polishing area 411, hollow fiber 420, sample tank 421, second single-mode fiber 430, second polishing area 431, fiber core 440, outgoing light lead 500, spectrometer 600, micro-injection pump 700. Detailed Implementation

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0046] The experimental methods used in the following embodiments 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 by a person of ordinary skill in the art through commercial channels.

[0047] Embodiment 1

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

[0049] S1. Prepare an endotoxin standard solution, and the content of endotoxin in the endotoxin standard solution is 10 -4 -10 -1 EU / mL, and the four concentrations selected are 10 -4 EU / mL, 10 -3 EU / mL, 10 -2 EU / mL, and 10 -1 EU / mL, respectively.

[0050] S2. Each time, 5 uL of the standard limulus reagent solution and 5 uL of the endotoxin standard solution with four different concentrations in S1 are injected into the microfluidic channel in which an optical fiber biochemical sensor is embedded, wherein the optical fiber biochemical sensor is provided with a sample containing area. After the endotoxin standard solution and the standard limulus reagent solution are injected each time, the standard mixed solution obtained by mixing the standard limulus reagent solution and the endotoxin standard solution through the rapid mixing action of the microfluidic channel can completely cover the sample containing area. In order to improve the accuracy and reliability of the test, two groups of negative control groups are additionally prepared, wherein the first negative control group uses 5 uL of the standard limulus reagent solution + 5 uL of limulus reagent test water injected into the microfluidic channel in which the optical fiber biochemical sensor is embedded, and the second negative control group uses 5 uL of limulus reagent test water + 5 uL of limulus reagent test water injected into the microfluidic channel in which the optical fiber biochemical sensor is embedded.

[0051] The optional detection limit of the limulus reagent solution is 0.005-0.1 EU / mL, and the detection limit of the limulus reagent solution used in this embodiment is 0.06 EU / mL. The greater the concentration of the limulus reagent solution used, the faster the detection speed, but it also leads to an increase in the use of limulus reagent and an increase in detection cost. The preparation method of the limulus reagent is to dissolve the standard limulus reagent solid product in the corresponding 100 μL standard limulus reagent test water to prepare a solution with a corresponding detection limit. The limulus reagent test water is a commercially available limulus reagent accessory, which is water after removing pyrogens.

[0052] In specific implementation, in order to improve the detection sensitivity, the optical fiber biochemical sensor is prepared according to embodiments 1-5 of the reference patent document CN114965359A (a refractive index optical fiber sensor and a manufacturing method thereof), that is, it is equivalent to a refractive index optical fiber sensor. Specifically, reference is made to Figures 2-3 The optical fiber biochemical sensor includes a first single-mode optical fiber 410, a hollow optical fiber 420, and a second single-mode optical fiber 430 connected in sequence, and the hollow optical fiber 420 is provided with a sample groove 421 as a sample containing area for containing a sample to be measured. The first single-mode optical fiber 410 and the second single-mode optical fiber 430 are both processed by side edge polishing to form a first polishing area 411 and a second polishing area 431, respectively. Thus, the hollow optical fiber 420 is fused between the first single-mode optical fiber 410 and the second single-mode optical fiber 430, and the first polishing area 411, the hollow optical fiber 420, and the second polishing area 431 jointly form a D-shaped groove. The D-shaped groove facilitates the convergence of the mixed solution in the microfluidic channel, enables the mixed solution to be quickly and fully filled in the sample groove 421, and also ensures the transmission power of the first single-mode optical fiber 410 and the second single-mode optical fiber 430, thereby improving the efficiency of exciting multiple modes in the mixed solution waveguide and improving the detection speed and accuracy. Figure 3 The inner diameter of the sample groove 421 is greater 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 and affecting the detection result.

[0053] It should be noted that the sensitivity of the detection system is independent of the volume of the filling solution, so as long as the mixed solution completely fills the sample groove, the detection purpose can be achieved, and the volume of other external solutions does not affect the sensitivity of the detection system. In specific implementation, the length of the hollow optical fiber 420 is 0.5-2.5mm, for example, 1.6mm, the remaining thickness of the hollow optical fiber 420 is between 62.5-75um, the inner diameter (equivalent to the inner diameter of the sample groove 421) is 10-100um, and preferably 30um, so as to ensure that the sensing principle of the optical fiber biochemical sensor is based on the anti-resonance effect, and also 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 as 8-9um, for example, 8.2um, and the cladding outer diameter is 125-150um, preferably 125um. As for the length size of the first single-mode optical fiber 410 and the second single-mode optical fiber 430, it can be adaptively set according to the length of the microfluidic channel actually used, so as to ensure that the microfluidic channel is sufficient to stably assemble the optical fiber biochemical sensor. The light source is a super-continuous 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, so as to provide the endotoxin detection system with high reliability, wide spectral range and high output power, which can meet the requirements of different specifications of optical fiber biochemical sensors for light sources.

[0054] S3. After injecting the endotoxin standard solution and the standard limulus 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, and the spectral data output by the optical fiber biochemical sensor when containing different concentrations of standard mixed solutions are acquired, as shown in Figure 5 which shows the corresponding curve of the wave trough shift change with time acquired by the spectral analyzer when the optical fiber biochemical sensor contains different concentrations of endotoxin standard solutions or negative control group reagents, in combination with Figure 6 It can be known that as the content of endotoxin increases, the slope of the curve becomes larger, that is, there is a positive correlation between the wave trough shift amount per unit time and the concentration of endotoxin. Further, as shown in Figure 6 the right, by extracting the time required for the spectral trough value in the spectral data to drift by 5nm, an endotoxin concentration logarithm-time fitting curve graph is drawn. Referring further to Figure 6 the right, in the present application, as the concentration of endotoxin in the test sample gradually increases, the response time y required for the wave trough to drift by 5nm is linearly correlated with the logarithm lgC of the test endotoxin concentration. The linear relationship of the optical fiber biochemical sensor to the endotoxin concentration of 10 -4 -10 -1 The linear relationship of the optical fiber biochemical sensor to the endotoxin concentration of 10

[0055] The principle of changing the trough position in the experiment is that when the standard limulus reagent reacts with the endotoxin in the test sample, the mixed solution will gradually appear a coagulation effect with time, specifically, the solution state changes from liquid to gel state, and the equivalent refractive index will increase. The optical fiber biochemical sensor used in the application is a device for sensing refractive index change dominated by the anti-resonance principle. With the gradual increase of the equivalent refractive index of the mixed solution, the trough value of the spectrum will gradually decrease. According to this principle, the relationship between the trough value and the endotoxin content is analyzed, so as to realize quantitative monitoring of the unknown endotoxin content of the test sample.

[0056] In specific operation, in order to simplify the environmental requirements of the reaction between the endotoxin standard solution and the standard limulus reagent, the experimental environment temperature is kept at room temperature of 25℃, and the test process is that the transmittance spectrum of the optical fiber biochemical sensor is recorded once every 3 seconds by the spectrum analyzer connected with the output end of the optical fiber biochemical sensor, and the change of the resonance wavelength in the selected range is continuously monitored. In order to ensure the reusability of the device, the longest recording time of the spectrum is not more than 6 min, and the recording time can be appropriately reduced when the spectrum changes rapidly. The specific spectrum analyzer 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 / s. A device with fast, high resolution, high dynamic range is provided to obtain the detection spectrum.

[0057] S4. The standard limulus reagent solution in step S2 and the test sample liquid are injected into the microfluidic channel embedded with the optical fiber biochemical sensor according to the specific volume ratio (1:1) in step 2, the mixed sample solution obtained by uniformly mixing the standard limulus reagent solution and the test sample liquid covers the sample containing area, the spectrum data output by the optical fiber biochemical sensor when containing the sample mixed solution is obtained, the time required for extracting the spectrum trough value drift at the specific wavelength (5nm) is compared with the fitting curve obtained in step S3, specifically, the concentration of endotoxin in the test sample is obtained by the concentration formula C=10 (y+3.8) / -84.9 to realize quantitative detection of endotoxin.

[0058] In addition, after each test is completed, the optical fiber biochemical sensor is taken out, washed with alcohol for three times to remove impurities on the surface of the optical fiber, then dried by using a heating plate, and then soaked in the prepared aiptasia solution for 30 min for cleaning residual endotoxin and organic impurities; after cleaning, the waste liquid is sucked by using a glass suction tube, and then a large amount of deionized water is repeatedly washed for three times, and finally placed in a 100℃ drying box for drying for 1h, so that the optical fiber biochemical sensor can be reused.

[0059] Example 2

[0060] As Figures 1-4As shown, the embodiment provides an endotoxin quantitative detection system, which is suitable for the endotoxin quantitative method in embodiment 1, and includes a light source emitting device 100, an incident light lead 200, a microfluidic channel 300, a fiber-optic biochemical sensor 400, an emergent light lead 500 and a spectrum analyzer 600.

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

[0062] One end of the incident light lead 200 is connected with the light source emitting device, the other end of the incident light lead 200 penetrates into the microfluidic channel 300 and accesses the fiber-optic biochemical sensor 400, one end of the emergent light lead 500 is connected with the fiber-optic biochemical sensor 400, and the other end of the emergent light lead 500 penetrates out of the microfluidic channel 300 and accesses the spectrum analyzer 600. The fiber-optic biochemical sensor 400 is set in the manner as described in embodiment 1.

[0063] In specific implementation, the incident light lead 200 is introduced from the front end opening of the microfluidic channel 300, the emergent light lead 500 is introduced from the rear end opening of the microfluidic channel 300, the incident light lead 200 and the emergent light lead 500 are straightened in the microfluidic channel 300, the incident light lead 200 is encapsulated in the front end opening of the microfluidic channel 300, and the emergent light lead 500 is encapsulated in the rear end opening of the microfluidic channel 300, so that the incident light lead 200, the fiber-optic biochemical sensor 400 and the emergent light lead 500 are all fixed well, the fiber-optic biochemical sensor 400 is stabilized in the area between the sample inlets and the sample outlet 330, and the fiber-optic biochemical sensor 400 can reliably receive the mixed solution after the sample liquid and the standard limulus reagent solution are mixed. Further, since the fiber-optic biochemical sensor 400 needs to be cleaned after each sample detection, in a preferred embodiment, the incident light lead 200 and the emergent light lead 500 are fixed with the microfluidic channel 300 in a detachable manner, so as to facilitate the loading and unloading of the fiber-optic biochemical sensor 400. In addition, in order to improve the signal light transmission stability, the incident light lead 200 and the emergent light lead 500 can be realized in the form of fiber-optic jumpers.

[0064] In detection, the sample liquid to be detected and the standard limulus reagent solution are respectively injected into the microfluidic channel 300 through two sample inlets, and then the two solutions are rapidly and uniformly mixed through the microfluidic channel 300. Since the sample groove of the optical fiber biochemical sensor 400 is arranged between the sample inlets and the sample outlet 330, the mixed solution can fully cover the sample groove before flowing out of the sample outlet 330. The mixed solution gradually solidifies to cause a change in the refractive index, thereby causing a change in the spectrum of the signal light output from the optical fiber biochemical sensor 400. Since the spectrum change rule is related to the content of endotoxin, according to the detection method of the reference example 1, the spectrum data of the emitted signal light can be analyzed by the spectrum analyzer 600, and thus the content of endotoxin in the sample liquid can be obtained.

[0065] Reference Figure 4 In some embodiments, the microfluidic channel 300 is a cuboid, 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 of 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, so that the solution is more easily injected into the microfluidic channel 300. In specific implementation, reference Figure 1 、 4 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] Reference Figure 1 、 4 The first sample inlet 310 and the second sample inlet 320 are oppositely arranged on both sides of the flow direction of the microfluidic channel 300, so that the sample liquid and the labeled limulus reagent respectively input through the first sample inlet 310 and the second sample inlet 320 have opposite incident directions, which helps to achieve rapid mixing.

[0067] As shown in Figure 1 , to realize precise sampling, a micro-injection pump 700 is further arranged in communication with the sample inlet. In specific implementation, the number of micro-injection pumps 700 can be a double-channel mode, so as to extend two connecting pipes connected with the first sample inlet 310 and the second sample inlet 320, so as to facilitate precise control of the sample amount of the two sample inlets to keep consistent. In some embodiments, the specification of the micro-injection pump 700 is 0.5-60 mL, the injection rate is 0.008 nL / h-1246 L / min, the injection accuracy is ±0.5%, and the reproducibility is ±0.2%.

[0068] In addition, referring to Figure 1 , in order to improve the connection strength of the micro-injection pump 700 and the sample inlet, the outer surface of the micro-fluidic channel 300 is further provided with a drainage pipeline 340 in communication with the sample inlet. In some embodiments, the drainage pipeline 340 is integrally formed on the surface of the micro-fluidic channel 300 and is made of the same material as the micro-fluidic channel 300, so that the drainage pipeline 340 and the micro-fluidic channel 300 are connected with high strength, and the solution input by the micro-injection pump 700 can be stably drained. Similarly, an output pipeline 360 can also be connected to the sample outlet 330 to facilitate the discharge of the sample liquid in the micro-fluidic pipeline.

[0069] Further, as shown in Figure 1 , the drainage pipeline 340 is arranged obliquely relative to the micro-fluidic channel 300, and the oblique direction is from the sample inlet to the direction away from the optical fiber biochemical sensor 400. In this way, the sample liquid and the standard limulus reagent solution have an initial velocity towards the optical fiber biochemical sensor 400 when they are input into the micro-fluidic channel 300, which can ensure that the sample liquid and the standard limulus reagent solution are guided to the optical fiber biochemical sensor 400 after being mixed uniformly.

[0070] Obviously, the above embodiments of the present application are only examples for clearly illustrating the technical solutions of the present application, and are not intended to limit the specific embodiments of the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the claims of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method for quantitative determination of endotoxin, characterized by, The method comprises the following steps: S1. Preparing at least four endotoxin standard solutions with different concentrations; S2. Injecting the standard limulus reagent solution and the endotoxin standard solution with different concentrations 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 containing area, the microfluidic channel is provided with at least two sample inlets and at least one sample outlet, the area of the microfluidic channel between any sample inlet and any sample outlet covers the sample containing area, and the standard mixed solution obtained by mixing the standard limulus reagent solution and the endotoxin standard solution covers the sample containing area; S3. Inputting signal light into the optical fiber biochemical sensor, obtaining the spectral data output by the optical fiber biochemical sensor when containing different concentrations of standard mixed solutions, extracting the time required for the spectral valley value to drift to a specific wavelength, and drawing a logarithmic endotoxin concentration-time fitting curve, wherein the optical fiber biochemical sensor comprises 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 groove serving as the sample containing area, and the signal light is input from one end of the first single-mode optical fiber by a light source emitting device, 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 is then output to a spectrum analyzer from one end of the second single-mode optical fiber, wherein when the signal light is input into the first single-mode optical fiber and output to the hollow optical fiber through the end face thereof, the mixed solution and the cladding layer of the hollow optical fiber form an anti-resonance reflection optical waveguide structure; S4. Injecting the standard limulus reagent solution and the sample liquid to be tested into the microfluidic channel embedded with the optical fiber biochemical sensor according to the specific volume ratio in step S2, mixing the standard limulus reagent solution and the sample liquid to be tested to obtain a sample mixed solution covering the sample containing area, obtaining the spectral data output by the optical fiber biochemical sensor when containing the sample mixed solution, extracting the time required for the spectral valley value to drift to a specific wavelength as described in step S3, and comparing with the fitting curve, so as to obtain the endotoxin content in the sample liquid to be tested.

2. The method of claim 1, wherein the endotoxin is quantitatively detected. The endotoxin standard solution has an endotoxin content of 10 -4 -10 -1 EU / mL.

3. The method of claim 1, wherein the endotoxin is quantitatively detected. The specific volume ratio is that the ratio of the standard limulus reagent solution to the endotoxin standard solution is 1:1-50:1, or the ratio of the standard limulus reagent solution to the sample liquid to be tested is 1:1-50:

1.

4. The method of claim 1, wherein the endotoxin is quantitatively detected. The specific wavelength is 2-20 nm.

5. The method of claim 1 to 4, wherein The wavelength range of the signal light is 450-2400 nm, and the output power is 5-20 dBm.

6. The method of claim 1-4, wherein, The output end of the optical fiber biochemical sensor is connected with a spectrum analyzer to obtain the spectral data output by the optical fiber biochemical sensor, the resolution of the spectrum analyzer is 10-20 pm, the test wavelength range is 600-1700 nm, the wavelength resolution is 0.03-1 nm, the dynamic range is 40-60 dB, and the scanning speed is 20-40 nm / s.

7. An endotoxin quantitation system, characterized by comprising: The detection system is suitable for the detection method according to any one of claims 1-6, and comprises a light source emitting device, an incident light lead, a microfluidic channel, an optical fiber biochemical sensor, an emergent light lead and a spectrum analyzer. The optical fiber biochemical sensor is embedded in the micro-fluidic channel and has a gap between the inner wall of the micro-fluidic channel, the optical fiber biochemical sensor is provided with a sample containing area, the micro-fluidic channel is provided with at least two sample inlets and at least one sample outlet, and the micro-fluidic channel area between any sample inlet and any sample outlet covers the sample containing area; One end of the incident light lead is connected with the light source emitting device, the other end of the incident light lead penetrates into the micro-fluidic channel and connects with the optical fiber biochemical sensor, one end of the outgoing light lead is connected with the optical fiber biochemical sensor, and the other end of the outgoing light lead penetrates out of the micro-fluidic channel and connects with the spectrum analysis instrument.

8. The endotoxin quantitation system according to claim 7, wherein The optical fiber biochemical sensor comprises 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 groove serving as the sample containing area, the light source emitting device inputs signal light from one end of the first single-mode optical fiber during detection, the signal light sequentially passes through the first single-mode optical fiber, the hollow optical fiber filled with the sample liquid to be detected and the second single-mode optical fiber, and then is output from one end of the second single-mode optical fiber to the spectrum analysis instrument.

9. The endotoxin quantitation system according to claim 8, wherein The length of the hollow optical fiber is 0.5-2.5 mm.

10. The endotoxin quantitation system according to claim 7, wherein The micro-injection pump in communication with the sample inlet is further included.

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