阅读说明：本技术 使用新型电化学传感器的氨基酸特异实时检测方法 (Amino acid specific real-time detection method using novel electrochemical sensor ) 是由 王江云 夏霖 韩明杰 杨朝雅 于 2019-09-06 设计创作，主要内容包括：本申请提供了一种使用新型电化学传感器的氨基酸特异实时检测方法,所述的电化学传感器包括掺入了非天然氨基酸的氧化还原酶。(The present application provides a method for the specific real-time detection of amino acids using a novel electrochemical sensor comprising an oxidoreductase incorporating an unnatural amino acid.)

1. An electrochemical sensor comprising an amino acid oxidase incorporating an unnatural amino acid.

2. Electrochemical sensor according to claim 1, wherein the amino acid oxidase is glycine oxidase GlyOx or L-tryptophan oxidase TrpOx.

3. Electrochemical sensor according to claim 2, wherein the amino acid oxidase is glycine oxidase GlyOx, which PDB encodes 1NG4, or L-tryptophan oxidase TrpOx, which PDB encodes 5G 3T.

4. An electrochemical sensor according to any one of claims 1 to 3, wherein the unnatural amino acid is 2-amino-3- (4-mercaptophenyl) propionic acid (p-thiol-phenylalanine, TF).

5. The electrochemical sensor according to claim 4, wherein the amino acid oxidases incorporating an unnatural amino acid are GlyOx-266TF and TrpOx-395 TF.

6. The electrochemical sensor according to claim 5, wherein the amino acid oxidase incorporating an unnatural amino acid is TrpOx-395 TF.

7. An electrochemical sensor according to any one of claims 1 to 6, wherein the electrochemical sensor comprises Bodipy373 as a connection linker.

8. An electrochemical sensor according to any one of claims 1 to 6, wherein the electrode used in the electrochemical sensor is a carbon-based electrode.

9. The electrochemical sensor according to claim 7, wherein the electrode used in the electrochemical sensor is selected from a glassy carbon electrode, an HOPG electrode, a carbon fiber electrode or a graphene electrode.

10. An electrochemical sensor according to claim 6 or 7, wherein the electrode is decorated with a carbon-based nanocomposite material.

11. The electrochemical sensor according to any one of claims 1 to 9, wherein the electrochemical sensor is manufactured by casting a Nafion-MWNTs composite on a glassy carbon electrode and drying at room temperature to obtain a Nafion-MWNTs composite membrane modified GC electrode, and then dropping a mixture including a Bodipy 373-modified enzyme mutant to the Nafion-MWNTs composite membrane modified GC electrode and standing until drying.

12. An electrochemical sensor according to any of claims 1-9, wherein the electrochemical sensor is prepared by a process comprising casting a cocktail of enzymes including a Bodipy 373-modified enzyme mutant onto a HOPG electrode surface and drying for 20 minutes at room temperature.

13. A method for amino acid specific real-time detection, wherein an electrochemical sensor according to any one of claims 1 to 11 is used.

14. The method according to claim 12, wherein the real-time detection method is for detecting amino acids in blood or sweat.

15. The method according to claim 13, wherein the method uses an electrochemical sensor comprising TrpOx-395TF-Bodipy373 to detect tryptophan in blood or sweat.

16. use of an electrochemical sensor according to any one of claims 1 to 11 for the preparation of an instrument for the specific real-time detection of amino acids.

Technical Field

The application belongs to the field of electrochemical sensors and the field of physiological parameter detection, and particularly provides a method for specifically detecting amino acid in real time by using a novel electrochemical sensor, wherein the electrochemical sensor comprises oxidoreductase doped with unnatural amino acid.

Background

Amino acids are essential metabolic intermediates and cell signaling molecules, and abnormal amino acid metabolism causes many serious diseases, so that real-time amino acid analysis is of great significance for diagnosis and medicine. Conventional analytical procedures for measuring amino acid concentrations are generally based on non-portable spectroscopic or chromatographic instruments, which are clearly not suitable for POCT and health care monitoring wearable biosensors.

Electrochemical sensors are critical for point-of-care testing (POCT) and wearable sensing devices. Establishing an efficient electron transport pathway between the oxidoreductase and the electrode is the key to converting the enzyme-catalyzed reaction into an electrochemical signal, and to constructing a robust, sensitive and selective biosensor. Enzymatic Electrochemical Biosensors (EEBs) provide a convenient, low cost and real-time method for measuring analyte concentrations. There are several key requirements for efficient EEB: high surface density between the highly active sites and the electrodes, long-term enzyme stability and potentially the most challenging efficient electron transport pathway (EET). Despite the tremendous efforts to improve enzyme robustness, selectivity and activity to meet the major requirements for practical sensing applications, highly selective and sensitive electrochemical sensors remain impractical for most identification of healthy biomolecules.

one of the important points for improving enzymatic electrochemical biosensors is to improve the electron transfer between the enzyme and the electrode. The use of mediators in EEBs is a robust method to improve electron transfer, but the use of mediators often results in an increased excess potential relative to the original redox potential of the enzyme, and redox mediators are often non-selective, facilitating not only electron transfer between the electrode and the protein, but also electron transfer of various interfering molecules. Furthermore, for in vivo applications, mediator immobilization is required to ensure biocompatibility. Enhancing electron transfer from enzyme electrodes using nanomaterials makes a great contribution to the implementation of 3 rd generation biosensors, but the random orientation of the enzyme with respect to the electrode surface results in a large variation in electron transfer efficiency. Attachment of enzyme cofactors to conducting nanoparticles has been shown to be useful for achieving higher catalytic conversion rates than unmodified systems, but this approach is not applicable to enzymes whose cofactors are completely buried within the protein. Therefore, there is a great need for a fully biocompatible and cost-effective method for preparing a widely applicable POCT biosensor.

Disclosure of Invention

To develop an efficient and widely applicable ligation strategy to convert oxidoreductases into electrochemical sensors, we specifically incorporated the unnatural amino acid, 2-amino-3- (4-mercaptophenyl) propionic acid (or p-thiol-phenylalanine, TF), as a unique anchor point for the enzyme into two different amino acid oxidases, glycine oxidase (GlyOx, PDB encodes 1NG4) and L-tryptophan oxidase (TrpOx, PDB encodes 5G 3T). Importantly, TF differs from tyrosine of only one atom, which introduces minimal activity interference to the target enzyme. We then used Bodipy373 as an enzyme/electrode linker that reacted specifically with TF via thiol-chlorine nucleophilic substitution reaction (S-Click reaction). Unlike other biorthogonal reactions, a significant red shift was produced following reaction with TF, which facilitates convenient characterization of the Bodipy 373-labeled oxidoreductase. The modified enzyme may be attached to the surface of a carbon electrode and produce an EET biocatalytic current towards its specific substrate at a potential close to the original redox potential of the enzyme, thereby increasing selectivity. Biosensors made accordingly showed real-time and selective monitoring of tryptophan (Trp) in blood and sweat samples, with a linear range of 0.02-0.8 mM. The use of the sensor for the measurement of various biomolecules can be further extended along the concept of the present invention.

In one aspect, the present application provides an electrochemical sensor comprising an amino acid oxidase incorporating an unnatural amino acid.

Further, the amino acid oxidase is glycine oxidase GlyOx or L-tryptophan oxidase TrpOx.

Further, the amino acid oxidase is glycine oxidase GlyOx, which PDB encodes 1NG4, or L-tryptophan oxidase TrpOx, which PDB encodes 5G 3T.

Further, the unnatural amino acid is 2-amino-3- (4-mercaptophenyl) propionic acid (p-thiol-phenylalanine, TF).

Further, the amino acid oxidases incorporating unnatural amino acids are GlyOx-266TF and TrpOx-395 TF.

Further, the amino acid oxidase incorporating an unnatural amino acid is TrpOx-395 TF.

Further, the electrochemical sensor includes Bodipy373 as a connection linker.

Further, the electrode used in the electrochemical sensor is a carbon-based electrode.

Further, the electrode used in the electrochemical sensor may be selected from a glassy carbon electrode, an HOPG electrode, or a graphene electrode.

Further, the electrode is decorated with a carbon-based nanocomposite material.

Further, the preparation process of the electrochemical sensor comprises the steps of casting the Nafion-MWNTs composite substance on a glassy carbon electrode, drying at room temperature to obtain a Nafion-MWNTs composite membrane modified glassy carbon electrode, then dropwise adding the mixture containing the Bodipy373 modified enzyme mutant to the Nafion-MWNTs composite membrane modified glassy carbon electrode, and standing until the mixture is dried.

Further, the electrochemical sensor was prepared by casting a mixture enzyme mixture including the Bodipy 373-modified enzyme mutant on the surface of the HOPG electrode and drying at room temperature for 20 minutes.

In another aspect, the present application provides a method for the specific real-time detection of amino acids, wherein the electrochemical sensor described above is used.

Further, the real-time detection method is used for detecting amino acids in blood or sweat.

Further, the method detects tryptophan in blood or sweat using an electrochemical sensor comprising TrpOx-395TF-Bodipy 373.

In another aspect, the application provides the application of the electrochemical sensor in the preparation of an amino acid specific real-time detection instrument.

The enzyme usable in the electrochemical sensor of the present application may be any of various enzymes having redox activity according to the detection requirements, and is not limited to glycine oxidase or tryptophan oxidase used in the examples of the present application.

the modifier/linker used in the electrochemical sensors of the present application is not limited to Bodipy373, and any modifier/linker with similar properties known/developed in the art may be used, e.g. based on Bodipy373 having different hydrophilic side chains.

Drawings

FIG. 1 is a diagram of the synthetic pathway for TF;

FIG. 2 is a schematic diagram of the formation of TrpOx-395TF-Bodipy 373: wherein A) shows the structure of TrpOx and the position of TF incorporation, and the distance between TF and FAD; B) is a schematic representation of the attachment of TrpOx from the TF395 site to the surface of carbon nanotubes via a Bodipy373 linker; C) is a schematic diagram of the S-Click reaction (S represents a sulfur atom in TF and Cl represents a chlorine atom in Bodipy 373).

FIG. 3 is a truncated graph of the genetic incorporation of TF into proteins and site-specific modifications using the S-Click reaction. A) SDS-PAGE gel images of Coomassie blue stained GlyOx and TrpOx mutants expressed in the absence or presence of 1mM TF; B) coomassie blue staining (top) and fluorescence images (bottom) of SDS-PAGE gels of TrpOx-395TF and GlyOx-266TF mutants incubated in the presence or absence of Bodipy 373; C) MS spectra are shown for TrpOx-395TF and GlyOx-266 TF.

FIG. 4 shows the results of experiments on the activity of wild-type and mutant TrpOX.

FIG. 5 is a kinetic analysis of the reaction of Bodipy373 with TF and N-acetylcysteine. A) The results were measured as a function of time for the absorption spectra of 27 μ M body 373 after addition of 10mM TF; B) kinetic curve of reaction of Bodipy373 with TF, absorption wavelength: 570nm, Bodipy 373: 27 μ M, TF: 10mM, buffer: HEPES (pH7.4, 200 mM)/5% CH₃And (C) CN. Second order rate constant of 5.6M^-1s^-1. C) Is the absorbance spectrum of 27 μ M body 373 measured as a function of time after addition of 100mM N-acetylcysteine (NAcCys); D) the kinetic profile for Bodipy373 reacted with NAcCys. Absorption wavelength: 570nm, Bodipy 373: 27 μ M, TF: 10mM, buffer: HEPES (pH7.4,200 mM)/5% CH₃And (C) CN. Second order rate constant of 0.0057M^-1.s^-1. TF reacts 1000 times faster with Bodipy373 dye than NAcCys at pH 7.4. Kinetic measurements were performed on an Agilent 8453 uv-vis spectroscopy system.

FIG. 6 is Bodipy373 in the absence of CNTs (grey) or in the presence of CNTs (green); TrpOx-395TF-Bodipy373 showed fluorescence spectra in the absence of CNT (black) or in the presence of CNT (red).

FIG. 7 is an atomic force microscope image and a Cyclic Voltammogram (CV) measured by performing electrochemical Trp. A) An AFM image absorbed on the HOPG surface for TrpOx-395 TF. B) Is an AFM image of TroPOX-395TF-Bodipy373 adsorbed on the HOPG surface. C) Cyclic Voltammogram (CV) of TrpOx-395TF adsorbed on CNT/GCE; D) the Cyclic Voltammogram (CV) of TrpOx-395TF-Bodipy373 adsorbed on CNT/GCE before (red) and after (black) addition of 2mM Trp. CV was performed in PBS buffer (pH7.4), scan rate: 5mV/s

FIG. 8 is a simulation and characterization diagram of bodipy373/CNT interactive molecules. (A, B) chemical structures of pyrene and Bodipy 373. (C, D) top view of pyrene/Bodipy 373 interaction with CNT surface; optimizing interaction graphs of the pyrene-CNT surface and the Bodipy373-CNT surface by a PM6-DH + semi-empirical QM method; (E, F) side view of pyrene/Bodipy 373 interacting with CNT surface. The atomic colors are as follows: carbon atom of CNT: carbon atoms of green, pyrene and Bodipy 373: blue, oxygen: red, nitrogen: purple, boron: pink, fluorine: light green, chlorine: green; hydrogen: white (not shown in full in the grey scale).

FIG. 9 is a graph of an MD simulation of a pyrene/Bodipy and CNT composite system. RMSD (A) and calculated interaction energy (B) of pyrene/Bodipy 373 and CNT complexes during 100ps MD simulation.

FIG. 10 is a graph showing the current response of nafion coating TrpOx-395TF-Bodipy373 CNT/GCE at-0.05V in PBS pH7.4 after the addition of Trp (30 μ M), ascorbic acid (AA, 50 and 100 μ M), uric acid (UA, 100 μ M) and dopamine (DA, 100 μ M) in one portion.

FIG. 11 is the results of real-time measurement of tryptophan concentration, wherein A) is the current response of TrpOx-395TF-Bodipy373/CNT/GCE upon addition of Trp, with a potential of-0.05V applied in PBS buffer pH 7.4; B) the relationship between the steady-state current and the Trp concentration; C) for in vitro measurement of Trp concentration after Trp, Tyr and IDO injection; D) from 48 hours to 54 hours for real-time and on-line Trp concentration measurements during HeLa cell growth. (ii) a Blue dots: blank culture, black spot: HeLa cells in the absence of IFN- γ stimulation, red dots: HeLa cells 24 hours after 50ng/ml IFN-. gamma.stimulation.

FIG. 12 is a graph of the stability results of TrpOx-395TF-Bodipy373 CNT/GCE under operating conditions, A) long term stability over 10 days; B) short term stability over 13 hours. Steady state currents from chronoamperograms to 50 μ MTrp were recorded from the graph, and in the long term stability test, the electrodes were washed with 0.01M PBS (pH7.4) and stored at 4 ℃ after each test; in a short-term test, the electrodes were washed with 0.01M PBS, pH7.4 buffer and mounted above the PBS solution at room temperature after each test.

FIG. 13-detailed NMR chart of the product of each step in the TF synthesis.

FIG. 21 is a CV of various TrpOx mutants with a Bodipy373 linkage on CNT/GCE, A) TrpOx-48TF-Bodipy373/CNT/GCE, B) TrpOx-129TF-Bodipy373/CNT/GCE C) TrpOx-343TF-Bodipy373/CNT/GCE, black curve: in the absence of Trp, red curve: CV was performed in 0.1M PBS buffer (pH7.4) in the presence of 2mM Trp, scan rate: 5mV/s

FIG. 22 is the steady state absorption spectra of TrpOx-395TF before (red curve) and after (green curve) addition of 100. mu. MTrp substrate. 100 μ MTrp solution was used as a control (blue curve). The enzyme concentration was 10. mu.M, and the reaction was carried out in PBS buffer pH 7.40.02M, in a total volume of 100. mu.L per well. B) EPR spectrum of the TrpOx-395TF semiquinone group (red trace) obtained during the reaction with its substrate. The signal was centered at g-2.0046, with a linewidth (peak-to-peak) of 11.2 gauss.

FIG. 23 is A) CV of Bodipy373 modified on CNT/GCE, B) CV of wt TrpOx/Bodipy373 mixture made on CNT/GCE, black curve: in the absence of Trp, red curve: in the presence of 2mM Trp. C) CV of GlyOx 266TF modified on CNT/GCE without Bodipy373 attachment in the absence (black curve) or in the presence (red curve) of 5mM glycine. D) CV of GlyOx-266TF-Bodipy373/CNT/GCE in the absence (black curve) or in the presence (red curve) of 5mM glycine. CV was performed in 0.1M PBS buffer (pH7.4), scan rate: 5 mV/s.

Examples

Main reagents, strains and plasmids:

All chemicals were purchased from Innochem without further purification;

Glassy carbon electrodes were purchased from Tianjin Aida;

The HOPG electrode of the guide substrate is purchased from Nanjing XFNano;

1H and 13C NMR spectra were recorded on a Bruker AMX-500 instrument;

Relative to CDCl₃At 7.26ppm(s) and D₂Residual signal of O at 4.79ppm, reporting chemical shift of 1H NMR; relative to CDCl₃The signal at 77.0ppm reports the chemical shift of 13C NMR.