Novel endotoxin sensing detection device and method based on tachypleus amebocyte lysate

Abstract

The invention discloses a novel endotoxin sensing detection device and method based on tachypleus amebocyte lysate, and relates to the field of endotoxin inspection.The device comprises a light source, a micro-nano fiber bragg grating sensor, a capillary tube, a reflected signal detection unit and a processing unit, and the light source inputs light signals into the micro-nano fiber bragg grating sensor; after the capillary tube extracts a to-be-detected sample solution, the to-be-detected sample solution is in contact with the micro-nano fiber bragg grating sensor; the turbidity of the to-be-detected sample solution is changed due to the reaction of endotoxin in the to-be-detected sample solution and the tachypleus amebocyte lysate, so that the resonant wavelength of the micro-nano fiber bragg grating sensor is changed, and the reflected signal detection unit receives a reflected light signal of the micro-nano fiber bragg grating sensor; the processing unit is used for detecting the concentration of endotoxin in the sample solution to be detected according to the resonant wavelength in the reflected light signal. According to the application, high-sensitivity quantitative detection of endotoxin can be realized under the condition of a small amount of tachypleus amebocyte lysate.

Description

Technical Field

[0001] The present application relates to the field of endotoxin detection, and in particular to a novel endotoxin sensing detection device and method based on horseshoe crab reagent. Background Art

[0002] Horseshoe crab is a marine animal with important scientific research significance and economic value. The horseshoe crab reagent method is also called the bacterial endotoxin test method. It is an in vitro analytical method based on the reaction between endotoxins produced by Gram-negative bacteria and horseshoe crab reagents, and the resulting gelation reaction to qualitatively or quantitatively detect bacterial endotoxins.

[0003] However, driven by economic interests, horseshoe crab resources have been subject to disorderly and excessive private fishing. In the past 30 years, the natural resources of Chinese horseshoe crab have declined rapidly. Therefore, developing a quantitative detection device that only requires a small amount of horseshoe crab reagent to achieve endotoxin concentration is of great significance to the protection of Chinese horseshoe crab resources and the healthy development of the horseshoe crab reagent industry. Summary of the invention

[0004] The purpose of the present application is to provide a novel endotoxin sensing detection device and method based on horseshoe crab reagent, which can ensure high-sensitivity quantitative detection of sample endotoxins while requiring one to two orders of magnitude less horseshoe crab reagent dosage.

[0005] To achieve the above objectives, this application provides the following solutions:

[0006] In a first aspect, the present application provides a novel endotoxin sensing detection device based on horseshoe crab reagent, comprising: a light source, a micro-nano fiber Bragg grating sensor, a capillary, a reflection signal detection unit and a processing unit;

[0007] The light source is connected to the micro-nano fiber Bragg grating sensor and is used to input an optical signal to the micro-nano fiber Bragg grating sensor;

[0008] The micro-nano fiber Bragg grating sensor is nested in the capillary tube, and the capillary tube is used to absorb the sample solution to be tested and the horseshoe crab reagent. The horseshoe crab reagent reacts with the endotoxin in the sample solution to be tested in the capillary tube, causing the turbidity of the sample solution to be tested to change, thereby changing the resonant wavelength of the micro-nano fiber Bragg grating sensor;

[0009] The reflection signal detection unit is connected to the micro-nano fiber Bragg grating sensor and is used to receive the reflection light signal of the micro-nano fiber Bragg grating sensor;

[0010] The processing unit is connected to the reflected signal detection unit and is used to detect the concentration of endotoxin in the sample solution to be tested according to the resonant wavelength in the reflected light signal.

[0011] In a second aspect, the present application provides a novel endotoxin sensing detection method based on horseshoe crab reagent, the method is applied to the novel endotoxin sensing detection device based on horseshoe crab reagent, the method comprising:

[0012] Obtaining the reflected light signal of the micro-nano fiber Bragg grating sensor;

[0013] Determining a resonance peak wavelength according to a resonance characteristic wavelength in the reflected light signal;

[0014] Determine the fluid refractive index of the sample solution to be tested by using a wavelength-refractive-index relationship curve and the resonance peak wavelength;

[0015] The endotoxin concentration of the sample solution to be tested is determined by the endotoxin concentration relationship curve, the resonance peak wavelength and the fluid refractive index.

[0016] According to the specific embodiments provided in this application, this application has the following technical effects:

[0017] The present application provides a novel endotoxin sensing detection device and method based on horseshoe crab reagent. Through the nested connection between the micro-nano fiber Bragg grating sensor and the capillary, only a small amount of horseshoe crab reagent and the sample solution to be tested need to enter the capillary through the capillary effect and contact the micro-nano fiber Bragg grating sensor, causing the resonant wavelength of the micro-nano fiber Bragg grating sensor to change, thereby completing the endotoxin detection process of the sample solution to be tested. The present application reduces the sample amount of horseshoe crab reagent and the sample solution to be tested, which is of great significance for the protection of Chinese horseshoe crab resources and the healthy development of the horseshoe crab reagent industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 A schematic diagram of the structure of a novel endotoxin sensing detection device based on horseshoe crab reagent provided in one embodiment of the present application;

[0020] Figure 2 A schematic diagram of a novel endotoxin sensing detection method based on horseshoe crab reagent provided in one embodiment of the present application;

[0021] Figure 3 is the refractive index response curve of the micro-nano fiber Bragg grating sensor;

[0022] Figure 4Schematic diagram of the resonance spectrum of the micro-nano fiber Bragg grating sensor when detecting sample solutions with different endotoxin concentrations;

[0023] Figure 5 This is the endotoxin concentration response curve of the micro-nano fiber Bragg grating sensor. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0025] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0026] In an exemplary embodiment, Figure 1 As shown, a new endotoxin sensing detection device based on horseshoe crab reagent is provided, including a light source (not shown in the figure), a micro-nano fiber Bragg grating sensor 1, a capillary 4, a reflection signal detection unit (not shown in the figure) and a processing unit (not shown in the figure).

[0027] The light source is connected to the micro-nano fiber Bragg grating sensor 1 and is used to input an optical signal 8 to the micro-nano fiber Bragg grating sensor 1 .

[0028] The micro-nano fiber Bragg grating sensor 1 is nested in the capillary 4, and the capillary 4 is used to absorb the sample solution 5 and the horseshoe crab reagent 6. The horseshoe crab reagent 6 reacts with the endotoxin 7 in the sample solution 5 to be tested in the capillary 4, causing the turbidity of the sample solution 5 to be tested to change, thereby changing the resonant wavelength of the micro-nano fiber Bragg grating sensor 1.

[0029] The reflection signal detection unit is connected to the micro-nano fiber Bragg grating sensor 1 and is used to receive the reflection light signal 9 of the micro-nano fiber Bragg grating sensor 1 .

[0030] The processing unit is connected to the reflection signal detection unit, and is used to detect the concentration of endotoxin in the sample solution 5 to be tested according to the resonance wavelength in the reflection light signal 9 .

[0031] In a specific embodiment, the micro-nano fiber Bragg grating sensor 1 includes a micro-nano fiber 2 and a fiber Bragg grating 3; the fiber Bragg grating 3 is written on the micro-nano fiber 2. The micro-nano fiber 2 is made by a heat source heating taper brittle fracture method or a single-end corrosion method.

[0032] In a specific embodiment, the micro-nano optical fiber 2 includes a uniform diameter region and a diameter variation region; the uniform diameter region is cylindrical, and the diameter variation region is conical; the fiber Bragg grating 3 is written on the uniform diameter region using a phase mask method. The diameter range of the uniform diameter region is 2μm-4μm, of which 3.5μm is the best, and the length range of the diameter variation region is 0.5cm-1cm.

[0033] It should be noted that the micro-nano optical fiber 2 may also be in a shape with uniform diameter.

[0034] In a specific embodiment, the interior of the micro-nano optical fiber 2 is doped with rare earth elements, and the exterior of the micro-nano optical fiber 2 is modified with gold nanostars or precious metals.

[0035] The reaction between horseshoe crab reagent 6 and endotoxin 7 requires the temperature to be controlled between 37-38 degrees Celsius. The optical fiber photothermal effect can be used to achieve temperature control of the sample solution 5 to be tested, so that the horseshoe crab reagent 6 and endotoxin 7 are at the optimal reaction temperature. Specifically, the optical fiber photothermal effect can be achieved by the doping inside the micro-nano optical fiber 2 and the photothermal conversion material arranged outside the micro-nano optical fiber 2 to absorb the injected laser. Specifically, the doping of rare earth elements inside the micro-nano optical fiber 2 and the arrangement of gold nanostars outside the micro-nano optical fiber 2 to receive the heating light generate the optical fiber photothermal effect to heat the sample solution 5 to be tested in the capillary 4, thereby achieving the in-situ heating reaction of the horseshoe crab reagent 6 and endotoxin 7.

[0036] By modifying the surface of the micro-nano optical fiber 2 with gold nanostars or noble metals, the Bragg reflection signal is enhanced during the detection of endotoxin 7 concentration, thereby improving the sensitivity of endotoxin concentration detection.

[0037] In a specific embodiment, the micro-nano optical fiber 2 is provided with a micro-nano fiber Bragg grating temperature sensor. By providing the micro-nano fiber Bragg grating temperature sensor in the micro-nano optical fiber 2 area, the real-time temperature of the heating reaction of the horseshoe crab reagent 6 and the endotoxin 7 can be monitored.

[0038] In a specific embodiment, the diameter of the micro-nano optical fiber 2 is smaller than the inner diameter of the capillary 4, or the inner wall of the capillary 4 is connected to the outer wall of the uniform diameter area of ​​the micro-nano optical fiber 2, and the empty space between the capillary 4 and the micro-nano optical fiber 2 is the detection area. The sample solution 5 to be tested that enters the capillary 4 is less than 10 μl. The sample solution 5 to be tested can be a biochemical solution, serum or blood.

[0039] The working principle of the novel endotoxin sensing detection device based on horseshoe crab reagent provided in the present application is:

[0040] An optical signal 8 is input into the fiber Bragg grating 3 through a light source. After the capillary 4 extracts the sample solution 5 to be tested, the sample solution 5 to be tested is brought into contact with the micro-nano fiber Bragg grating sensor 1. The reaction between the endotoxin 7 in the sample solution 5 and the horseshoe crab reagent 6 causes the turbidity of the sample solution 5 to be tested to change, thereby changing the resonant wavelength of the fiber Bragg grating 3. The fiber Bragg grating 3 reflects the optical signal 8, and the reflected optical signal 9 is analyzed to achieve specific detection.

[0041] The micro-nano fiber Bragg grating sensor 1 provided in the present application utilizes the refractive index of the fluid as a transducer parameter through the refractive index response characteristic, and can be combined with functional means to achieve specific detection of biochemical samples, and can be applied to sensing fields such as biology and chemistry that require micro sampling. The fiber Bragg grating sensor 1 can achieve rapid taper pulling and batch production, and its diameter is thinner than the uniform micro-nano fiber Bragg grating, and the structure is more compact. In addition, the fiber Bragg grating 3 of the micro-nano fiber Bragg grating sensor 1 is in a uniform diameter region, has good sensitivity, and can achieve the determination of sample volume through the spatial diameter chirp effect.

[0042] The novel endotoxin sensing detection device based on horseshoe crab reagent provided in the present application adopts a combination design of a micro-nano fiber Bragg grating sensor 1 and a capillary 4, which greatly reduces the dosage of the required liquid and can realize the detection of trace samples. Since the center of gravity of the micro-nano fiber Bragg grating sensor 1 is at the end with a thicker diameter, its mechanical properties are better and it is not easy to be damaged; the capillary 4 itself also acts as a protective cover, which can reduce the possibility of damage to the sensing structure and facilitate storage and transportation.

[0043] In an exemplary embodiment, Figure 2 As shown, a novel endotoxin sensing detection method based on horseshoe crab reagent is provided, comprising:

[0044] S1: Obtain the reflected light signal of the micro-nano fiber Bragg grating sensor.

[0045] The horseshoe crab reagent 6 and the sample solution 5 to be tested enter the detection area through the capillary effect, causing the effective refractive index of the fiber Bragg grating 3 to change, the light source inputs the light signal 8, and the reflection signal detection unit receives the reflection light signal 9.

[0046] S2: Determine a resonance peak wavelength according to a resonance characteristic wavelength in the reflected light signal.

[0047] S3: Determine the fluid refractive index of the sample solution to be tested through the wavelength-refractive-index relationship curve and the resonance peak wavelength.

[0048] The wavelength-refractive-index relationship curve represents the corresponding relationship between the fluid refractive index of the sample solution 5 to be tested and the resonance peak wavelength in the reflected light signal 9, such as Figure 3 shown.

[0049] S4: Determine the endotoxin concentration of the sample solution to be tested through the endotoxin concentration relationship curve, the resonance peak wavelength and the fluid refractive index.

[0050] The endotoxin concentration relationship curve is characterized by the corresponding relationship between the endotoxin 7 concentration of the sample solution 5 to be tested and the resonance peak value in the reflected light signal 9, such as Figure 4 and Figure 5 shown. Figure 5 The formula y=0.028-0.023x+0.004x 2 -0.002x 3 , is the cubic polynomial fitting function of the independent variable x (endotoxin concentration) and the dependent variable y (resonance wavelength), R 2 The fitting effect of the response function.

[0051] The horseshoe crab reagent 6-endotoxin 7 reaction is a competitive mechanism, that is, when the content of endotoxin 7 in the sample solution 5 to be tested is small, the residual horseshoe crab reagent 6 increases, the fluid refractive index of the sample solution 5 to be tested increases, and the Bragg reflection signal (i.e., the reflected light signal 9) has a larger drift, while when the content of endotoxin 7 is high, the Bragg reflection signal drift is smaller. The endotoxin concentration of the sample solution 5 to be tested is detected by combining the endotoxin concentration relationship curve with the resonance peak wavelength and the fluid refractive index.

[0052] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. At the same time, for those skilled in the art, according to the ideas of this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting this application.

Claims

1. A novel endotoxin sensing detection device based on horseshoe crab reagent, characterized in that: include: Light source, micro-nano fiber Bragg grating sensor, capillary, reflection signal detection unit and processing unit; The light source is connected to the micro-nano fiber Bragg grating sensor and is used to input an optical signal to the micro-nano fiber Bragg grating sensor; The micro-nano fiber Bragg grating sensor is nested in the capillary tube, and the capillary tube is used to absorb the sample solution to be tested and the horseshoe crab reagent. The horseshoe crab reagent reacts with the endotoxin in the sample solution to be tested in the capillary tube, causing the turbidity of the sample solution to be tested to change, thereby changing the resonant wavelength of the micro-nano fiber Bragg grating sensor; The reflection signal detection unit is connected to the micro-nano fiber Bragg grating sensor and is used to receive the reflection light signal of the micro-nano fiber Bragg grating sensor; The processing unit is connected to the reflection signal detection unit, and is used to detect the concentration of endotoxin in the sample solution to be tested according to the resonance wavelength in the reflection light signal.

2. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 1 is characterized in that: The micro-nano fiber Bragg grating sensor comprises a micro-nano optical fiber and a fiber Bragg grating; the fiber Bragg grating is written on the micro-nano optical fiber.

3. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 2 is characterized in that: The micro-nano optical fiber comprises a uniform diameter region and a diameter variation region; the uniform diameter region is cylindrical, and the diameter variation region is conical; the fiber Bragg grating is written in the uniform diameter region.

4. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 2, characterized in that: The fiber Bragg grating is written on the micro-nano optical fiber by using a phase mask method.

5. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 2, characterized in that: The micro-nano optical fiber is manufactured by adopting a heat source heating taper brittle fracture method or a single-end corrosion method.

6. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 2, characterized in that: The micro-nano optical fiber is doped with rare earth elements.

7. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 2, characterized in that: The exterior of the micro-nano optical fiber is modified with gold nanostars or precious metals.

8. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 2, characterized in that: The micro-nano optical fiber is provided with a micro-nano optical fiber Bragg grating temperature sensor.

9. The novel endotoxin sensing detection device based on horseshoe crab reagent according to claim 1, characterized in that: The sample solution to be tested is a biochemical solution, serum or blood.

10. A novel endotoxin sensing detection method based on horseshoe crab reagent, characterized in that: The method is applied to the novel endotoxin sensing detection device based on horseshoe crab reagent according to any one of claims 1 to 9, and the method comprises: Obtaining the reflected light signal of the micro-nano fiber Bragg grating sensor; Determining a resonance peak wavelength according to a resonance characteristic wavelength in the reflected light signal; Determine the fluid refractive index of the sample solution to be tested by using a wavelength-refractive-index relationship curve and the resonance peak wavelength; The endotoxin concentration of the sample solution to be tested is determined by the endotoxin concentration relationship curve, the resonance peak wavelength and the fluid refractive index.

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