A gas ethanol optical fiber sensor based on fluorescence detection

Abstract

The present invention provides a gas ethanol optical fiber sensor based on fluorescence detection and a preparation method thereof. The sensor is composed of a Teflon tube and a tapered optical fiber. The Teflon tube is fixed with an immobilized enzyme carrier and alcohol dehydrogenase, and a tapered optical fiber is placed at one end of the tube. The two ends of the Teflon tube are sealed, and the side of the Teflon tube is provided with NAD + The device preparation method includes: using a heating softening method to fix the immobilized enzyme carrier on the inner wall of the Teflon tube; using a covalent bonding method to combine the alcohol dehydrogenase and the immobilized enzyme carrier; using a taper method to prepare a tapered optical fiber; using a laser ablation method to process a liquid inlet and outlet hole on the side of the Teflon tube; and using a hot melt method to seal the Teflon tube to obtain the device. + The enzymatic reaction generates NADH, and the fluorescence characteristics of NADH cause the transmission spectrum of the tapered optical fiber to change. The transmission spectrum can be monitored to detect ethanol gas. The present invention has the advantages of repeatability, simple operation and low detection cost.

Description

Technical Field

[0001] The invention relates to a gas ethanol sensor, in particular to a gas ethanol sensor based on optical fiber sensing of fluorescence detection. Background Art

[0002] Ethanol is a commonly used fuel and disinfectant in life, and it is also widely used in traditional Chinese medicine. At present, most Chinese herbal medicines are extracted mainly using high-concentration ethanol. As an organic solvent, ethanol can extract more useful elements from Chinese herbal medicines, and its solubility in useful elements of Chinese herbal medicines is better than that of water. After soaking Chinese herbal medicines in ethanol, most of them use heating and evaporation to separate ethanol and drugs. The currently widely used standard method to judge whether ethanol has been completely separated is "no alcohol taste", that is, to use the sense of smell to feel whether ethanol is still present. This method has low accuracy. There are also electrochemical sensing methods, but this method is only applicable when the ethanol concentration is high enough, and the detection speed is slow. It is necessary to design a miniaturized and high-precision gas ethanol fast detector.

[0003] NAD (nicotinamide adenine dinucleotide) is a biological term, Chinese name: Nicotinamide adenine dinucleotide, referred to as coenzyme I. NAD exists in two forms: NAD + and NADH. These two forms of NAD are called a "redox pair"; NAD + It is a coenzyme of dehydrogenases, such as alcohol dehydrogenase (ADH), which is used to oxidize ethanol. It plays an irreplaceable role in glycolysis, gluconeogenesis, tricarboxylic acid cycle and respiratory chain. The intermediate product will transfer the hydrogen removed to NAD + , making it NADH, which is a strong fluorescent substance, and the fluorescence intensity signal is proportional to the amount of NADH present.

[0004] Alcohol dehydrogenase (ADH), also known as alcohol dehydrogenase, is a group of compounds that exist in the digestive system of humans or other animals. More than 90-95% of alcohol is decomposed in the human body through oxidation, and the dehydrogenation process is the most important step, which is responsible for this alcohol dehydrogenase and aldehyde dehydrogenase. Summary of the invention

[0005] In view of the above technical defects, the present invention provides a gas ethanol optical fiber sensor based on fluorescence detection. The gas permeability of the Teflon tube is used to achieve indirect contact between the ethanol gas and the reactants in the tube. The chitosan and alcohol dehydrogenase can be covalently bonded to achieve enzyme immobilization. The ethanol and NAD +The enzymatic reaction of the solution in the environment of alcohol dehydrogenase generates NADH with fluorescent properties to achieve fluorescence detection, and finally obtains a gas ethanol detection system with high sensitivity, repeatability, simple operation and low detection cost.

[0006] The first object of the present invention is to disclose a gas ethanol optical fiber sensor based on fluorescence detection, comprising a Teflon tube, a single-ended tapered multimode optical fiber, an immobilized enzyme carrier, alcohol dehydrogenase, NAD + Buffer, liquid inlet, and liquid outlet; NAD + The buffer solution is injected into the Teflon tube from the liquid inlet, and the ethanol gas to be tested penetrates from the wall of the Teflon tube into the tube and interacts with the NAD + It reacts in the environment of alcohol dehydrogenase to produce NADH. Since NADH has fluorescence characteristics, the emitted fluorescence signal is received by the single-ended tapered multimode optical fiber and transmitted to the spectrometer to analyze the intensity and wavelength of the fluorescence signal to detect the ethanol concentration of the gas to be tested.

[0007] The enzymatic reaction of the present invention is:

[0008]

[0009] The Teflon tube of the present invention has good gas permeability, an inner diameter of 203 μm, an outer diameter of 254 μm, a length of 40 mm, and a refractive index of 1.29.

[0010] The single-ended tapered multimode optical fiber of the present invention has a cladding diameter of 125 μm, a core diameter of 105 μm, and a length of 80 mm. The tapered sensing area is made by a large-scale taper drawing machine, and the tapered sensing length is 1000 μm.

[0011] The immobilized enzyme carrier of the present invention is made of chitosan after surface functionalization; the diameter of chitosan is preferably 30 μm to 50 μm; the preparation process of the immobilized enzyme carrier is as follows: 20 mL of 5% glacial acetic acid is added to 0.6 g of chitosan, 80 mL of liquid paraffin and 3 mL of span-80 solution are added after stirring and dissolving, and stirred for 30 minutes at 25°C, and then 10 mL of 7% glutaraldehyde solution is added, stirred for 1 hour at 40°C, and pH is adjusted to 10-11 with 8 mol / L NaOH solution, and stirred for 2 hours at 70°C. Then the solution is poured into a beaker, 30 mL of petroleum ether solution and 30 mL of acetone solution are added, and the mixture is allowed to stand for stratification after stirring, the upper oil phase is discarded, and the mixture is washed with petroleum ether and acetone in batches and filtered, and dried at 4°C, so that the surface functionalization of the chitosan is completed, thereby obtaining the immobilized enzyme carrier. The immobilized enzyme carrier helps the alcohol dehydrogenase to be indirectly fixed on the inner wall of the Teflon tube by covalently bonding with the alcohol dehydrogenase, and its highest inactivation temperature is 315.74℃.

[0012] The NAD of the present invention + The concentration of the buffer solution is 1.0mmol / L. NAD + The buffer mainly provides NAD for the enzymatic reaction in the Teflon tube + , the fluorescent substance NADH is generated in the enzymatic reaction, and the emission wavelength of the fluorescent substance is 490nm.

[0013] Preferably, the liquid inlet is used for NAD + The buffer solution was injected into the sensor, which was located 2 mm from the end of the optical fiber of the Teflon tube and had a diameter of 90 μm.

[0014] Preferably, the liquid outflow port is used for the waste liquid discharge sensor, is located 2 mm from the other end of the Teflon tube, and has a diameter of 90 μm.

[0015] The second purpose of the present invention is to disclose a preparation method of a gas ethanol optical fiber sensor based on fluorescence detection, the steps are as follows: first, a liquid inlet and a liquid outlet are processed on the side of a Teflon tube by a laser ablation method; then, an immobilized enzyme carrier is placed in the Teflon tube, and an ultrasonic vibration method is used to evenly disperse the immobilized enzyme carrier on the inner wall of the Teflon tube, and then the Teflon tube is softened by heating. The heating temperature of the Teflon tube is 200-210°C, and the temperature is maintained for 8-10 minutes, so that the immobilized enzyme carrier is slightly sunk into the softened inner surface of the Teflon tube. After cooling and hardening, the immobilized enzyme carrier can be fixed on the inner wall of the Teflon tube, and the immobilized enzyme carrier does not lose activity under this environment; then, a covalent bond method is used to fix alcohol dehydrogenase on the immobilized enzyme carrier; then, a single-ended tapered multimode optical fiber is extended into the Teflon tube; then, the other end of the Teflon tube is sealed by a melting method; finally, a syringe is used to inject NAD + The buffer solution is injected into the Teflon tube so as to fill the entire interior of the sensor, thereby obtaining a gas ethanol optical fiber sensor based on fluorescence detection.

[0016] The single-ended tapered multimode optical fiber of the present invention is prepared by a large-scale taper machine. First, a coating layer of 40 mm in length is stripped off, and then the stripped area is subjected to discharge heating to form a taper. The tapered sensing length is set to 1000 μm, and the waist length is set to 1000 μm. A blade is then used for cutting to obtain a single-ended tapered multimode optical fiber.

[0017] The alcohol dehydrogenase is fixed by a covalent bond method, and the process is as follows: after the immobilized enzyme carrier is fixed on the inner wall of a Teflon tube, 50 mL of a Tris-HCl buffer solution with a concentration of 0.05 mol / L and a pH value of 6.8 is added, and then 50 mL of an alcohol dehydrogenase solution is added, and after stirring at room temperature for 2.5 hours, the solution is washed with water and filtered to obtain the alcohol dehydrogenase fixed on the immobilized enzyme carrier.

[0018] The beneficial effects of the present invention are as follows: the gas ethanol optical fiber sensor has high sensitivity, the Teflon tube has good gas permeability and can detect ethanol gas at low concentrations, and because the immobilization function of alcohol dehydrogenase is utilized, the enzyme catalytic function can be repeatedly used, saving detection costs, and overcoming the shortcomings of traditional gas ethanol detectors, such as limited detection range, low sensitivity and high detection cost. In the field of traditional Chinese medicine, the use of the gas ethanol optical fiber sensor can realize the absorption and detection of low-concentration gas ethanol after Chinese herbal medicines are soaked in ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the structure of the gas ethanol optical fiber sensor.

[0020] Figure 2 A schematic diagram of a specific embodiment of the system. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] like Figure 1 , which is a schematic diagram of the structure of the gas ethanol fiber sensor, where (1) is a Teflon tube, (2) is a single-ended tapered multimode optical fiber, (3) is an immobilized enzyme carrier, (4) is alcohol dehydrogenase, and (5) is NAD + Buffer, (6) is the liquid inlet, and (7) is the liquid outlet.

[0023] like Figure 1The present invention provides a method for preparing a gas ethanol optical fiber sensor based on fluorescence detection, comprising: using a laser ablation method to process a liquid inlet (6) and a liquid outlet (7) with a diameter of 90 μm on the side of a Teflon tube (1) with a length of 40 mm; then placing an immobilized enzyme carrier (3) in the Teflon tube (1), using an ultrasonic vibration method to evenly disperse the immobilized enzyme carrier (3) on the inner wall of the Teflon tube (1), and then heating the Teflon tube to soften it, the heating temperature is 200-210° C., and the temperature is maintained for 8-10 minutes, so that the immobilized enzyme carrier (3) The immobilized enzyme carrier (3) is slightly sunken into the softened inner surface of the Teflon tube (1), and after cooling and hardening, the immobilized enzyme carrier (3) can be fixed on the inner wall of the Teflon tube (1); the preparation process of the immobilized enzyme carrier is as follows: 20 mL of 5% glacial acetic acid is added to 0.6 g of chitosan, and after stirring and dissolving, 80 mL of liquid paraffin and 3 mL of span-80 solution are added, and stirred at 25°C for 30 minutes, and then 10 mL of 7% glutaraldehyde solution is added, and stirred at 40°C for 1 hour, and the pH is adjusted to 10-11 with 8 mol / L NaOH solution, and stirred at 70°C for 2 hours. Then the solution is poured into a beaker, 30 mL of petroleum ether solution and 30 mL of acetone solution are added, and after stirring, the solution is allowed to stand for stratification, and the upper oil phase is discarded, and the chitosan surface is functionalized by washing with petroleum ether and acetone in batches and filtered, and dried at 4°C, thereby completing the surface functionalization of the chitosan and obtaining the immobilized enzyme carrier. Then, the alcohol dehydrogenase (4) is fixed on the immobilized enzyme carrier (3) by using a covalent bond method; the fixing process is as follows: add 50 mL of 0.05 mol / L Tris-HCl buffer solution with a pH value of 6.8 into the Teflon tube (1), then add 50 mL of alcohol dehydrogenase (4) solution, stir at room temperature for 2.5 hours, wash with water and filter, and the alcohol dehydrogenase (4) fixed on the immobilized enzyme carrier (3) can be obtained. Then, after stripping the 40 mm multimode optical fiber coating layer using a wire stripper, the stripped area is placed in the processing area of ​​a large taper machine for discharge heating taper, and the taper sensing length is set to: 1000 μm, and the waist length is set to: 1000 μm, and then a blade is used for cutting to obtain a single-ended tapered multimode optical fiber (2). Then, the single-ended tapered multimode optical fiber (2) is inserted into the Teflon tube (1) with an insertion length of 20 mm; then, the other end of the Teflon tube (1) is sealed by a melting method; finally, a syringe is used to inject NAD + The buffer solution (5) is injected into the Teflon tube (1) so as to fill the entire interior of the sensor, thereby obtaining a gas ethanol optical fiber sensor based on fluorescence detection.

[0024] like Figure 2As shown, it is a specific embodiment of a gas ethanol fiber optic sensing system based on fluorescence detection, (8) is a gas ethanol fiber optic sensor based on fluorescence detection, which is used to detect the ethanol concentration of the external environment gas, (9) is a multimode transmission optical fiber, which is used to transmit optical signals, (10) is an optical circulator, which is used to transmit optical signals, (11) is an ultraviolet LED light source, which is used to emit 340nm ultraviolet light to excite NADH to emit light, (12) is an LED light source driver, which is used to stabilize the ultraviolet LED light source (11), (13) is a bandpass filter, which is used to select optical signals with wavelengths around 490nm to enter the spectrometer, (14) is an optical fiber spectrometer, which is used to detect the fluorescence signal detected by the sensor, and (15) is a PC monitor, which is used to detect the spectral intensity signal.

[0025] When implementing this example, it is necessary to change (5)NAD + Buffer solution is injected from the liquid inlet (6) until the entire sensor is filled, and then the liquid inlet (6) and the liquid outlet (7) are closed. Subsequently, (8) a gas ethanol optical fiber sensor based on fluorescence detection is placed in the gas ethanol environment to be tested. The gas ethanol penetrates into the system through the inner wall of the Teflon tube (1) and reacts with (5) NAD + The buffer undergoes an enzymatic reaction under the catalysis of (4) alcohol dehydrogenase to produce the fluorescent substance NADH, and then (11) the ultraviolet LED light source and (12) the LED light source driver are turned on to generate a light signal with a wavelength of 340nm, which is transmitted through (10) the optical circulator and (9) the multimode transmission optical fiber to (8) a gas ethanol optical fiber sensor based on fluorescence detection. NADH is excited by (2) the single-ended tapered multimode optical fiber to emit fluorescence with a wavelength of 490nm, and the fluorescence signal is transmitted to (13) the bandpass filter through (2) the single-ended tapered multimode optical fiber, (9) the multimode transmission optical fiber and (10) the optical circulator. The light signal with a wavelength around 490nm is selected to enter (14) the optical fiber spectrometer, and is displayed on (15) the PC display. The concentration of the gas ethanol to be tested is determined according to the intensity of the spectrum, thereby realizing the detection of the concentration of low-concentration gas ethanol. After the system is used, the liquid inlet (6) and the liquid outlet (7) are opened, and the waste liquid is discharged from the system from the liquid outlet (7) by air pressure.

[0026] Those skilled in the art clearly know that according to the method of the present invention, low-concentration gas ethanol can be detected with high precision and in real time, and can be combined and optimized in design when there is no conflict in technical features.

Claims

1. A gas ethanol optical fiber sensor based on fluorescence detection, characterized in that: The sensor comprises a Teflon tube (1), a single-end tapered multimode optical fiber (2), an immobilized enzyme carrier (3), an alcohol dehydrogenase (4), NAD + The sensing end of the single-ended tapered optical fiber (2) extends into the Teflon tube (1) at a distance of 30 mm from one end, and the liquid inlet (6) and the liquid outlet (7) are processed at both ends of the Teflon tube (1) at a distance of 2 mm from the port. The immobilized enzyme carrier (3) is evenly fixed on the inner wall of the Teflon tube (1), the alcohol dehydrogenase (4) is fixed on the immobilized enzyme carrier (3), and the two ends of the Teflon tube are sealed. + The buffer solution (5) is injected into the Teflon tube (1) from the liquid inlet (6) and fills the entire device; The ethanol gas to be measured penetrates from the wall of the Teflon tube (1) into the tube and interacts with the NAD + Under the catalysis of alcohol dehydrogenase, acetaldehyde and NADH are produced. Since NADH has fluorescence characteristics, the emitted fluorescence signal is received by the single-ended tapered multimode optical fiber and transmitted to the spectrum analyzer, and the intensity and wavelength of the fluorescence signal are analyzed to detect the ethanol concentration of the gas to be tested. After the system is used, the liquid inlet (6) and the liquid outlet (7) are opened, and the waste liquid is discharged from the system from the liquid outlet (7) by using air pressure.

2. A gas ethanol optical fiber sensor based on fluorescence detection according to claim 1, characterized in that: The Teflon tube (1) has good gas permeability, an inner diameter of 203 μm, an outer diameter of 254 μm, a length of 40 mm, and a refractive index of 1.

29.

3. A gas ethanol optical fiber sensor based on fluorescence detection according to claim 1, characterized in that: The single-ended tapered multimode optical fiber (2) has a cladding diameter of 125 μm, a core diameter of 105 μm, and a length of 80 mm. The tapered sensing area is made by a large-scale taper machine, and the tapered sensing length is 1000 μm.

4. A gas ethanol optical fiber sensor based on fluorescence detection according to claim 1, characterized in that: The immobilized enzyme carrier (3) is prepared by surface functionalization of chitosan; the diameter of chitosan is 30 μm to 50 μm; the preparation process of the immobilized enzyme carrier (3) is as follows: 20 mL of 5% glacial acetic acid is added to 0.6 g of chitosan, and after stirring and dissolving, 80 mL of liquid paraffin and 3 mL of span-80 solution are added, and the mixture is stirred at 25° C. for 30 minutes, and then 10 mL of 7% glutaraldehyde solution is added, and the mixture is stirred at 40° C. for 1 hour, and the pH value is adjusted to 10-11 with 8 mol / L NaOH solution, and the mixture is stirred at 70° C. for 2 hours, and then the solution is poured into a beaker, and 30 mL of petroleum ether solution and 30 mL of acetone solution are added, and the mixture is allowed to stand for stratification after stirring, and the upper oil phase is discarded, and the mixture is washed with petroleum ether and acetone in batches and filtered, and dried at 4° C., so that the surface functionalization of the chitosan is completed, thereby obtaining the immobilized enzyme carrier (3).

5. A gas ethanol optical fiber sensor based on fluorescence detection according to claim 1, characterized in that: The NAD + The concentration of buffer (5) was 1.0 mmol / L.

6. A gas ethanol optical fiber sensor based on fluorescence detection according to claim 1, characterized in that: The liquid inlet (6) is used for NAD + The injection of the buffer solution is located 2 mm from the optical fiber insertion end of the Teflon tube (1) and has a diameter of 90 μm.

7. The gas ethanol optical fiber sensor based on fluorescence detection according to claim 1, characterized in that: The liquid outflow port (7) is used for the outflow of waste liquid and is located 2 mm from the other end of the Teflon tube (1) and has a diameter of 90 μm.

8. The method for preparing the optical fiber sensor according to claim 1, characterized in that: First, a liquid inlet (6) and a liquid outlet (7) are processed on the side of the Teflon tube (1) by laser ablation; then, an immobilized enzyme carrier (3) is placed in the Teflon tube (1), and an ultrasonic vibration method is used to evenly disperse the immobilized enzyme carrier (3) on the inner wall of the Teflon tube (1); then, the Teflon tube (1) is softened by heating. The heating temperature for softening the Teflon tube (1) is 200-210° C., and the temperature is maintained for 8-10 minutes, so that the immobilized enzyme carrier (3) is slightly sunken into the softened inner surface of the Teflon tube (1). After cooling and hardening, the immobilized enzyme carrier (3) can be fixed on the inner wall of the Teflon tube (1); then, a covalent bond method is used to fix the alcohol dehydrogenase (4) on the immobilized enzyme carrier (3); then, a single-ended tapered multimode optical fiber (2) is extended into the Teflon tube (1); then, the other end of the Teflon tube (1) is sealed by melting; finally, a syringe is used to inject NAD + The buffer solution (5) is injected into the Teflon tube (1) so as to fill the entire interior of the sensor.

9. The preparation method according to claim 8, characterized in that: The single-ended tapered multimode optical fiber (2) is prepared by a large-scale tapering machine. First, a coating layer of 40 mm in length is stripped off, and then the stripped area is subjected to discharge heating to form a tapered surface. The tapered sensing length is set to 1000 μm, and the waist length is set to 1000 μm. A blade is then used for cutting to obtain the single-ended tapered multimode optical fiber (2).

10. The preparation method according to claim 8, characterized in that: The alcohol dehydrogenase (4) is fixed by a covalent bond method, and the process is as follows: after the immobilized enzyme carrier (3) is fixed on the inner wall of the Teflon tube (1), 50 mL of a 0.05 mol / L Tris-HCl buffer solution with a pH value of 6.8 is added, and then 50 mL of an alcohol dehydrogenase (4) solution is added. After stirring at room temperature for 2.5 hours, the solution is washed with water and filtered to obtain the alcohol dehydrogenase (4) fixed on the immobilized enzyme carrier (3).

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