Optical fiber biosensor and preparation method and application thereof

Abstract

The invention discloses an optical fiber biosensor, which is prepared by coating the surface of an amino-functionalized waist-tapered optical fiber sensor with a graphene oxide layer and adsorbing an aptamer NE-AP or an aptamer LF-AP on the surface of the graphene oxide layer. The aptamer NE-AP can be specifically combined with the elastinase ELANE; the aptamer LF-AP can be specifically combined with the lactoferrin LF. After the optical fiber biosensor captures ELANE and LF proteins, the roughness and thickness of the surface of the optical fiber at the waist-cone-shaped part are changed, when light penetrates through the waist-cone-shaped part covered by biomolecules, the effective refractive index of the surface of the optical fiber biosensor is changed due to the fact that the diameter is gradually reduced and an evanescent field outside a core is enhanced, and the optical fiber biosensor can be used for detecting the optical fiber in the waist-cone-shaped part. Therefore, red shift occurs according to a transmitted spectrum induced after aptamer-protein combination is tracked, and high-sensitivity and low-detection-limit detection on the activity of the neutrophil is realized. The method has the characteristics of high sensitivity and low detection limit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection technology of biotechnology, and particularly to a fiber-optic biosensor for rapid in-vitro detection of neutrophil activity and a preparation method thereof. BACKGROUND

[0002] Infectious diseases caused by sepsis and chronic inflammation seriously endanger human health, and early diagnosis and early treatment play an important role in reducing mortality. At present, neutrophil activity is mainly determined by flow cytometry and plasma marker methods. However, the release amounts of flexible protease (ELANE) and lactoferrin (LF) released by neutrophils in patients with sepsis and inflammatory infection are different in different periods. These methods have the problems of complex operation, low throughput and insufficient sensitivity, and at the same time, the performance in complex biological samples is often affected by non-specific adsorption and matrix effect, which cannot distinguish neutrophil subpopulations in different activity states. In recent years, the application of fiber-optic sensing technology based on molecular recognition in neutrophil activity detection has become a research hotspot.

[0003] Fiber-optic sensing technology is a real-time and remote analysis microanalysis technology, which has the characteristics of small volume, low cost, anti-electromagnetic interference, anti-shock, large information transmission capacity, high sensitivity, rapid response, etc., and is widely used in biological sensing detection applications. The fiber core is prepared into a waist-shaped structure by a hydrogen flame heating method, the radial refractive index of the tapered optical fiber is changed, and the penetration depth and penetration energy of the evanescent field in the sensing area are enhanced; then the tapered optical fiber is combined with functional materials, and the interaction between the functional material film and the evanescent wave is used to improve the detection sensitivity, which has the characteristics of simple structure and small volume, and has high sensitivity, which can meet the requirements of online analysis, real-time analysis and in-vivo analysis of optical signals in different fields, and realizes the improvement of the detection sensitivity of the fiber-optic biosensor.

[0004] Graphene oxide (GO) is a new two-dimensional nanomaterial with excellent biocompatibility and high surface area, which can improve the sensitivity and selectivity of sensors and show great potential in biosensing applications. For example, in patent No. CN202510634222.7, entitled "Optical fiber biosensor and its preparation method, and optical fiber sensing system", it is mentioned that AuNPs@MOF composite film is coated on the outer surface of the optical fiber sensing part by chemical modification method to form a surface plasmon resonance layer, which is used to excite the optical fiber sensing part to generate surface plasmon waves, and to bind or adsorb sensing target DNA, miRNA and protein, thereby realizing biosensing detection of target detection objects. For example, in patent No. CN202410612053.2, entitled "Tuberculosis Mycobacterium in vitro rapid detection system based on graphene oxide coated microfiber sensor", it is mentioned that the aptamer specific to Mycobacterium tuberculosis protein and the graphene oxide GO coated tapered optical fiber are used as a sensor after incubation, and the aptamer can effectively capture Mycobacterium tuberculosis protein in vitro; the micro optical fiber coated with graphene oxide can detect Mycobacterium tuberculosis protein in real time, thereby realizing rapid in vitro detection of Mycobacterium tuberculosis. SUMMARY

[0005] Based on the above description, the present application provides an optical fiber biosensor, in which graphene oxide is fixed on the surface of an amino-functionalized tapered optical fiber sensor, and aptamer NE-AP and aptamer LF-AP specific to ELANE and LF are electrostatically adsorbed on the surface of the graphene oxide, so as to highly sensitively, lowly detectably and specifically detect the key markers ELANE and LF of neutrophil activity.

[0006] The technical scheme adopted is as follows: coating a graphene oxide layer on the surface of an amino-functionalized optical fiber sensor, and adsorbing aptamer NE-AP or aptamer LF-AP on the surface of the graphene oxide layer to obtain an aptamer NE-AP optical fiber biosensor and an aptamer LF-AP optical fiber biosensor; the aptamer NE-AP can specifically bind to elastase ELANE; and the aptamer LF-AP can specifically bind to lactoferrin LF.

[0007] Preferably, the optical fiber sensor is a tapered optical fiber sensor, and the tapered portion is amino-functionalized.

[0008] Preferably, the tapered portion of the optical fiber sensor is coated with a graphene oxide layer, and the surface of the graphene oxide layer is adsorbed with aptamer NE-AP or aptamer LF-AP.

[0009] A preparation method of an optical fiber biosensor, comprising the following steps: 1) Preparation of an amino-functionalized tapered optical fiber sensor: a) Fabrication of a waist-cone fiber optic sensor: The two ends of an optical fiber with the coating removed are fixed to stepper motors. A local area of ​​the optical fiber is heated using a hydrogen flame lamp. The speed of the two stepper motors moving towards each other is set. The heated and stretched part of the optical fiber shows characteristic structural peaks on a spectrometer, that is, the heated part of the optical fiber shows a conical structure, forming a waist-cone fiber optic sensor. By changing the flame size and the stretching time and speed, waist-cone fiber optic sensors with different transmission spectra can be obtained. b) Preparation of piranha solution: Mix 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 and stir with a glass rod to make them evenly mixed to obtain a piranha solution; then drip the piranha solution onto the waist-cone part of the waist-cone fiber optic sensor prepared in 1) through a glass dropper, leave it for 10-30 minutes to remove foreign matter from the surface of the waist-cone part of the waist-cone fiber optic sensor, and then clean it with deionized water to make the waist-cone part of the waist-cone fiber optic sensor rich in hydroxide ions; c) Aminated waist-cone fiber: 3-aminopropyltriethoxysilane (APTES) and ethanol are mixed uniformly in a volume ratio of 1:4 to obtain a mixture; the mixture is dripped onto the waist-cone fiber sensor obtained in step b), and left to stand for 25-35 minutes. The hydrolyzed groups form covalent bonds with the hydroxide ions on the surface of the waist-cone fiber, exposing the amino groups, thus achieving the aminated treatment of the waist-cone fiber sensor and obtaining an aminated waist-cone fiber sensor. 2) Surface adsorption of GO graphene oxide on the surface of the amination-modified waisted conical fiber optic sensor: The final aminated waist-cone fiber optic sensor was immersed in a graphene oxide dispersion and sonicated for 10-20 minutes. The concentration of the graphene oxide dispersion was diluted to 0.25-0.5 mg / mL with deionized water. The graphene oxide dispersion was then spin-coated onto the waist-cone portion and allowed to stand for 25-40 minutes. Graphene oxide was coated onto the surface of the aminated waist-cone fiber optic sensor through electrostatic interactions between the oxygen-containing groups on the graphene oxide and the amino groups on the aminated waist-cone fiber optic sensor, thus obtaining a graphene oxide-coated waist-cone fiber optic sensor. Graphene oxide and double-distilled water were mixed at a 1:1 ratio and ultrasonically dispersed to obtain a graphene oxide dispersion. 3) Preparation of NE-AP and LF-AP probes The surface of the graphene oxide-coated waist-cone fiber optic sensor obtained in step 2) was placed in an incubation box. The NE-AP aptamer and LP-AP aptamer were incubated on the surface of the waist-cone part of the waist-cone fiber optic sensor for 2 hours, thereby obtaining fiber optic biosensors with NE-AP probe and LF-AP probe, respectively.

[0010] Preferably, the waist-cone fiber optic sensor has a waist-cone portion with a diameter of φ8μm to 10μm, i.e., the diameter of the waist column is 8μm to 10μm.

[0011] An application of a fiber optic biosensor for the in vitro detection of neutrophil activity.

[0012] This invention discloses a fiber optic biosensor, belonging to the field of biodetection. It comprises lipopolysaccharide-induced inflammatory mice, sepsis patients, and healthy humans as test samples; aptamers NE-AP and LF-AP that specifically bind to elastase (ELANE) and lactoferrin (LF); and graphene oxide immobilized on the surface of an amino-functionalized conical optical fiber. The aptamers NE-AP and LF-AP, which specifically bind to ELANE and LF, are electrostatically adsorbed onto the GO graphene oxide surface to fabricate the fiber optic biosensor, thereby enabling highly sensitive, low-limit detection, and specific detection of key biomarkers of neutrophil activity (ELANE and LF). This invention overcomes the insensitivity of existing methods in capturing biomarkers in early, low-abundance stages. The detection system is simple to operate and easily miniaturized, providing a new technical approach for the early diagnosis and real-time clinical monitoring of diseases such as sepsis and chronic inflammation. It also plays an important role in patient prognosis assessment.

[0013] Commercial elastase (ELANE) and lactoferrin (LF) sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis: 1. Centrifuge 15000g of commercial ELANE and LF protein samples for 5 minutes, collect the precipitates and dissolve them in a specific sample buffer; the sample buffer contains 10mM Tris-HCl (pH 6.8), 10% glycerol, 0.05% bromophenol blue, 2% SDS and 5% 2-mercaptoethanol; 2. The dissolved ELANE and LF proteins were labeled with known molecular weights and analyzed by SDS-PAGE. At the end of electrophoresis, the gel was stained with Thomas Brilliant Blue to observe the protein bands. The molecular weights of ELANE and LF were 52.6 kDa and 77 kDa, respectively.

[0014] Surface plasmon resonance (SPR) measurements of NE-AP and LF-AP aptamers To accurately determine the binding affinity of specific aptamers, surface plasmon resonance (SPR) analysis was performed using the BIAcore T200 biosensor system.

[0015] 1. Pre-equilibrate the carboxymethylated sensor chip (Cytiva) with running buffer (10mM HEPES, pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM CaCl2, 2.7 mM KCl); 2. The chip surface was activated by a mixture of 100 mM N-hydroxysuccinimide and 400 mM N-ethyl-N'(dimethylaminopropyl)carbodiimide to immobilize His-labeled ELANE and LF proteins onto the chip; after protein immobilization, the chip surface was deactivated using 1 M ethanolamine hydrochloride (pH 8.5) to block any residual activating groups. 3. Samples of six different concentrations of NE-AP and LF-AP aptamers were introduced into the flow cell. After each sample injection, the protein surface was regenerated using 10 mM NaOH solution, and the chip was reequilibrated with running buffer. Finally, the SPR sensor spectrum was analyzed using BIA evaluation software to determine the binding affinity between the aptamer and the target protein.

[0016] Sample preparation All samples were prepared in accordance with the Declaration of Helsinki and approved by the Institutional Review Committee (or Ethics Committee) of Renji Hospital, Shanghai Jiao Tong University School of Medicine (Approval No. KY2021-060-A).

[0017] (1) Human sample preparation Plasma samples used for testing were obtained from patients with sepsis and healthy volunteers. First, peripheral venous blood samples from all subjects were collected into sterile EDTA anticoagulant tubes and processed within 2 hours of collection. The samples were centrifuged at 1500 × g for 15 minutes at 4°C to obtain the supernatant (plasma).

[0018] (2) Preparation of septicemic mouse samples LPS at a dose of 10 mg / kg was injected into the peritoneal cavity of mice, and peripheral blood was collected at 1, 24 and 96 hours after injection to obtain serum.

[0019] Table 1: Comparison of clinical inflammation between patients with sepsis and healthy individuals using fiber optic sensors.

[0020] Table 1 shows a comparison of clinical inflammation between patients with sepsis and healthy individuals using the fiber optic sensor prepared in this invention. This demonstrates the potential of the proposed aptamer-based fiber optic sensor in non-invasive early sepsis diagnosis and provides an effective platform for developing rapid, real-time sepsis screening technology. Attached Figure Description

[0021] Figure 1 This is a functionalized schematic diagram of the fiber optic biosensor of the present invention; Figure 2 This is a graph showing the specificity and purity analysis of SDS-PAGE on the purified ELANE and LF recombinant proteins; Figure 3 Secondary structure diagrams of the NE-AP aptamer for the ELANE protease and the LF-AP aptamer for the LF protein, as predicted by NUPACK in this invention. Figure 4 High-resolution three-dimensional structural diagrams of ELANE and LF proteins and their corresponding NE-AP and LF-AP aptamers in this invention, predicted by RNAComposer. Figure 5 This is a diagram showing the potential binding sites between ELANE and LF proteins and their corresponding NE-AP and LF-AP aptamers in this invention; Figure 6 This is a diagram showing the binding affinity test results of ELANE and LF proteins and their corresponding NE-AP and LF-AP aptamers in this invention; Figure 7 This diagram demonstrates the effectiveness of the fiber optic biosensor prepared in this invention for detecting targeted ELANE and LF proteins. Figure 8 The images show SEM images of fiber optic sensors under different conditions; specifically, A is the SEM image of the fiber optic sensor; B is the SEM image of the fiber optic sensor coated with a graphene oxide layer; C is the SEM image of the graphene oxide layer with NE-AP aptamers adsorbed on its surface; and D is the SEM image of the graphene oxide layer with LE-AP aptamers adsorbed on its surface. Figure 9 The image shows the detection spectra of the fiber optic biosensor at the same target protein concentration when graphene oxide adsorbs different aptamers of NE-AP and LF-AP in this invention. Figure 9 Transmission spectra of NE-AP probes immobilized on GO-modified optical fibers for detecting ELANE protein (A and B) and the corresponding relationship between wavelength shift and concentration; transmission spectra of LF-AP probes immobilized on GO-modified optical fibers for detecting LF protein (C and D) and the corresponding relationship between wavelength shift and concentration; E and F are comparison graphs of the detection limits of NE-AP (E) / LF-AP (F) fiber optic biosensors and traditional ELISA assays. Figure 10 This invention provides a test of the specificity of the fiber optic biosensor for targeting proteins. Figure 11 The results of the fiber optic biosensor of the present invention on the detection of ELANE protease and LF protein released by mouse neutrophils are shown. Detailed Implementation

[0022] The technical solution of the present invention is described in detail below. The embodiments of the present invention are for illustrative purposes only, and the scale of the structure is not limited by the embodiments.

[0023] like Figures 1 to 11 As shown, a fiber optic biosensor is prepared by coating an aminated fiber optic sensor surface with a graphene oxide layer, and adsorbing aptamer NE-AP or aptamer LF-AP onto the surface of the graphene oxide layer, thereby obtaining an aptamer NE-AP fiber optic biosensor and an aptamer LF-AP fiber optic biosensor. The aptamer NE-AP can specifically bind to elastase ELANE; the aptamer LF-AP can specifically bind to lactoferrin LF.

[0024] A method for fabricating an optical fiber biosensor, comprising the following steps; 1) Fabrication of an amination-modified waist-cone fiber optic sensor: a) Fabrication of waist-conical optical fiber: The two ends of the optical fiber with the coating removed are fixed to stepper motors. A local area of ​​the optical fiber is heated using a hydrogen flame lamp. The speed of the two stepper motors moving towards each other is set. The heated and stretched part of the optical fiber shows characteristic structural peaks on the spectrometer, that is, the heated part of the optical fiber shows a conical structure, forming a waist-conical optical fiber. By changing the flame size and the stretching time and speed, waist-conical optical fiber sensors with different transmission spectra can be obtained. The diameters of the waist column 1 of the waist-conical optical fiber sensor prepared by the above steps are φ9μm and φ9.6μm, respectively.

[0025] b) Preparation of piranha solution: Mix 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 and stir with a glass rod until they are evenly mixed to obtain a piranha solution; then drip the piranha solution onto the waist-cone part of the waist-cone fiber optic sensor prepared in step 1) with waist column diameters of 9μm and φ9.6μm, and leave it for 10-30 minutes to remove foreign matter from the surface of the waist-cone part of the waist-cone fiber optic sensor, and then clean it with deionized water to enrich the waist-cone part of the waist-cone fiber optic sensor with waist column diameters of 9μm and φ9.6μm with hydroxide ions; c) Aminated waist-cone fiber: 3-aminopropyltriethoxysilane (APTES) and ethanol are mixed uniformly in a volume ratio of 1:4 to obtain a mixture; the mixture is dripped onto the waist-cone portion of the two waist-cone fibers prepared in step b), and left to stand for 25-35 minutes. The hydrolyzed groups form covalent bonds with the -OH hydroxide ions on the surface of the waist-cone portion, exposing the amino groups, thus achieving the aminated treatment of the waist-cone portion of the waist-cone fiber, and obtaining two aminated waist-cone fiber sensors. 2) Adsorption of GO graphene oxide on the surface of an amination-modified waisted conical fiber optic sensor: The two aminated waist-conical fiber optic sensors finally obtained in step 1) were immersed in a graphene oxide dispersion and sonicated for 10-20 minutes. The concentration of the graphene oxide dispersion was diluted to 0.25-0.5 mg / mL with deionized water. The graphene oxide dispersion was then spin-coated onto the waist-conical portion and allowed to stand for 25-40 minutes. Through the electrostatic interaction between the oxygen-containing groups on the graphene oxide and the amino groups on the aminated waist-conical fiber optic sensor, the graphene oxide was coated onto the surface of the aminated waist-conical fiber optic, thus obtaining two graphene oxide-coated waist-conical fiber optic sensors. The graphene oxide and double-distilled water were mixed at a ratio of 1:1 and sonicated to obtain a graphene oxide dispersion. 3) Preparation of NE-AP and LF-AP probes The graphene oxide-coated waist-cone fiber optic sensor with a waist column diameter of 9 μm obtained in step 2) was placed in an incubation box. The NE-AP aptamer was incubated on the surface of the waist-cone part of the waist-cone fiber optic sensor for 2 hours to obtain the NE-AP probe fiber optic biosensor, i.e., the aptamer NE-AP fiber optic biosensor. Another waist-cone fiber optic sensor with a diameter of 9.6 μm and coated with graphene oxide was placed in an incubation box. The LP-AP aptamer was incubated on the surface of the waist-cone part of the waist-cone fiber optic sensor for 2 hours to obtain the fiber optic biosensor of the LF-AP probe, that is, the aptamer LF-AP fiber optic biosensor.

[0026] like Figure 10 As shown, the fiber optic biosensor of the present invention is used to test the specificity of the target protein, which verifies the reliability of the fiber optic biosensor and confirms that the detection signal comes only from the specific binding between the target protein and its corresponding aptamer, rather than environmental noise or other non-specific interference factors.

[0027] like Figure 11 The image shows the detection results of ELANE protease and LF protein released by mouse neutrophils using the fiber optic biosensor of the present invention. This demonstrates the ability of the fiber optic biosensor to quantitatively monitor the dynamics of neutrophil activation in real time through ELANE and LF. Different time patterns—immediate ELANE release and delayed LF response—can distinguish between acute and chronic inflammatory stages, further confirming the feasibility of the prepared fiber optic biosensor.

[0028] The fiber optic biosensor prepared by this invention can be used for in vitro detection of neutrophil activity.

[0029] A graphene oxide layer coating the surface of a waist-conical fiber optic sensor adsorbs aptamers NE-AP and / or LF-AP. Immersed in a solution of flexible protease (ELANE) and lactoferrin (LF), the fiber optic biosensor captures ELANE and LF proteins, causing changes in the roughness and thickness of the fiber surface at the waist-conical region. When light propagates through the fiber, total internal reflection occurs at the core-cladding interface. As light passes through the waist-conical region covered by biomolecules, the gradually decreasing diameter enhances the evanescent field outside the core, altering the effective refractive index of the fiber optic sensor surface. This results in a redshift in the transmission spectrum induced by aptamer-protein binding, enabling highly sensitive detection of neutrophil activity with a low detection limit.

Claims

1. An optical fiber biosensor, characterized by, Coating a graphene oxide layer on the surface of the amino-functionalized optical fiber sensor, and adsorbing aptamer NE-AP or aptamer LF-AP on the surface of the graphene oxide layer to obtain an aptamer NE-AP optical fiber biosensor and an aptamer LF-AP optical fiber biosensor; the aptamer NE-AP can specifically bind to elastase ELANE; and the aptamer LF-AP can specifically bind to lactoferrin LF.

2. The optical fiber biosensor according to claim 1, wherein, The optical fiber sensor is a waist-tapered optical fiber sensor, and the waist-tapered part is amino-functionalized.

3. A fiber-optic biosensor according to claim 2, wherein, The waist-tapered part of the optical fiber sensor is coated with a graphene oxide layer, and the surface of the graphene oxide layer is adsorbed with aptamer NE-AP or aptamer LF-AP.

4. A method for the production of an optical fibre biosensor according to any one of claims 1-3, characterised in that, The steps are as follows: 1) Preparation of an amino-functionalized waist-tapered optical fiber sensor: a) Preparation of a waist-tapered optical fiber sensor: fixing both ends of the optical fiber with the coating layer removed on a stepper motor, heating the local area of the optical fiber by using a hydrogen flame lamp, setting the speed of the two stepper motors moving towards each other, and the heated and stretched part of the optical fiber appears a characteristic structure peak on the spectrometer, that is, the tapered part of the optical fiber appears a tapered structure, forming a waist-tapered optical fiber sensor; by changing the size of the flame and the time and speed of stretching, waist-tapered optical fiber sensors with different transmission spectra can be obtained; b), preparation of piranha solution: 98% concentrated sulfuric acid and 30% hydrogen peroxide are mixed in a volume ratio of 3:1, and stirred with a glass rod to make them well mixed to obtain a piranha solution; then the piranha solution is dropped on the waist taper part of the waist taper optical fiber sensor prepared in 1) through a glass dropper, placed for 10-30 minutes, the surface foreign matter of the waist taper part of the waist taper optical fiber sensor is removed, and then cleaned with deionized water, so that the waist taper part of the waist taper optical fiber sensor is rich in hydroxyl ion OH - ; c) Amino-functionalization of the waist-tapered part of the waist-tapered optical fiber sensor: uniformly mixing 3-aminopropyltriethoxysilane APTES and ethanol at a volume ratio of 1:4 to obtain a mixed solution; dropping the mixed solution on the waist-tapered part of the waist-tapered optical fiber sensor prepared in step b), and standing for 25-35 minutes, so that the hydroxyl groups form covalent bonds with the hydroxyl ions on the surface of the waist-tapered part and expose the amino groups, achieving amino-functionalization of the waist-tapered part of the waist-tapered optical fiber sensor, and obtaining an amino-functionalized waist-tapered optical fiber sensor; 2) Adsorption of GO graphene oxide on the surface of the amino-functionalized waist-tapered optical fiber sensor: immersing the finally prepared amino-functionalized waist-tapered optical fiber sensor in a graphene oxide dispersion solution, and ultrasonically treating for 10-20 minutes; diluting the concentration of the graphene oxide dispersion solution to 0.25-0.5 mg / mL with deionized water; spin coating the graphene oxide dispersion solution on the amino-functionalized waist-tapered optical fiber sensor, and standing for 25-40 minutes; through electrostatic interaction between the oxygen-containing groups on the graphene oxide and the amino groups on the amino-functionalized waist-tapered optical fiber sensor, the graphene oxide is coated on the surface of the amino-functionalized waist-tapered optical fiber sensor, and a graphene oxide-coated waist-tapered optical fiber sensor is prepared; wherein the graphene oxide dispersion solution is prepared by mixing graphene oxide and double-distilled water at a volume ratio of 1:1 and then uniformly ultrasonically dispersing the mixture; 3) Preparation of NE-AP probes and LF-AP probes placing the finally prepared graphene oxide-coated waist-tapered optical fiber sensor in an incubation box, and incubating NE-AP aptamer and LP-AP aptamer on the surface of the waist-tapered part of the waist-tapered optical fiber sensor, respectively, for 2 hours to prepare NE-AP probe and LF-AP probe optical fiber biosensors, respectively, that is, an aptamer NE-AP optical fiber biosensor and an aptamer LF-AP optical fiber biosensor.

5. The method of claim 2, wherein the step of applying a coating of a material to the optical fiber is performed by a process selected from the group consisting of: dip coating, spin coating, and sputter coating. The waist taper fiber sensor, the waist taper part is 8-10 μm in diameter, i.e. the waist column is 8-10 μm in diameter.

6. Use of an optical fibre biosensor as claimed in any one of claims 1-3, characterized in that, For in vitro detection of neutrophil activity.

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