Application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of the critical quality attribute of spiciness

Abstract

The present invention provides an application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of a key quality attribute of pungent taste. It is characterized in that the specific steps include: (1) construction of a TRPC4 molecular sensory artificial intelligence biosensor; (2) parameter optimization and methodological investigation of a TRPC4 molecular sensory artificial intelligence biosensor; (3) application of a TRPC4 molecular sensory artificial intelligence biosensor in the key quality attribute of pungent taste. The pungent molecular sensory artificial intelligence biosensor constructed by the present invention has a strong affinity with pungent substances, and the available dissociation constant K D The present invention provides a method for constructing and testing a TRPC4 molecular sensory artificial intelligence biosensor for its performance, as well as its application in the critical quality attribute of pungent taste, providing important technical support for the identification of critical quality attributes of pungent taste in traditional Chinese medicine and traditional Chinese medicine compound prescriptions.

Description

Technical Field

[0001] The present invention belongs to the field of biosensors and relates to the application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of key quality attributes of pungent taste. Background Art

[0002] Transient receptor potential channel (TRP) is a type of channel that can activate the cell membrane by changing the membrane potential or intracellular Ca 2+ TRP channels are cation channel proteins that act as signal transducers based on the concentration of cations. TRPs are divided into seven TRP protein families: TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML. TRP channels act as molecular sensors responsible for detecting external and internal environments, participating in various physiological processes such as vision, taste, smell, hearing, touch, pain, and itching, and are associated with the development and progression of diseases such as cardiovascular and tumor diseases.

[0003] TRPCs are non-selective Ca 2+ Channels can be divided into four categories: (1) TRPC1 (2) TRPC2 (3) TRPC3 / 6 / 7 (4) TRPC4 / 5, of which TRPC2 is a pseudogene in humans. TRPC is expressed in tracheal / bronchial smooth muscle cells, lung endothelial cells / epithelial cells, B and T lymphocytes, eosinophils, and mast cells. Among them, TRPC4 ion channels are widely expressed in human tissues and can assemble into homomeric channels by themselves, or assemble into heteromeric channels with TRPC1 and TRPC5. Its wide distribution suggests that it may play an important role in lung diseases. TRPC can activate VGCC of smooth muscle cells and endothelial cells, causing intracellular calcium ions Ca 2+ The concentration increased, and Ca 2+ It is a powerful agonist of various effector cells involved in the pathogenesis of asthma. For example, in smooth muscle cells and endothelial cells, Ca 2+ It can affect the release of vasoactive substances. 2+ dependent manner to concentrate in the lungs, when Ca 2+ During influx, cells degranulate and release various inflammatory mediators. Studies in TRPC4-deficient mice have shown that TRPC4 affects endothelium-dependent regulation of vascular tone, endothelial permeability, and neurotransmitter release from thalamic interneurons. This discovery of the TRPC4 ion channel has important implications for the development of anti-heart failure and antidepressant drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of the key quality attribute of pungent taste.

[0005] To overcome the shortcomings of the existing technology, the present invention first provides a method for constructing a TRPC4 molecular sensory artificial intelligence biosensor, the specific steps of which are as follows:

[0006] Step 1: Add a thiol reagent to the AlGaAs / GaAs HEMT biosensor chip and soak it at room temperature for 17-24 hours to generate Au-S bonds on the surface of the HEMT device and form a self-assembled monolayer;

[0007] Step 2: After the self-assembled monolayer is formed, the excess thiol reagent in (1) is washed away with water, and an equal volume mixture of carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is added to the sample cell and soaked at room temperature for 15-20 minutes to generate a stable amine activation product for activating the carboxyl group;

[0008] Step 3: Wash the HEMT device with phosphate buffer solution (PBS), add TRPC4 pungent taste receptor protein, and place it in a 0-4°C environment for reaction for 2-5 hours to obtain a TRPC4-HEMT biosensor.

[0009] According to some specific embodiments of the present invention, the present invention provides a detection method for a TRPC4 molecular sensory artificial intelligence biosensor, characterized in that the electrochemical workstation used for detection includes but is not limited to 2400, CHI660E devices that provide current and voltage, the given constant voltage is 0-2V, and the current sensitivity can reach the μA level, and the detection method includes but is not limited to linear sweep voltammetry (LSV), cyclic voltammetry (CV), and current-time curve (Amperometric it Curve).

[0010] According to some specific embodiments of the present invention, the present invention provides a parameter optimization process for a TRPC4 molecular sensory artificial intelligence biosensor, as follows:

[0011] The characteristic IT curves of the protein after modification for 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, and 5h were collected using an electrochemical workstation. DS -V DS The curve changes with protein modification time as shown in Figure 1 As shown in (a), it can be seen from the figure that when TRPC4 protein is added for 0.5h, I DS -V DSThe curve remained essentially unchanged. When TRPC4 protein was added for 1 hour, the absolute current offset fluctuated significantly. After 2 hours, the fluctuation range of the absolute current offset stabilized. Considering the loss of protein activity, a 2-hour TRPC4 protein functionalization time was optimal.

[0012] (2) Using the mother solution of L-borneol standard as the carrier, the characteristic IT curve of the TRPC4-HEMT artificial intelligence biosensor after 0-10 minutes of reaction with the mother solution of standard was collected using an electrochemical workstation. The IT curve of the biosensor changes with the sample reaction time as shown in the figure below. Figure 1 As shown in (b), it can be seen from the figure that when the sample is just added and after a period of reaction, I DS -V DS The curve changes significantly; when the sample reacts for 3 minutes, the fluctuation range of the absolute offset value of the current tends to be stable. Therefore, the optimal detection time for the TRPC4 molecular sensor artificial intelligence biosensor to react with the sample should be greater than 3 minutes.

[0013] The pungent substance is the pungent substance in traditional Chinese medicine theory, and the pungent, bitter and slightly cold L-borneol is preferably used as the pungent standard. The preparation method of the L-borneol standard sample solution is as follows: accurately weigh 3.084 mg of L-borneol, add 20 mL of 1% DMSO PBS solution, mix well as the mother solution, dilute 1000 times, and then dilute 6 times according to a 10-fold concentration gradient to prepare 7 samples as test samples.

[0014] The electrochemical workstation includes but is not limited to 2400 and CHI660E devices that provide current and voltage, with a given constant voltage of 0-2V and a current sensitivity of up to μA level. The scanning method is linear sweep voltammetry (LSV), and the collected signals include: current-voltage curve (Amperometric iv Curve) and current-time curve (Amperometric it Curve).

[0015] The present invention further provides an application of a TRPC4 molecular sensory artificial intelligence biosensor in identifying the key quality attribute of pungent taste, characterized in that the specific construction steps are as follows:

[0016] Step 1: Prepare a TRPC4-HEMT biosensor. Standard solutions (ferulic acid, menthol, L-borneol, gingerol, and musk ketone) with concentrations ranging from low (1 pmol / L) to high (1 mmol / L) were added sequentially. The current intensity between the source and drain electrodes was recorded using an electrochemical device.

[0017] Step 2: Take the logarithm of the concentration of the test compound (Lg[A g]) as the abscissa and the relative value of current change ((I-I0) / I0) as the ordinate, a linear fit was performed to determine the concentration range of the TRPC4-HEMT biosensor for detecting the corresponding test compound;

[0018] Step 3: The concentration of the compound to be tested ([A g ]) as the horizontal axis, with the concentration range determined in step 2 ([A g ]) / current change I-I0,ΔI) is used as the vertical coordinate, and linear fitting is performed to determine whether the compound interacts with TRPC4 and the strength of the interaction (dissociation constant) according to formula (1-3), thereby screening the key quality attributes of pungent taste.

[0019]

[0020]

[0021]

[0022] Among them [A b ] is the concentration of TRPC4, [A g ] is the concentration of the test compound [C], K and K A is the binding constant, K D is the dissociation constant, ΔI is the current change value, ΔI max is the maximum current change.

[0023] In summary, the present invention provides an application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of the key quality attribute of pungent taste. The method of the present invention has the following advantages: the present invention constructs a TRPC4-HEMT artificial intelligence biosensor, which, through performance optimization, can be used to identify the key quality attribute of pungent taste in traditional Chinese medicine and traditional Chinese medicine compound, with high sensitivity (sensitivity can reach μA level), strong specificity (clear response to the key quality attribute of pungent taste), detection concentration as low as 1pmol / L, detection range of 1pmol / L-10μmol / L, and good linear range (R 2 The coefficient can reach 0.9) and other advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (a) Curve graph of TRPC4 protein at different modification times; (b) Changes in biosensor current of ferulic acid standard solution at different reaction times.

[0025] Figure 2 (a1)-(e1) I of ferulic acid, menthol, L-borneol, gingerol, and musk ketone standards at different concentrations DS -V DSCurve changes; (a2)-(e2) Logarithm of the concentration of standard solutions of ferulic acid, menthol, L-borneol, gingerol and musk ketone (Lg[A g ]) and the linear fitting relationship between the relative value of current change ((I-I0) / I0); (a3)-(e3) Concentrations of standard solutions of ferulic acid, menthol, L-borneol, gingerol and musk ketone ([A g ]) and the linear fitting relationship between concentration / current change (I-I0, ΔI). DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific examples and accompanying drawings. Where specific conditions are not specified in the examples, conventional conditions were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0027] Example 1: A method for constructing a TRPC4 molecular sensory artificial intelligence biosensor

[0028] (1) Adding a thiol reagent to an AlGaAs / GaAs HEMT biosensor chip and soaking it at room temperature for 20 hours to generate Au-S bonds on the surface of the HEMT device and form a self-assembled monolayer;

[0029] (2) After the self-assembled monolayer is formed, the excess thiol reagent in (1) is washed away with water, and an equal volume mixture of carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is added to the sample cell and soaked at room temperature for 15 minutes to generate a stable amine activation product for activating the carboxyl group;

[0030] (3) Accurately measure 10 μg of TRPC4 protein at a concentration of 1 mg / mL, add the purified TRPC4 buffer solution to 100 μL, and mix thoroughly to obtain the TRPC4 pungent taste receptor protein solution.

[0031] (4) The HEMT device was washed with phosphate buffer solution (PBS), and a TRPC4 pungent taste receptor protein solution was added. The device was placed at 4°C for reaction for 2.5 hours to obtain a TRPC4-HEMT biosensor.

[0032] Example 2: Parameter Optimization Method for a TRPC4 Molecular Sensory Artificial Intelligence Biosensor

[0033] Parameter optimization method for TRPC4 molecular sensory artificial intelligence biosensor, including but not limited to: protein modification time (h) and sample reaction time (min).

[0034] (1) Optimization of TRPC4 protein modification time

[0035] The TRPC4 pungent taste receptor protein was added to the carboxyl-activated AlGaAs / GaAs HEMT biosensor chip for modification and expression. The characteristic IT curves after 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, and 5h of protein modification were collected using a CHI660E electrochemical workstation. The results are shown in Figure 2. Figure 1 As shown in (a), biosensor I DS -V DS The curve changes with protein modification time as shown in Figure 1 As shown in Figure (a), when TRPC4 pungent protein was added for 0.5h, I DS -V DS The curve remained essentially unchanged. When TRPC4 pungent protein was added for 1 hour, the absolute current offset fluctuated significantly. After 2 hours, the fluctuation range of the absolute current offset stabilized. Therefore, further optimization of the TRPC4 pungent taste receptor protein functionalization time in the biosensor should be greater than 2 hours.

[0036] (2) Optimization of sample addition time

[0037] Accurately weigh 3.084 mg of L-borneol, add 20 mL of 1% DMSO in PBS solution, mix well as the mother solution, dilute 1000 times, and dilute 6 times in a 10-fold concentration gradient to prepare 7 samples as L-borneol standard solution. Using the L-borneol standard mother solution as the carrier, the CHI660E electrochemical workstation was used to collect the characteristic IT curve of the TRPC4-HEMT artificial intelligence biosensor after 0-10 minutes of reaction with the standard mother solution. The IT curve of the biosensor changes with the sample reaction time as shown in the figure below. Figure 1 As shown in (b), the fluctuation range of the absolute offset value of the current tends to be stable when the sample reacts for 3 minutes. Therefore, the optimal detection time for the TRPC4 molecular sensor artificial intelligence biosensor to react with the sample should be greater than 3 minutes.

[0038] According to the above results, the modification time of TRPC4 pungent protein is 2h and the sample reaction time is 3min.

[0039] Example 3: Application of a TRPC4 molecular sensory artificial intelligence biosensor in identifying the key quality attribute of pungent taste (1) Sample solution preparation

[0040] Preparation of ferulic acid solution: Accurately weigh 0.97 mg of ferulic acid standard (molecular weight 194.18) into a beaker and add an appropriate amount of 5% DMSO aqueous solution to dissolve it; the solution is injected into a 5 mL volumetric flask along a glass rod, the beaker is washed 2-3 times with 5% DMSO aqueous solution, and the solution is poured into the volumetric flask and shaken for initial mixing; 5% DMSO aqueous solution is added to the volume until the concave liquid surface is parallel to the scale line, the bottle is stoppered, and the solution is inverted and shaken to obtain a ferulic acid standard solution with a concentration of approximately 1.0 mmol / L; after diluting 1000 times, the solution is diluted 6 times in a 10-fold concentration gradient to obtain a ferulic acid standard solution ranging from 1.0 μmol / L to 1.0 pmol / L, which is then set aside.

[0041] Preparation of musk ketone solution: Accurately weigh 1.19 mg of musk ketone standard (molecular weight 238.42) into a beaker and add an appropriate amount of 5% DMSO aqueous solution to dissolve it; the solution is poured into a 5 mL volumetric flask along a glass rod, the beaker is washed 2-3 times with 5% DMSO aqueous solution, and the solution is poured into the volumetric flask and shaken for initial mixing; 5% DMSO aqueous solution is added to the volume until the concave liquid surface is parallel to the scale line, the bottle is stoppered, and the solution is inverted and shaken to obtain a musk ketone standard solution with a concentration of approximately 1.0 mmol / L; after diluting 1000 times, the solution is diluted 6 times in a 10-fold concentration gradient to obtain a musk ketone standard solution ranging from 1.0 μmol / L to 1.0 pmol / L, which is set aside.

[0042] Preparation of menthol solution: Accurately weigh 0.78 mg of menthol standard (molecular weight 156.26) into a beaker and dissolve it in an appropriate amount of 5% DMSO aqueous solution. Pour the solution into a 5 mL volumetric flask along a glass rod. Wash the beaker 2-3 times with 5% DMSO aqueous solution and pour it into the volumetric flask, shake it, and initially mix it. Add 5% DMSO aqueous solution to the volume until the concave meniscus is aligned with the scale line. Secure the bottle with the stopper, invert it, and shake it to obtain a menthol standard solution with a concentration of approximately 1.0 mmol / L. After diluting it 1000-fold, dilute it 6 times in a 10-fold concentration gradient to obtain a menthol standard solution ranging from 1.0 μmol / L to 1.0 pmol / L, which is then set aside.

[0043] Preparation of borneol solution: accurately weigh 0.77 mg of borneol standard (molecular weight 154.25) in a beaker, add an appropriate amount of 5% DMSO aqueous solution to dissolve it; the solution is injected into a 5 mL volumetric flask along a glass rod, the beaker is washed 2-3 times with a 5% DMSO aqueous solution, and poured into a volumetric flask, shaken, and initially mixed; 5% DMSO aqueous solution is added to make the volume until the concave liquid surface is aligned with the scale line, the bottle is covered with a stopper, turned upside down, and shaken to obtain a borneol standard solution with a concentration of about 1.0 mmol / L; after dilution 1000 times, it is diluted 6 times according to a 10-fold concentration gradient to obtain a borneol standard solution of 1.0 μmol / L to 1.0 pmol / L, and set aside.

[0044] Preparation of 6-gingerol solution: Accurately weigh 1.47 mg of 6-gingerol standard (molecular weight 294.39) into a beaker and dissolve it in an appropriate amount of 5% DMSO aqueous solution; the solution is poured into a 5 mL volumetric flask along a glass rod, the beaker is washed 2-3 times with 5% DMSO aqueous solution, and the solution is poured into the volumetric flask and shaken for initial mixing; 5% DMSO aqueous solution is added to the volume until the concave meniscus is aligned with the scale line, the bottle is stoppered, and the solution is inverted and shaken to obtain a 6-gingerol standard solution with a concentration of approximately 1.0 mmol / L; after diluting 1000-fold, the solution is diluted 6 times in a 10-fold concentration gradient to obtain a 6-gingerol standard solution ranging from 1.0 μmol / L to 1.0 pmol / L, and set aside.

[0045] (2) Sample testing

[0046] 5% DMSO aqueous solution was selected as blank control, and each sample solution was added to the TRPC4-HEMT biosensor from low concentration to high concentration, and 60 μL was added to the sensor. After reacting at 4 °C for 3 minutes, the detection was started and the collected I DS -V DS Curve and analyze.

[0047] (3) Binding strength of the key quality attribute of pungent taste and TRPC4 protein

[0048] The current-voltage curves collected in step (2) were integrated to calculate the binding strength of each chemical substance with TRPC4 protein using formula (1)(2)(3), using K D The calculation formula is as follows:

[0049]

[0050]

[0051]

[0052] Among them [A b ] is the concentration of TRPC4 protein, [A g ] is the concentration of the test compound [C], K and K A is the binding constant, K D is the dissociation constant, ΔI is the current change value, ΔI max is the maximum current change.

[0053] like Figure 2 As shown in (a1), the current response signal gradually decreases as the concentration of ferulic acid decreases. g ]) is the horizontal axis and I DSThe linear fitting results between the relative values ​​of change (I-I0) / I0 are as follows Figure 2 As shown in (a2), a good linear relationship was observed in the range of 1.0 pmol / L–10 nmol / L, and the linear equation was y = -0.005x-0.0064, R 2 =0.8636; further, the concentration of ferulic acid ([A g ]) and its corresponding concentration / current change (I-I0, ΔI) also show a good linear relationship, and the linear equation obtained by fitting is y=7×10 6 x+0.0004,R 2 =0.9998. The dissociation constant K of the interaction between ferulic acid and TRPC4 protein was calculated according to the formula D =3.5×10 -10 mol / L, the results showed that ferulic acid had a strong interaction with TRPC4 protein.

[0054] Similarly, the above formulas (1-3) were used to calculate the dissociation constant of the interaction between menthol and TRPC4 protein, which was 6.1×10 -9 mol / L; the dissociation constant of the interaction between L-borneol and TRPC4 protein is 2.1×10 -10 mol / L; the dissociation constant of the interaction between 6-gingerol and TRPC4 protein is 5.175×10 -9 mol / L; the dissociation constant of the interaction between musk ketone and TRPC4 protein is 3.82×10 -9 mol / L.

[0055] According to the survey results analysis, K D The value is 2×10 -2 -100 mol / L, indicating that the binding force is very weak or even non-existent; in the range of 10 -3 -10 -2 mol / L range, indicating that the binding force is weak; in the range of 10 -5 -10 -3 mol / L range, indicating that the binding force is in an intermediate state; in the range of 10 -8 -10 -5 mol / L range, indicating that the binding force is very strong; in the range of 10 -13 -10 -8 mol / L range, indicating that the binding force is very strong.

[0056] The dissociation constants K of the interactions between musk ketone, ferulic acid, menthol, borneol, and 6-gingerol and TRPC4 protein D The values ​​are all 10 -8 ~10 -13mol / L range, indicating that musk ketone, ferulic acid, menthol, borneol, and 6-gingerol all have strong binding forces with TRPC4 protein, which indicates that the constructed TRPC4 molecular sensory artificial intelligence biosensor has a good application effect in identifying the key quality attributes of pungent taste.

[0057] Finally, it should be noted that those skilled in the art will understand that the embodiments described above are only some of the embodiments of the present invention, not all of them, and are intended to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, all other embodiments derived by those skilled in the art without inventive effort are also within the scope of protection of the present invention.

Claims

1. Application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of pungent key quality attributes, characterized in that: The specific steps are as follows: Step 1: Expression and purification of TRPC4 protein; Step 2: The purified TRPC4 protein was coupled to a biosensor device to construct a TRPC4 sensory artificial intelligence biosensor, and its parameters were optimized and its performance was investigated; Step 3: Using the molecular sensory artificial intelligence biosensor constructed in step 2, the pungent key quality attribute is identified through the affinity interaction between the analyte and the pungent taste receptor TRPC4 at gradient concentrations. The key quality attributes of pungent taste are musk ketone, ferulic acid, menthol, L-borneol and 6-gingerol.

2. The application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of pungent key quality attributes according to claim 1, characterized in that: Artificial intelligence biosensors include field-effect transistors, high electron mobility field-effect transistors, surface plasmon resonance, quartz crystal microbalance sensors, and microfluidic chip biosensors.

3. The application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of pungent key quality attributes according to claim 1, characterized in that: The biosensor parameters in step 2 include protein modification time and sample addition time, wherein the protein modification time is 2-5 hours and the sample addition time is 3-8 minutes.

4. The application of a TRPC4 molecular sensory artificial intelligence biosensor in the identification of pungent key quality attributes according to claim 1, characterized in that: The key quality attribute of pungent taste is the specific binding of TRPC4 protein, that is, the affinity between the sample and the protein is essentially a non-covalent force, and the magnitude of the affinity is expressed by the dissociation constant K. d Value representation.

Images