A quantum dot olfactory sensor, its preparation method and application

Abstract

The application belongs to the field of intelligent sensors, and more particularly relates to a quantum dot olfactory sensor, a preparation method and application thereof. The application adopts an odor receptor protein with a target gas specific binding site obtained by a natural enzyme molecule or a biological synthesis method, realizes coupling of the odor receptor and quantum dots through electrical labeling, and obtains an artificial olfactory receptor material. A planar three-electrode or transistor device is used as a conversion element, the olfactory receptor material is fixed on the conversion element, gas information is converted into an electrical signal through catalysis of the odor receptor protein molecule on the target gas, and identification, qualitative and quantitative detection of the gas are realized. The quantum dot olfactory sensor preparation method is simple, and can realize specific and sensitive detection of the target gas in a complex atmosphere environment.

Description

Technical Field

[0001] This application belongs to the field of intelligent sensors, and more specifically, relates to a quantum dot olfactory sensor, its preparation method and application. Background Technology

[0002] Olfactory sensors are intelligent sensors that mimic the structure and function of a biological olfactory system, converting gas information into electrical signals. They can quickly and cost-effectively identify simple or complex odors. The demand for olfactory sensors in environmental monitoring, process industries, and the tobacco, alcohol, and food industries is growing, creating an urgent need for the development of highly sensitive and specific gas sensors. However, current common gas sensors, such as metal-oxide-semiconductor gas sensors, typically have high operating temperatures (>100°C). o C) High power consumption and cross-sensitivity to multiple gases with poor selectivity have become major challenges in the development of artificial olfaction technology. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this application is to provide a quantum dot olfactory sensor, its preparation method, and its application. The method involves using natural enzymes or biologically synthesized odor receptors to label quantum dots to prepare artificial olfactory receptor materials, and constructing a quantum gas sensor using a three-electrode or high-mobility field-effect transistor as a transducer. This enables the specific detection of target gases, thereby solving the technical problems of existing gas sensors, such as the high operating temperature of gas-sensitive materials and insufficient specificity.

[0004] To achieve the above objectives, in a first aspect, this application provides a quantum dot olfactory sensor, characterized in that it comprises a quantum dot film formed on a conversion element by a quantum dot solution, and an odor receptor protein incubated on the quantum dot film;

[0005] The odor receptor protein is a natural enzyme molecule or a biologically synthesized odor receptor protein; the amino acid sequence of the natural enzyme molecule contains an active binding site that can specifically bind to the gas to be tested, and the biologically synthesized odor receptor protein contains the active binding site.

[0006] During detection, the odor receptor protein specifically binds to the gas to be tested, thereby collecting an electrical signal on the conversion element and detecting the gas to be tested.

[0007] Preferably, the quantum dots are lead sulfide colloidal quantum dots, cadmium sulfide colloidal quantum dots, or bismuth sulfide colloidal quantum dots; the quantum dot solution is obtained by dispersing the quantum dots in an organic solvent; and / or,

[0008] The conversion element is a planar three-electrode device or a transistor device.

[0009] Preferably, when the gas to be tested is ethyl acetate, the natural enzyme molecule is a natural esterase, wherein the natural esterase is an esterase derived from porcine liver or from Bacillus subtilis; and / or,

[0010] When the gas to be tested is ethanol, the natural enzyme molecule is alcohol dehydrogenase; and / or,

[0011] When the gas to be tested is formaldehyde, the natural enzyme molecule is formaldehyde dehydrogenase.

[0012] Preferably, the odor receptor protein is incubated on the quantum dot film, specifically by: adding a solution of the odor receptor protein dropwise onto the quantum dot film and incubating it to obtain quantum dot-labeled odor receptor protein; and / or,

[0013] The odor receptor protein solution is obtained by dissolving the odor receptor protein in PBS buffer solution, with a concentration of 0.5-5 mg / mL; the incubation temperature is 25-37℃, and the incubation time is 0.5-1h.

[0014] Preferably, the surface of the incubated odor receptor protein is further coated with a sensing aid reagent to ensure the activity of the odor receptor protein and the conductivity of the sensor surface. The sensing aid reagent is a mixture of PBS, glycerol and chloride.

[0015] According to another aspect of the present invention, a method for fabricating the quantum dot olfactory sensor is provided, comprising the following steps:

[0016] (1) Obtaining a quantum dot thin film formed on a conversion element from a quantum dot solution;

[0017] (2) The odor receptor protein solution is transferred onto the quantum dot film and incubated to prepare a quantum dot olfactory sensor.

[0018] Preferably, the preparation method further includes the step of:

[0019] (3) A sensing aid reagent is dropped onto the surface of the odor receptor protein on the quantum dot olfactory sensor to ensure the activity of the odor receptor protein and the conductivity of the sensor surface for the detection of the gas to be tested; wherein:

[0020] The sensing aid reagent is a mixture of PBS, glycerol, and a solute; the solute is a chloride.

[0021] Preferably, the volume ratio of PBS to glycerol in the mixed reagent is 1:1 to 10:1; and the molar concentration of the chloride is 1 to 1000 mM.

[0022] According to another aspect of the present invention, an application of the quantum dot olfactory sensor described herein is provided in the field of gas detection.

[0023] Overall, the technical solutions conceived in this application have the following beneficial effects compared with the prior art:

[0024] (1) This invention uses natural enzyme molecules or synthetic biology methods to obtain odor receptors with specific binding sites for target gases. The odor receptors are coupled to quantum dots via electrical labeling to obtain artificial olfactory receptor materials. Using a planar three-electrode or transistor device as the conversion element, the olfactory receptor material is fixed onto the conversion element. The gas information is converted into an electrical signal through the catalytic effect of the odor receptor molecules on the target gas, enabling the identification, qualitative, and quantitative detection of the gas. The quantum dot olfactory sensor fabrication method described in this invention is simple and can achieve specific and sensitive detection of target gases in complex atmospheric environments.

[0025] (2) This invention uses an electrochemical planar three-electrode / HEMT chip as an electrical signal transduction device to achieve efficient electron transduction during the gas reaction catalyzed by odor receptor proteins. This method is not only applicable to the specific catalysis of enzymes and gases, but also to odor receptor proteins and their derived short-chain peptides. The quantum dot olfactory sensor proposed in this invention can artificially design and synthesize corresponding odor receptor proteins based on the gene sequence of the odor binding site of the natural enzyme corresponding to the gas to be tested, and fix them on the conversion element to realize the detection of specific types of gas to be tested. It is applicable to the detection of a variety of gases.

[0026] (3) The quantum dot olfactory sensor for ethyl acetate prepared in some embodiments of the present invention has a response of 0.6 μA to 50 ppm ethyl acetate gas and has high detection specificity. Attached Figure Description

[0027] Figure 1 This is the fabrication process of the quantum dot olfactory sensor of the present invention;

[0028] Figure 2 The gas-sensitive cycling stability of the quantum dot olfactory sensors prepared in Examples 1 and 2 against 50 ppm ethyl acetate;

[0029] Figure 3 These are the selective test results of the quantum dot olfactory sensor prepared in Example 1;

[0030] Figure 4 The results are from testing the quantum dot olfactory sensor prepared in Example 1 with different concentrations of ethyl acetate (3-100 ppm);

[0031] Figure 5The gas-sensing properties of Comparative Examples 1 and 2 against 50 ppm ethyl acetate are shown. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0033] This invention provides a quantum dot olfactory sensor for detecting a target gas, comprising a quantum dot film formed on a conversion element by a quantum dot solution, and an odor receptor protein incubated on the quantum dot film; the odor receptor protein is a natural enzyme molecule or a biologically synthesized odor receptor protein; the amino acid sequence of the natural enzyme molecule contains an active binding site capable of specifically binding to the target gas, and the biologically synthesized odor receptor protein contains the active binding site; during detection, the odor receptor protein specifically binds to the target gas, thereby collecting an electrical signal on the conversion element, and thus detecting the target gas.

[0034] In some embodiments, the quantum dot surface contains oleic acid, and the odor receptor protein has abundant active binding sites that specifically bind to the gas to be tested.

[0035] In some embodiments, the quantum dots are lead sulfide colloidal quantum dots, cadmium sulfide colloidal quantum dots, or bismuth sulfide colloidal quantum dots; the quantum dot solution is obtained by dispersing the quantum dots in an organic solvent. The organic solvent can be toluene, n-octane, etc.

[0036] In some embodiments, colloidal quantum dot materials are synthesized via an organic thermal injection method; for example, lead sulfide quantum dots are synthesized by a nucleation reaction between a lead source and bis(trimethylsilyl)sulfide to form PbS colloidal quantum dots with an oleic acid-containing surface. Furthermore, colloidal quantum dots of different sizes can be synthesized by controlling the thermal injection temperature. A preferred thermal injection temperature is 95–150 °C. o C, the hot injection reaction time is 30-50 s. The concentration of colloidal quantum dots in the colloidal quantum dot solution is 1-30 mg / mL, preferably 1-10 mg / mL.

[0037] In some embodiments, the conversion element is a planar three-electrode or a transistor device. When the conversion element is a planar electrochemical three-electrode, the quantum dot film is formed by coating a quantum dot solution onto the working electrode of the planar electrochemical three-electrode. Alternatively, when the conversion element is a high electron mobility transistor chip, the quantum dot film is formed by coating a quantum dot solution onto the gate surface of the high electron mobility transistor chip.

[0038] In some embodiments, when the test gas is ethyl acetate, the natural enzyme molecule is a natural esterase, wherein the natural esterase is an esterase derived from pig liver or from Bacillus subtilis; when the test gas is ethanol, the natural enzyme molecule is alcohol dehydrogenase; and when the test gas is formaldehyde, the natural enzyme molecule is formaldehyde dehydrogenase.

[0039] In some embodiments, the amino acid sequence of the natural enzyme molecule contains an active binding site that specifically binds to the analyte gas. The biologically synthesized odor receptor protein is an odor receptor protein biologically synthesized based on the active binding site and obtained through heterologous expression using an *E. coli* expression system. Therefore, the biologically synthesized odor receptor protein contains the active binding site, and thus can specifically interact with the corresponding analyte gas during detection, thereby enabling the detection of the analyte gas using the sensor of the present invention. The active binding site in the amino acid sequence of the natural enzyme molecule that specifically binds to the analyte gas can be obtained through self-analysis or by consulting literature, and then an odor receptor protein containing the above-mentioned active binding site can be synthesized using conventional biological synthesis methods.

[0040] In some embodiments, the biological synthesis of odorant receptor proteins with target gas-specific active binding sites for different test gases includes the following steps:

[0041] S1. Based on the active binding sites in the amino acid sequences of natural enzyme molecules corresponding to different test gases that specifically interact with the gas, design an odor receptor protein gene sequence containing the active binding sites, and design corresponding primers to amplify the target fragment sequence corresponding to the odor receptor protein gene.

[0042] S2. The target fragment sequence is ligated into the multiple cloning site region of the recombinant protein expression vector to obtain the recombinant DNA sequence;

[0043] S3. The recombinant DNA sequence was mixed with chemically competent Escherichia coli BL21(DE3), heat-shocked, and then spread on a solid culture medium for culture and screening.

[0044] S4. Select normally growing transformants and culture them in LBK medium until the OD600 reaches 0.4-0.6. Add isopropyl thio-β-D-galactoside and induce culture.

[0045] S5. Centrifuge to recover cells, sonicate, centrifuge to collect supernatant; elute stepwise, dialyze to obtain purified recombinant odor receptor protein.

[0046] In some embodiments, in step S1, the natural enzyme molecule is an esterase (such as an esterase derived from pig liver or a natural esterase derived from Bacillus subtilis), alcohol dehydrogenase, formaldehyde dehydrogenase, etc. The odor receptor protein is a protein with a target gas-specific active binding site, such as ethyl acetate-binding protein, ethanol-binding protein, or formaldehyde-binding protein. In step S3, the heat shock temperature is 40-45°C. o C, the heat shock time is 40-50 s. In step S3, the solid culture medium is LB (LBK) solid culture medium containing kanamycin. In step S4, the culture temperature is 37°C. o C, The induction culture temperature is 14-18°C. o C, the final concentration of the isopropyl thio-β-D-galactoside is 0.5-2 mM. In step S5, the elution buffer used is an imidazole solution.

[0047] In some embodiments, odor receptor proteins are incubated on the quantum dot film, specifically by adding the odor receptor protein solution dropwise onto the quantum dot film and incubating it to obtain quantum dot-labeled odor receptor proteins.

[0048] In some embodiments, the odor receptor protein solution is obtained by dissolving the odor receptor protein in PBS buffer solution, with a concentration of 0.5-5 mg / mL. The incubation is performed at a temperature of 25-37°C for 0.5-1 h. This incubation treatment can replace some of the oleic acid on the surface of the colloidal quantum dot film with natural enzyme molecules or synthetic odor receptor protein molecules. When preparing this sensor, the volume ratio of the added odor receptor protein solution to the coated quantum dot solution is 1:1-20:1, more preferably 3:1-10:1.

[0049] In some embodiments, the surface of the incubated odor receptor protein is further coated with a sensing aid to ensure the activity of the odor receptor protein and the conductivity of the sensor surface. The sensing aid is a mixture of PBS, glycerol, and chloride.

[0050] This invention also provides a method for preparing the aforementioned quantum dot olfactory sensor, such as... Figure 1 As shown, it includes the following steps:

[0051] (1) Obtaining a quantum dot thin film formed on a conversion element from a quantum dot solution;

[0052] (2) The odor receptor protein solution is transferred onto the quantum dot film and incubated to prepare a quantum dot olfactory sensor.

[0053] In some embodiments, step (1) involves coating the quantum dot solution onto the conversion element and drying it to form the quantum dot film; in some embodiments, the coating is performed by drop coating, spin coating, or inkjet printing, etc. The thickness of the formed quantum dot film is preferably 100–600 nm, more preferably 300–600 nm, to facilitate electron transport between the odor receptor protein modification layer and the conversion element.

[0054] In some embodiments, the preparation method further includes the step of:

[0055] (3) The sensing aid reagent is dropped onto the surface of the odor receptor protein on the quantum dot olfactory sensor to ensure the activity of the odor receptor protein and the conductivity of the sensor surface for the detection of the gas to be tested; wherein: the sensing aid reagent is a mixture of PBS, glycerol and solute; the solute is chloride.

[0056] In some embodiments, the volume ratio of PBS to glycerol in the mixed reagent is 1:1 to 10:1, more preferably 2:1 to 6:1; the molar concentration of the chloride is 1 to 1000 mM, more preferably 200 to 600 mM. Preferably, the chloride can be potassium chloride, sodium chloride, etc.

[0057] During detection, the gas to be tested is introduced, and the test is performed using an electrochemical workstation or a semiconductor parameter analyzer. The quantum dot olfactory sensor of this invention can perform both qualitative and quantitative gas detection. First, the sensor is calibrated using a standard gas to obtain a standard curve. Then, the gas to be tested is detected to obtain the quantitative detection result.

[0058] The quantum dot olfactory sensor provided by this invention can be used in the field of gas detection. Depending on the type of gas to be detected, a suitable odor receptor protein can be selected. The odor receptor protein can be a natural enzyme molecule or a biologically synthesized odor receptor protein.

[0059] The specific catalytic interaction between odor receptors and gas molecules in this invention provides a new paradigm for constructing highly sensitive and specific olfactory sensors. Constructing gas sensors using colloidal quantum dots labeled with odor receptor molecules holds promise for achieving specific recognition of target gases, offering a new direction for research into specific room-temperature olfactory sensors.

[0060] This invention utilizes colloidal quantum dot materials to label odor receptor proteins (natural or biologically synthesized) to prepare artificial olfactory receptor materials, and fabricates a quantum dot olfactory sensor using a planar three-electrode or transistor as the conversion element. This sensor converts the specific binding reaction between the target gas and the artificial olfactory receptor material into an electrical signal output, enabling specific identification and quantitative detection of the target gas. This invention utilizes the specific catalytic interaction between odor receptors and gas molecules to construct an olfactory sensor with high sensitivity and specificity, providing a new approach for research on room temperature gas sensors.

[0061] The following are the sources of the reagents and instruments used in the embodiments of this invention:

[0062] Electrochemical planar three-electrode: purchased from Botan Technology (Weihai) Co., Ltd.

[0063] CHI760E electrochemical workstation: purchased from Shanghai Chenhua Instrument Co., Ltd.

[0064] Esterase (porcine liver or Bacillus subtilis): Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0065] Glycerin solution (100%, sterile): purchased from Beijing Innocare Technology Co., Ltd.

[0066] Phosphate-buffered saline (PBS): purchased from Beijing Innocare Technology Co., Ltd., 10×PBS (0.1M, pH 7.2-7.4).

[0067] n-Octane: Purchased from Sinopharm Chemical Reagent Co., Ltd.

[0068] Potassium chloride: Purchased from Beijing Innocare Technology Co., Ltd.

[0069] V-1530T constant temperature heating platform from Dongguan V-1530T Automation Technology Co., Ltd.

[0070] Semiconductor parameter analyzer: Keysight B1500A is a Keysight semiconductor device parameter analyzer.

[0071] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0072] The embodiments of this application are described below with reference to the accompanying drawings.

[0073] Example 1

[0074] This embodiment describes the fabrication of a quantum dot olfactory sensor based on natural esterases and a three-electrode system, specifically including the following steps:

[0075] S1. Preparation of PbS colloidal quantum dots based on the hot-injection method. 1.8 g of lead oxide, 6 mL of oleic acid, and 20 mL of octadecene were mixed in a three-necked flask and heated at 120 °C. o Under vacuum and stirring at C for 8 hours, a lead precursor was obtained. A sulfur precursor was prepared by dissolving 280 μL of bis(trimethylsilyl)sulfide in 10 mL of vacuum-sealed octadecene solution in a glove box. Then, under a nitrogen atmosphere, the sulfur precursor was rapidly added to the lead precursor, and the reaction was allowed to proceed for 30 s. The reactants were then rapidly cooled in a cold water bath. The precipitate was washed several times with toluene and acetone, centrifuged, and vacuum dried before being dispersed in n-octane to obtain a 5 mg / mL quantum dot solution.

[0076] S2. Dissolve 2 mg of lyophilized esterase powder (derived from pig liver) in 1 mL of PBS solution and shake for 10 min until dissolved to obtain the esterase solution. Dissolve 372.7 mg of KCl in 10 mL of PBS-glycerol mixture (PBS:glycerol = 8:2) and shake for 10 min until dissolved to obtain the sensing aid reagent.

[0077] S3. Drop 5 μL of quantum dot solution onto the working electrode of the planar three-electrode system. After drying, a quantum dot film with a thickness of approximately 500 nm is formed. Drop 26 μL of the prepared esterase solution onto the quantum dot film and incubate at a constant temperature of 37°C. o Incubate at C for 1 hour to form an enzyme-quantum dot interface, thus obtaining a quantum dot olfactory sensor.

[0078] S4. Place the prepared quantum dot olfactory sensor in the test chamber, and add sensing aid reagent until it completely covers the working electrode, reference electrode, and counter electrode. Connect to an electrochemical workstation, and obtain the redox peak position through cyclic voltammetry to determine the working voltage applied during the gas-sensitive test. Use a dynamic gas mixing system to introduce a certain concentration of ethyl acetate gas into the test chamber, and test the cyclic voltammetry curve, concentration gradient, and selectivity.

[0079] Example 2

[0080] This embodiment describes the fabrication of a quantum dot olfactory sensor based on a synthetic ethyl acetate odor receptor and a three-electrode system, specifically including the following steps:

[0081] S1. The preparation of quantum dots is the same as step S1 in Example 1.

[0082] S2. Based on the active binding site of the natural esterase specifically binding to ethyl acetate gas disclosed in Biochemistry 2010, 49, 1931–1942, an ethyl acetate binding protein gene sequence was designed. Based on the synthesized gene sequence, corresponding primers were designed, and the ethyl acetate binding protein sequence (EACBinder) was amplified in large quantities using PCR technology. Using a seamless cloning kit, the target fragment sequence was ligated into the multiple cloning site (MCS) region of the recombinant protein expression vector pET-28a(+), obtaining the recombinant DNA sequence pET-28a-EACBinder. pET-28a-EACBinder was mixed with chemically competent Escherichia coli BL21(DE3) and incubated at 42°C. o After heat shock at 37°C for 45 seconds, the transformants were plated onto LB (LBK) solid medium containing kanamycin for culture and screening. Normally growing transformants were picked and cultured in LBK medium at 37°C. o After culturing at C, the culture medium was inoculated at a ratio of 2% into a large bottle of LBK medium at 37°C. o Cultured at C until OD600 reaches 0.4-0.6, then add 1 mM isopropyl thio-β-D-galactoside (IPTG) and transfer to 16... o Cells were induced and cultured at C. After centrifugation, the cells were recovered and resuspended in PBS buffer. Sonication was performed for 15 min to release the induced protein expression within the cells. The supernatant was collected by centrifugation. The supernatant was mixed with nickel ion affinity chromatography media and eluted stepwise with imidazole solution to obtain a protein solution. Imidazole was removed by dialysis to obtain purified recombinant protein.

[0083] S3. The preparation process of the sensor-aided reagent is the same as step S2 in Example 1.

[0084] S4. Drop 5 μL of quantum dot solution onto the working electrode of a planar three-electrode system, and allow it to dry to form a quantum dot film. Then, drop an ethyl acetate-bound protein solution onto the quantum dot film and incubate at a constant temperature of 37°C. o Incubate at C for 1 hour to form an enzyme-quantum dot interface, thus obtaining a quantum dot olfactory sensor.

[0085] S5. The specific gas-sensing test of the quantum dot sensor for ethyl acetate is the same as step S4 in Example 1.

[0086] Example 3

[0087] This embodiment describes the fabrication of a quantum dot HEMT olfactory sensor based on a synthetic ethyl acetate odor receptor and a high-mobility field-effect transistor, specifically including the following steps:

[0088] S1. The preparation of quantum dots is the same as step S1 in Example 1.

[0089] S2. The synthesis of ethyl acetate-bound protein is the same as step S2 in Example 2.

[0090] S3. The preparation process of the sensor-aided reagent is the same as step S2 in Example 1.

[0091] S4. Drop 5 μL of quantum dot solution onto the gate of a high-mobility field-effect transistor, and dry to form a quantum dot film. Then, drop an ethyl acetate-bound protein solution onto the quantum dot film and incubate at a constant temperature of 37°C. o Incubate at C for 1 hour to form an enzyme-quantum dot interface, thus obtaining a quantum dot olfactory sensor.

[0092] S5. Place the prepared quantum dot HEMT gas sensor in the test chamber, and add sensing aid reagent until it completely covers the gate, source, and drain. Connect the semiconductor parameter analyzer and apply a voltage of -6 V to -1.2 V to test its performance under different concentrations of ethyl acetate. I DS - V DS curves and I DS -V GS Characteristic curves.

[0093] Following the above method, the quantum dot olfactory sensors of Examples 1, 2, and 3 were tested against 50 ppm ethyl acetate (a mixture of ethyl acetate and air, wherein the concentration of ethyl acetate gas was 50 ppm) to examine the response and recovery capabilities of the gas sensors to ethyl acetate. A voltage of -0.8 V was applied to the prepared quantum dot gas sensors during the test. The cycling stability of Examples 1 and 2 at 50 ppm ethyl acetate was tested, and the results are as follows: Figure 2 As shown, Examples 1 and 2 exhibit good cyclic stability with 50 ppm ethyl acetate.

[0094] The specificity of the sensor described in Example 1 for ethyl acetate was tested. Different gases of the same concentration (50 ppm each) were sequentially introduced into the sensor's test chamber, and the sensor's sensitivity to each gas was observed. The results are as follows: Figure 3 As shown, it can be seen that it has the strongest response to ethyl acetate and almost no response to other gases, that is, it has a specific recognition ability for ethyl acetate gas.

[0095] The quantum dot olfactory sensor from Example 2 was used to test different concentrations of ethyl acetate (3-100 ppm) to examine the gas sensor's response and recovery capabilities to different concentrations of ethyl acetate. The results are as follows: Figure 4 As shown, the sensor response increases with increasing ethyl acetate concentration. By fitting the curve of the relationship between ethyl acetate concentration and current, it can be used for the quantitative detection of ethyl acetate.

[0096] Comparative Example 1

[0097] This comparative example prepares a quantum dot olfactory sensor without odor receptors. The difference between this example and Examples 1 and 2 is that no esterase solution or synthetic ethyl acetate odor receptor solution is added. Specifically, the following steps are included:

[0098] S1. Preparation of PbS colloidal quantum dots based on the hot injection method is the same as in Example 1.

[0099] S2. Dissolve 372.7 mg KCl in 10 mL of PBS-glycerol mixture (PBS:glycerol = 8:2), shake for 10 min until dissolved, and obtain the sensing aid reagent.

[0100] S3. Take 5 μL of quantum dot solution and drop it onto the working electrode of the planar three-electrode system. After drying, a quantum dot film is formed, and a quantum dot olfactory sensor is obtained.

[0101] S4. Place the prepared quantum dot olfactory sensor in the test chamber, and add sensing auxiliary reagent until it completely covers the working electrode, reference electrode, and counter electrode. Connect the electrochemical workstation, apply the working voltage, and use a dynamic gas mixing system to introduce a certain concentration of ethyl acetate gas into the test chamber. Test the cyclic voltammetry curve, concentration gradient, and selectivity.

[0102] Comparative Example 2

[0103] This comparative example prepares an olfactory sensor without quantum dots, the difference from Example 2 being that no quantum dot solution is added. Specifically, it includes the following steps:

[0104] S1. The biological synthesis of ethyl acetate odor receptors is the same as step S2 in Example 2.

[0105] S2, the preparation of the sensing auxiliary reagent is the same as step S2 in Comparative Example 1.

[0106] S3. Drop the ethyl acetate odor receptor solution onto the working electrode of the planar three-electrode system and maintain a constant temperature of 37°C. o After incubating at C for 1 hour, an olfactory sensor was obtained.

[0107] S4. The gas-sensing test of the sensor for ethyl acetate is the same as step S4 in Comparative Example 2.

[0108] Following the above method, the olfactory sensors of Comparative Example 1 and Comparative Example 2 were used to test 50 ppm ethyl acetate to examine the response and recovery capabilities of the gas sensors to ethyl acetate. A voltage of -0.8 V was applied to the prepared gas sensors during the test. The cycling stability of Comparative Example 1 at 50 ppm ethyl acetate was tested, and the results are as follows: Figure 5As shown, Comparative Example 1 showed no obvious response signal to 50 ppm ethyl acetate, indicating that the ethyl acetate odor receptor synthesized through biosynthetic design can respond to ethyl acetate. The cyclic stability of Comparative Example 2 at 50 ppm ethyl acetate was tested, and the results are as follows... Figure 5 As shown, Comparative Example 2 showed a significant response signal to 50 ppm ethyl acetate, but it was prone to odor receptor inactivation, indicating the important role of quantum dots in constructing the receptor protein-electrode interface.

[0109] Comparative Example 3

[0110] The rest is the same as in Example 1, except that the sensing aid reagent prepared in step S2 contains only phosphate buffer solution PBS and does not contain potassium chloride and glycerol.

[0111] Comparative Example 4

[0112] The rest is the same as in Example 1, except that the sensing aid reagent prepared in step S2 contains only phosphate buffer solution PBS and glycerol, does not contain potassium chloride, and the volume ratio of PBS to glycerol is 8:2.

[0113] Comparative Example 5

[0114] The rest is the same as in Example 1, except that the sensing aid reagent prepared in step S2 contains only phosphate buffer solution PBS and potassium chloride, does not contain glycerol, and the concentration of potassium chloride in the composite reagent is 500mM.

[0115] The quantum dot sensors obtained in Comparative Examples 3, 4, and 5 were subjected to ethyl acetate gas-sensing performance tests (3 cycles of 50 ppm ethyl acetate testing). It was found that the baseline drift of the sensors prepared in Comparative Examples 3 and 5 was relatively severe. The baseline of the sensor prepared in Comparative Example 4 was more stable than that of Comparative Example 2, but the baseline current of the sensor was smaller.

[0116] Example 4

[0117] The process is the same as in Example 1, except that the quantum dot solution obtained in step S1 is 10 mg / mL. In step S2, 5 mg of lyophilized esterase powder (derived from pig liver) is dissolved in 1 mL of PBS solution and shaken for 10 min until dissolved to obtain the esterase solution. 372.7 mg of KCl is dissolved in 20 mL of a PBS-glycerol mixture (PBS:glycerol = 6:1) and shaken for 10 min until dissolved to obtain the sensing aid reagent. In step S3, 5 μL of the quantum dot solution is drop-coated onto the working electrode of a planar three-electrode system. After drying, a quantum dot film with a thickness of approximately 500 nm is formed. 15 μL of the prepared esterase solution is drop-coated onto the quantum dot film and incubated at 37°C. o Incubate at C for 1 hour to form an enzyme-quantum dot interface, thus obtaining a quantum dot olfactory sensor.

[0118] Example 5

[0119] The process is the same as in Example 1, except that the quantum dot solution obtained in step S1 is 1 mg / mL. In step S2, 0.5 mg of lyophilized esterase powder (derived from pig liver) is dissolved in 1 mL of PBS solution and shaken for 10 min until dissolved to obtain the esterase solution. 372.7 mg of KCl is dissolved in 10 mL of a PBS-glycerol mixture (PBS:glycerol = 2:1) and shaken for 10 min until dissolved to obtain the sensing aid reagent. In step S3, 5 μL of the quantum dot solution is drop-coated onto the working electrode of a planar three-electrode system. After drying, a quantum dot film with a thickness of approximately 500 nm is formed. 50 μL of the prepared esterase solution is drop-coated onto the quantum dot film and incubated at 37°C. o Incubate at C for 1 hour to form an enzyme-quantum dot interface, thus obtaining a quantum dot olfactory sensor.

[0120] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A quantum dot olfactory sensor, characterized in that, The quantum dot film formed by the quantum dot solution on the conversion element, and the odor receptor protein incubated on the quantum dot film; The odor receptor protein is a natural enzyme molecule or a biologically synthesized odor receptor protein; The natural enzyme molecule contains an active binding site capable of specifically binding to the gas to be detected in the amino acid sequence, and the biologically synthesized odor receptor protein contains the active binding site; During detection, the odor receptor protein specifically binds to the gas to be detected, so that an electrical signal is collected on the conversion element, thereby detecting the gas to be detected.

2. The sensor of claim 1, wherein, The quantum dot is a lead sulfide colloidal quantum dot, a cadmium sulfide colloidal quantum dot, or a bismuth sulfide colloidal quantum dot; and the quantum dot solution is obtained by dispersing the quantum dot in an organic solvent.

3. The sensor of claim 1, wherein, The conversion element is a planar three-electrode or a transistor device.

4. The sensor of claim 1, wherein, When the gas to be detected is ethyl acetate, the natural enzyme molecule is a natural esterase, wherein the natural esterase is an esterase derived from pig liver or an esterase derived from Bacillus subtilis; and / or, When the gas to be detected is ethanol, the natural enzyme molecule is ethanol dehydrogenase; and / or, When the gas to be detected is formaldehyde, the natural enzyme molecule is formaldehyde dehydrogenase.

5. The sensor of claim 1, wherein, The odor receptor protein is incubated on the quantum dot film, specifically: a solution of the odor receptor protein is added dropwise to the quantum dot film for incubation treatment to obtain quantum dot-labeled odor receptor protein; and / or, The solution of the odor receptor protein is obtained by dissolving the odor receptor protein in a PBS buffer solution, and the concentration is 0.5-5 mg / mL; the incubation temperature is 25-37°C, and the incubation time is 0.5-1 h.

6. The sensor of claim 1, wherein, The surface of the incubated odor receptor protein is also covered with a sensing auxiliary reagent to ensure the activity of the odor receptor protein and the conductivity of the sensor surface, and the sensing auxiliary reagent is a mixed reagent containing PBS, glycerol, and a chloride.

7. The method for fabricating a quantum dot olfactory sensor as described in any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) obtaining a quantum dot film formed by a quantum dot solution on a conversion element; (2) transferring an odor receptor protein solution to the quantum dot film and incubating to prepare a quantum dot olfactory sensor.

8. The method of claim 7, wherein the step of preparing is characterized by, The method further comprises the following step: (3) adding a sensing auxiliary reagent to the surface of the odor receptor protein on the quantum dot olfactory sensor to ensure the activity of the odor receptor protein and the conductivity of the sensor surface for detecting the gas to be detected; wherein: The sensing auxiliary reagent is a mixed reagent containing PBS, glycerol, and a solute; and the solute is a chloride.

9. The production method according to claim 8, wherein The volume ratio of PBS to glycerol in the mixed reagent is 1:1-10:1; and the molar concentration of the chloride is 1-1000 mM.

10. Application of the quantum dot olfactory sensor according to any one of claims 1-6 in the field of gas detection.

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