CN114324540A - Guanosine tetraphosphate electrochemical type nano enzyme sensor and preparation method thereof - Google Patents
Guanosine tetraphosphate electrochemical type nano enzyme sensor and preparation method thereof Download PDFInfo
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Images
Abstract
The invention discloses a guanosine tetraphosphate electrochemical type nano enzyme sensor and a preparation method thereof, and aims to provide a sensor capable of performing biological detection on ppGpp and a preparation method thereof. The sensor is characterized in that nano-gold particles and guanosine tetraphosphate hydrolase MESH1 molecules are adsorbed on a substrate through bridging agent chitosan, and are sealed through sealing liquid, so that a chitosan film layer, a nano-gold sol layer, an MESH1 enzyme layer and a sealing layer are formed on the substrate. The preparation method comprises the following steps: adding a low-concentration chitosan solution on the pretreated electrode, and forming a chitosan film on the surface of the electrode after drying; drying the electrode with the chitosan film, placing the electrode into the nano gold sol, and self-assembling for at least 12 hours at 4 ℃; washing with ultrapure water, drying, and self-assembling in a MESH1 enzyme protein solution at 4 ℃ for at least 12 h; after cleaning, sealing the non-specific sites by using a sealing solution; and cleaning and airing to obtain the sensor. The sensor has the advantages of high sensitivity, strong specificity, quick quantification and short response time.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a guanosine tetraphosphate electrochemical nano enzyme sensor, a preparation method thereof, application of the guanosine tetraphosphate electrochemical nano enzyme sensor and a method for biologically detecting guanosine tetraphosphate by using the guanosine tetraphosphate electrochemical nano enzyme sensor.
Background
Guanosine tetraphosphate (ppGpp) is an important compound for transmitting nitrogen starvation signals in organisms, and plays a vital role in growth and survival of the organisms. The currently known ppGpp detection methods include: based on liquid chromatography-mass spectrometry (UPLC-MS) and chemo-fluorescent probe technology. The liquid chromatography-mass spectrometry technology has high detection sensitivity and good accuracy, but needs to depend on expensive instruments and equipment and professional testers, and has relatively long analysis time. The chemical fluorescent probe technology has extremely high detection sensitivity, but the sensor is complex to prepare and has instability.
Disclosure of Invention
The invention aims to provide an electrochemical nano enzyme sensor capable of performing biological detection on ppGpp, aiming at the technical defects in the prior art.
The invention also aims to provide a preparation method of the ppGpp electrochemical nano enzyme sensor, which has the advantages of simple preparation method, high sensitivity, strong specificity and quick quantification.
The invention further aims to provide an application of the guanosine tetraphosphate electrochemical type nanoenzyme sensor in the aspect of detecting ppGpp.
Still another object of the present invention is to provide a high sensitivity, strong specificity, and rapid quantification method for ppGpp.
The technical scheme adopted for realizing the purpose of the invention is as follows:
a guanosine tetraphosphate electrochemical type nanoenzyme sensor takes an electrode as a substrate, gold nanoparticles and guanosine tetraphosphate hydrolase MESH1 molecules are adsorbed on the substrate through bridging agent chitosan, and the substrate is sealed through sealing liquid, so that a chitosan membrane layer, a nanogold sol layer, a MESH1 enzyme layer and the sealing layer are formed on the substrate, and the sealing liquid is bovine serum albumin.
A preparation method of a guanosine tetraphosphate electrochemical type nano enzyme sensor comprises the following steps:
(1) adding a low-concentration chitosan solution on the pretreated electrode, and forming a chitosan film on the surface of the electrode after drying; then, eliminating acid radicals contained in the chitosan membrane by using a low-concentration NaOH solution;
(2) drying the electrode with the chitosan film, placing the electrode into the nano gold sol, and self-assembling for at least 12 hours at 4 ℃;
(3) taking out the electrode obtained in the step (2), washing with ultrapure water and drying, and then self-assembling in a guanosine tetraphosphate hydrolase MESH1 enzyme protein solution at 4 ℃ for at least 12 h; the concentration of the MESH1 enzyme protein solution is not less than 0.5 mg/mL.
(4) Taking out the electrode obtained in the step (3), and sealing the non-specific site by using a sealing liquid after cleaning;
(5) and (4) cleaning and airing the electrode obtained in the step (4) to obtain the guanosine tetraphosphate electrochemical type nano enzyme sensor.
The electrode is a glassy carbon electrode, and the pretreatment method of the glassy carbon electrode comprises the following steps: polishing the glassy carbon electrode on a chamois leather by using alumina slurry with the particle size of 0.03 mu m in an 8-shaped manner, cleaning the glassy carbon electrode by using water, and then cleaning the glassy carbon electrode in an ultrasonic water bath until the surface is smooth and clean; the electrode after pretreatment meets the following requirements: the peak potential difference of cyclic voltammetry curve in the treatment solution is 64-80mV, and the treatment solution adopts K with the concentration of 1mmol/L3Fe(CN)6Solution of said K3Fe(CN)6The solution contains KNO with the concentration of 0.20mol/L3(ii) a The scanning range of the cyclic voltammetry curve is 0.6 to-0.1V, and the scanning speed is 50 mV/s.
In the step (1), the concentration of the chitosan solution is 0.5%; the concentration of the NaOH solution used was 0.5mol/L, and the immersion time in the NaOH solution was 5 minutes.
The nano gold sol in the step (2) is prepared by the following method: mixing 1% sodium citrate solution and 0.01% chloroauric acid solution according to the ratio of 1: 25, keeping the mixture for 5 to 10 minutes by using microwaves with medium fire power until the reaction solution turns bright red; the nano gold in the nano gold sol is spherical, the particles are uniformly dispersed, and the particle size is 20 +/-2 nm.
The confining liquid adopts a bovine serum albumin solution with the concentration of 5%, and the confining condition is that the incubation is carried out for at least 1h at 37 ℃.
An application of a guanosine tetraphosphate electrochemical type nanoenzyme sensor is used for detecting the content of ppGpp.
A biological detection method for guanosine tetraphosphate by using a guanosine tetraphosphate electrochemical nano enzyme sensor comprises the following steps:
(1) establishing a three-electrode system: the guanosine tetraphosphate electrochemical type nano enzyme sensor is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; adopting ultrapure water as a blank control;
(2) measuring the response current of the ppGpp standard substance with different concentrations under the action of the optimized voltage by adopting the three-electrode system through a time-current method, obtaining the change rate of the ppGpp response current by utilizing a formula (1), and establishing a relation curve between the change rate of the ppGpp response current and the concentration of the ppGpp; determining a linear concentration range from the ppGpp-responsive current rate of change versus ppGpp concentration curve; fitting a linear equation according to the linear relation between the ppGpp response current change rate and the ppGpp concentration;
in formula (1): Δ I represents a rate of change of the response current; i is1Response current value representing blank control, unit: a, I2Representing the response current value of ppGpp solution, in units: a;
(3) and (3) determining the sample to be detected by adopting the three-electrode system, and detecting the ppGpp in the sample to be detected by using the obtained linear equation to obtain the content of the ppGpp in the sample to be detected.
Pretreating a sample to be detected before detection: addition of NADP phosphatase to the sample solution to be assayed to avoid NADPH or NADP+And (4) interference.
Compared with the prior art, the invention has the beneficial effects that:
1. the guanosine tetraphosphate electrochemical nanoenzyme sensor can carry out biological detection on ppGpp, and has the advantages of high detection sensitivity, strong specificity, good stability, rapid quantification and short response time.
2. The preparation method of the guanosine tetraphosphate electrochemical nano enzyme sensor is simple, and the obtained sensor is high in detection sensitivity, strong in specificity, rapid in quantification and short in response time.
3. The detection method is a biological detection method, adopts a biosensor with high sensitivity and strong specificity to detect, does not need to carry out complex pretreatment on a sample, is simple to operate, does not depend on expensive instruments and equipment, has short response time and good stability, can specifically detect ppGpp, and has very important significance and application prospect for researching signal transmission mechanisms of amino acid or nucleotide starvation and rigorous reaction of animals, plants and microorganisms.
Drawings
FIG. 1 is a schematic diagram of the bioassay method of the present invention;
FIG. 2 shows a graph of the dependence of the sensor response current on the concentration for determining ppGpp;
FIG. 3 is a graph showing the dependence of the response current change rate of ppGpp, GTP, GMP, ADP and ATP on the concentration.
Detailed Description
The invention is described in detail below with reference to the figures and specific examples.
Example 1
1. Pretreating a glassy carbon electrode:
polishing the glassy carbon electrode on a chamois leather in a shape of 8 by using alumina slurry with the particle size of 0.03 mu m, cleaning the glassy carbon electrode in an ultrasonic water bath for tens of seconds after cleaning with water until the surface is smooth and clean; the electrode is at 1X 10-3mol/L K3Fe(CN)6Solution (containing 0).20mol/L KNO3) The electrode is activated by cyclic voltammetry with the scanning range of 0.6 to-0.1V and the scanning speed of 50mV/s, and the scanning is repeated until a stable cyclic voltammetry curve appears. The stable cyclic voltammogram meets the following requirements: the peak potential difference is below 80mV and as close to 64mV as possible, the electrode can be used, and finally the electrode is placed in a nitrogen environment for drying and standby. Compared with the conventional glassy carbon electrode pretreatment method, the method has the advantages of simpler operation and shorter time under the condition of meeting the same requirements.
2. Preparation of nanogold (GNPs) sols:
adding 4mL of 1% sodium citrate solution into 100mL of 0.01% chloroauric acid solution, mixing, placing in a microwave oven, maintaining with middle fire for 5-10min until the solution becomes transparent wine red to obtain nanometer gold sol, and storing at 4 deg.C in dark place. Scanning the prepared nano gold sol in a visible light range (400-700nm) by using a spectrophotometer, wherein the light absorbing colloidal gold has a single light absorbing peak in the visible light range, and the wavelength lambda of the light absorbing peak ismaxAround 518 nm. The size, shape and dispersion condition of the prepared nano gold sol particles are further accurately characterized by a transmission electron microscope, the synthesized nano gold has a regular shape, the nano gold is spherical, the particle size is uniform, the average particle size is about 18-20nm, and the aggregation phenomenon is avoided.
3. Preparing a guanosine tetraphosphate (ppGpp) electrochemical nanoenzyme sensor:
(1) 0.5g of chitosan is dissolved in 100mL of 1% acetic acid solution, and the solution is stirred until the chitosan solution with the concentration of 0.5% is completely dissolved. Dripping 10 mu L of chitosan solution with the concentration of 0.5% on the center of the surface of the pretreated glassy carbon electrode, placing the glassy carbon electrode on an ultra-clean workbench for drying for about 1 hour, and taking out the glassy carbon electrode after the chitosan solution on the surface of the electrode core is dried to form a film; then, the chitosan membrane is immersed into NaOH solution with the concentration of 0.5mol/L for about 5 minutes, and then is repeatedly cleaned by ultrapure water, and the purpose of eliminating acid radicals contained in the chitosan membrane and neutralizing the acid radicals is achieved;
(2) then, airing the glassy carbon electrode with the chitosan film obtained in the step (1), placing the glassy carbon electrode into the prepared nano gold sol, and carrying out self-assembly for 12 hours at 4 ℃;
(3) taking out the electrode treated in the step (2), washing the electrode with ultrapure water for several times, drying the electrode in the air, dripping 15 mu L of MESH1 enzyme protein solution with the concentration of not less than 0.5mg/mL on the surface of the electrode, and continuously performing self-assembly at 4 ℃ for 12 hours;
(4) taking out the electrode treated in the step (3), softly rinsing the electrode for several times by using ultrapure water, and then placing the electrode in a Bovine Serum Albumin (BSA) solution with the mass-volume ratio concentration of 5% for incubation for 1h at 37 ℃ to block the non-specific sites;
(5) and (4) taking out the electrode treated in the step (4), rinsing with ultrapure water, and airing to obtain the ppGpp electrochemical nano enzyme sensor, placing the sensor above Phosphate Buffer Solution (PBS), and storing in a refrigerator at 4 ℃ for later use.
4. Determining ppGpp by using a ppGpp electrochemical nanoenzyme sensor:
the principle diagram of the biological detection method is shown in figure 1, and the technical scheme of the invention is that the protease MESH1 can catalyze the hydrolysis of a substrate ppGpp to perform the following reactions:
MESH1 catalyzes the ppGpp reaction to generate products GDP and pyrophosphoric acid, and the reaction is accompanied by the generation and change of an electric signal and is amplified by the electric signal of the nano gold particles. The change of the electric signal can be captured by the electrochemical workstation through the glassy carbon electrode, and a standard curve is established according to the magnitude of the response current and the concentration of ppGpp, so that the detection of the ppGpp in the unknown sample is realized. The specific technical scheme is as follows:
(1) a three-electrode system is adopted, the prepared ppGpp electrochemical nano enzyme sensor is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode. Ultrapure water was used as a blank, and the response current of ppGpp solutions of different concentrations under the action of the optimized voltage (the optimized voltage in this example is-0.4V) was measured by a time-current method, and the change rate of the response current was used as an index for detection, and the measurement was performed in parallel three times for each concentration. The formula for calculating the rate of change of the response current is as follows:
in formula (2): Δ I represents a change in response current; i is1Response current value representing blank control, unit: a, I2Representing the response current value of ppGpp solution, in units: a;
(2) measuring the response current of the ppGpp standard substance with different concentrations under the action of the optimized voltage by adopting the three-electrode system through a time-current method, obtaining the change rate of the ppGpp response current by utilizing a formula (2), and establishing a relation curve between the change rate of the ppGpp response current and the concentration of the ppGpp; determining a linear concentration range from the ppGpp-responsive current rate of change versus ppGpp concentration curve; and fitting a linear equation according to the linear relation between the ppGpp response current change rate and the ppGpp concentration.
(3) And (3) determining the sample to be detected by adopting the three-electrode system, and detecting the ppGpp in the sample to be detected by using the obtained linear equation to obtain the content of the ppGpp in the sample to be detected.
In order to avoid the influence of other components on the detection, the sample to be detected is pretreated before the detection: adding NADP phosphatase to the sample solution to be assayed in an amount sufficient to remove NADPH/NADP that may be present in the sample+And (4) interference.
5. Selectivity/specificity, stability and lifetime of the sensor:
the detection method has the advantages that the ppGpp is selectively detected, the Mesh1 enzyme specifically hydrolyzes the ppGpp, the Mesh1 and the ppGpp are combined to generate GDP and pyrophosphoric acid, and the change is transmitted to a computer through an electrochemical sensor and an electrochemical workstation in the form of an electric signal to cause the change of the current value. The prepared ppGpp nano enzyme sensor is respectively placed in ppGpp, GTP, GMP, ADP and ATP solutions with different concentrations, and the concentrations are sequentially from 1 multiplied by 10-12mol/L、1×10- 11mol/L、……、1×10-6The mol/L was measured. As shown in FIG. 3, the rate of change of the response current of the electrochemical nanoenzyme sensor for ppGpp was very high, and the electrochemical nanoenzyme sensor for ppGpp was found to be similar to GTP, GMP,ADP and ATP were not responsive, indicating that the sensor had good selectivity (specificity) for ppGpp.
The manufactured ppGpp nano enzyme sensor is continuously measured in ppGpp solution with the same concentration for 10 times, and the RSD of the current change rate is 4.73%, which shows that the nano enzyme sensor has good stability. The nanoenzyme sensor is placed above a phosphoric acid buffer solution at 4 ℃ for storage, and is measured in a ppGpp solution with the same concentration every day, the response current value of the sensor is stable in the first 8 days, and the response current value in the 8 th day is 73.52% of that in the first day, which indicates that the nanoenzyme sensor can be stably stored for at least 7 days.
6. The following details are given by taking the detection of ppGpp in Arabidopsis thaliana as an example:
(1) the three-electrode system was used to measure ppGpp standards at different concentrations and establish a standard curve of ppGpp concentration versus rate of change of response current, as shown in FIG. 2. Calculated, the sensor response current rate of change (Δ I) and ppGpp concentration are 1 × 10-12-1×10-11Linear correlation in the mol/L range, and the standard curve equation is fitted as follows:
(2) placing two kinds of Arabidopsis (Arabidopsis wild type WT and GCR1 defective Arabidopsis KO) in a refrigerator of-80 deg.C for freezing for 1h, respectively weighing a certain mass of WT Arabidopsis leaf and GCR1 KO Arabidopsis leaf, placing into a mortar for grinding, placing the Arabidopsis leaf homogenate into a 1.5ml enzyme-free centrifuge tube, centrifuging at 8000 rpm for 10min, taking the supernatant, adding ultrapure water to make the volume to 10ml, adding NADP phosphatase (NADP phosphatase) microspheres into the solution to eliminate NADPH/NADP+And (4) influence, standing for about 5 minutes, filtering, and preparing the ppGpp sample buffer solution to be tested.
Wherein, the preparation process of the NADP phosphatase microspheres is as follows: dissolving 0.2g sodium alginate in 10mL distilled water, melting by bath heat, cooling, adding NADP phosphatase 0.1mg, dissolving, sucking into syringe, and adding into 2% CaCl under stirring2Dispersing in solution to form ballsThe application is as follows.
(3) The three-electrode system is adopted to determine the sample to be detected, the response time is only 50s, and as a result, the ppGpp content in each 100mg of WT arabidopsis leaves is 0.330 multiplied by 10-12mol/L, ppGpp content of 0.452 × 10 per 100mg GCR1 KO Arabidopsis thaliana leaf-12mol/L。
The sensor has high sensitivity, is not influenced by the turbidity and the color of the sample, has relatively simple required equipment and instruments, and does not need to carry out complex pretreatment on the sample. Compared with the detection method reported in the literature (liquid chromatography HPLC, liquid chromatography-mass spectrometer GC-MS and a chemical fluorescence sensor), the detection sensitivity of the detection method is improved by about four orders of magnitude, and the response time is only 50 s. Has important application value in research of determining the ppGpp content in strict reactions of animals, plants, microorganisms and the like.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A guanosine tetraphosphate electrochemical type nanoenzyme sensor is characterized in that an electrode is used as a substrate, nanogold particles and guanosine tetraphosphate hydrolase MESH1 molecules are adsorbed on the substrate through bridging agent chitosan, and the substrate is sealed through sealing liquid, so that a chitosan film layer, a nanogold sol layer, an MESH1 enzyme layer and the sealing layer are formed on the substrate, and the sealing liquid is bovine serum albumin.
2. The method for preparing the guanosine tetraphosphate electrochemical type nanoenzyme sensor as claimed in claim 1, which comprises the following steps of:
(1) adding a low-concentration chitosan solution on the pretreated electrode, and forming a chitosan film on the surface of the electrode after drying; then, eliminating acid radicals contained in the chitosan membrane by using a low-concentration NaOH solution;
(2) drying the electrode with the chitosan film, placing the electrode into the nano gold sol, and self-assembling for at least 12 hours at 4 ℃;
(3) taking out the electrode obtained in the step (2), washing with ultrapure water and drying, and then self-assembling in a guanosine tetraphosphate hydrolase MESH1 enzyme protein solution at 4 ℃ for at least 12 h; the concentration of the MESH1 enzyme protein solution is not less than 0.5 mg/mL.
(4) Taking out the electrode obtained in the step (3), and sealing the non-specific site by using a sealing liquid after cleaning;
(5) and (4) cleaning and airing the electrode obtained in the step (4) to obtain the guanosine tetraphosphate electrochemical type nano enzyme sensor.
3. The method for preparing a guanosine tetraphosphate electrochemical type nanoenzyme sensor according to claim 2, wherein the electrode is a glassy carbon electrode, and the pretreatment method of the glassy carbon electrode comprises the following steps: polishing the glassy carbon electrode on a chamois leather by using alumina slurry with the particle size of 0.03 mu m in an 8-shaped manner, cleaning the glassy carbon electrode by using water, and then cleaning the glassy carbon electrode in an ultrasonic water bath until the surface is smooth and clean; the electrode after pretreatment meets the following requirements: the peak potential difference of cyclic voltammetry curve in the treatment solution is 64-80mV, and the treatment solution adopts K with the concentration of 1mmol/L3Fe(CN)6Solution of said K3Fe(CN)6The solution contains KNO with the concentration of 0.20mol/L3(ii) a The scanning range of the cyclic voltammetry curve is 0.6 to-0.1V, and the scanning speed is 50 mV/s.
4. The method for preparing a guanosine tetraphosphate electrochemical type nanoenzyme sensor according to claim 2, wherein in the step (1), the concentration of the chitosan solution is 0.5%; the concentration of the NaOH solution used was 0.5mol/L, and the immersion time in the NaOH solution was 5 minutes.
5. The method for preparing an electrochemical guanosine tetraphosphate nanoenzyme sensor according to claim 2, wherein the nanogold sol obtained in the step (2) is prepared by a method comprising the following steps of: mixing 1% sodium citrate solution and 0.01% chloroauric acid solution according to the ratio of 1: 25, keeping the mixture for 5 to 10 minutes by using microwaves with medium fire power until the reaction solution turns bright red; the nano gold in the nano gold sol is spherical, the particles are uniformly dispersed, and the particle size is 20 +/-2 nm.
6. The method for preparing guanosine tetraphosphate electrochemical type nanoenzyme sensor according to claim 2, wherein the blocking solution is bovine serum albumin solution with a concentration of 5%, and the blocking condition is incubation at 37 ℃ for at least 1 h.
7. Use of the guanosine tetraphosphate electrochemical-based nanoenzyme sensor of claim 1 for detecting the content of ppGpp.
8. A method for biologically detecting guanosine tetraphosphate using the electrochemical guanosine tetraphosphate nanoenzyme sensor according to claim 1, comprising the steps of:
(1) establishing a three-electrode system: the guanosine tetraphosphate electrochemical type nano enzyme sensor is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; adopting ultrapure water as a blank control;
(2) measuring the response current of the ppGpp standard substance with different concentrations under the action of the optimized voltage by adopting the three-electrode system through a time-current method, obtaining the change rate of the ppGpp response current by utilizing a formula (1), and establishing a relation curve between the change rate of the ppGpp response current and the concentration of the ppGpp; determining a linear concentration range from the ppGpp-responsive current rate of change versus ppGpp concentration curve; fitting a linear equation according to the linear relation between the ppGpp response current change rate and the ppGpp concentration;
in formula (1): Δ I represents a rate of change of the response current; i is1Response current value representing blank control, unit: a, I2Representing the response current value of ppGpp solution, in units: a;
(3) and (3) determining the sample to be detected by adopting the three-electrode system, and detecting the ppGpp in the sample to be detected by using the obtained linear equation to obtain the content of the ppGpp in the sample to be detected.
9. The method for biologically detecting guanosine tetraphosphate by using the guanosine tetraphosphate electrochemical type nanoenzyme sensor as claimed in claim 8, wherein a sample to be detected is subjected to pretreatment before detection: addition of NADP phosphatase to the sample solution to be assayed to avoid NADPH or NADP+And (4) interference.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110114511A1 (en) * | 2009-11-17 | 2011-05-19 | Sjong Angele | Apparatus for detecting volatile organic compounds and related methods |
| CN107966560A (en) * | 2017-11-22 | 2018-04-27 | 江南大学 | A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle |
| CN109073590A (en) * | 2016-03-30 | 2018-12-21 | 池田食研株式会社 | Amino acid quantitative approach and amino acid reagents for quantitatively box |
| CN109709179A (en) * | 2019-03-01 | 2019-05-03 | 天津商业大学 | A kind of GPR120 electrochemical receptor sensor and its preparation method and application |
| US20190169630A1 (en) * | 2016-02-08 | 2019-06-06 | Centre National De La Recherche Scientifique (Cnrs ) | Improvement of photosynthetic organisms through the modulation of guanosine tetraphosphate homeostatis |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110114511A1 (en) * | 2009-11-17 | 2011-05-19 | Sjong Angele | Apparatus for detecting volatile organic compounds and related methods |
| US20190169630A1 (en) * | 2016-02-08 | 2019-06-06 | Centre National De La Recherche Scientifique (Cnrs ) | Improvement of photosynthetic organisms through the modulation of guanosine tetraphosphate homeostatis |
| CN109073590A (en) * | 2016-03-30 | 2018-12-21 | 池田食研株式会社 | Amino acid quantitative approach and amino acid reagents for quantitatively box |
| CN107966560A (en) * | 2017-11-22 | 2018-04-27 | 江南大学 | A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle |
| CN109709179A (en) * | 2019-03-01 | 2019-05-03 | 天津商业大学 | A kind of GPR120 electrochemical receptor sensor and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| CHIEN-KUANG CORNELIA DING等: "MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis", 《NATURE METABOLISM》, pages 1 - 21 * |
| 卢文珺 等: "信号分子ppGpp与微生物环境适应性", 《生命科学》, vol. 24, no. 4, pages 385 - 389 * |
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