Preparation method of cyclic adenosine monophosphate molecular imprinting electrochemical sensor

Abstract

This invention discloses a method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP). The sensor uses reduced graphene oxide (RGO) and gold nanoparticles as modifying materials. RGO is drop-coated onto the surface of a glassy carbon electrode and dried to obtain a modified electrode RGO / GCE. The RGO / GCE is then placed in a sulfuric acid deposition solution containing chloroauric acid, and gold nanoparticles are used to modify the RGO / GCE surface using a potentiostatic method to form a gold substrate, resulting in a deposited electrode AuNPs / RGO / GCE. The AuNPs / RGO / GCE is then electropolymerized in an electrolyte polymerization solution containing the template substance cAMP and the functional monomer o-phenylenediamine to obtain a dense and insulating polymer film. The polymerized electrode is immersed in an elution solution and eluted using cyclic voltammetry to obtain MIP / AuNPs / RGO / GCE. This sensor exhibits specific recognition selectivity for cAMP, high detection sensitivity, low detection limit, good reusability, and stability.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance detection technology, and relates to a method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP). Background Technology

[0002] Cyclic adenosine-3',5'-monophosphate (cAMP) was first discovered by Sutherland in 1971 and was the first intracellular second messenger to be found. It is widely distributed in animal, plant, and microbial cells. cAMP is a white or off-white powder with a relative molecular mass of 329.21, a melting point close to 220°C, a near-neutral pH, is slightly soluble in water, and insoluble in organic solvents. Its chemical formula is C6H5O6. 10 H 12 N5O6P is composed of one molecule of adenine, one molecule of ribose, and one molecule of phosphate group. cAMP is a ubiquitous bioactive component in the biological world, widely regulating physiological and biochemical processes in organisms. At least 40 human diseases (including major diseases such as myocardial infarction, coronary heart disease, cardiogenic shock, and hypertension) are related to the metabolic regulation of cAMP. Applications such as the deep processing of functional foods, health care, and high-purity extraction for medical use based on cAMP have high requirements for real-time and rapid quantitative detection of cAMP. Therefore, the detection and development of cyclic adenosine monophosphate (cAMP) has a promising future.

[0003] Currently, my country lacks national standards for cAMP detection, only having enterprise standards, all of which employ high-performance liquid chromatography (HPLC). Literature primarily utilizes HPLC, protein binding assays, thin-layer chromatography (TLC), and enzyme-linked immunosorbent assays (ELISA). Among these, HPLC suffers from drawbacks such as expensive equipment, complex and time-consuming pretreatment, and difficulty in achieving rapid detection; protein binding assays are relatively complex to operate; TLC requires a certain concentration of the analyte to be used; and ELISA methods are susceptible to acid and alkali instability, easy inactivation, and the potential for false positives.

[0004] Molecular imprinting is a technique for the artificial synthesis of polymers that specifically bind to template molecules, characterized by high efficiency and selectivity. It involves preparing molecularly imprinted polymers with binding sites that match the template molecules in structure, shape, size, and functional groups. Molecularly imprinted electrochemical sensors, using these polymers as recognition elements, offer advantages such as high specificity, high sensitivity, simple operation, and ease of miniaturization. However, its application in the detection of cAMP in jujube peel is limited; to date, this technique is primarily used for the determination of substances in pharmaceuticals, the environment, and biological agents.

[0005] Therefore, there is a need to develop a simple, specific, and sensitive cyclic adenosine monophosphate (cAMP) detection technology to meet the practical needs of detecting jujube peel and other samples. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to provide a method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP). This method uses cAMP as a template material and o-phenylenediamine as a functional monomer, and sequentially proceeds through electrode modification, polymer preparation, and elution steps to prepare the molecularly imprinted electrochemical sensor. The preparation process of this invention is simple and low-cost, and the resulting sensor has advantages such as high selectivity and high sensitivity, and can be used to detect the cAMP content in a sample.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP) comprises the following steps in sequence:

[0009] (1) Preparation of RGO / GCE

[0010] Reduced graphene oxide was added to dimethylformamide, and after stirring and sonication, a light black dispersion was obtained, denoted as A. Chitosan was added to a 1% acetic acid solution, and after magnetic stirring and sonication, the resulting solution was denoted as B. Under stirring, B was added dropwise to A, and after the two were mixed, a reduced graphene oxide modifier was obtained.

[0011] 5 μL of reduced graphene oxide modifier was drop-coated onto the surface of a glassy carbon electrode and dried to obtain the modified electrode RGO / GCE.

[0012] (2) Preparation of AuNPs / RGO / GCE

[0013] RGO / GCE was placed in a sulfuric acid deposition solution containing chloroauric acid and deposited for 100-500 s at a constant potential of (-0.5)-(-0.1) V for 100-500 s to modify the surface of RGO / GCE with gold nanoparticles to form a gold substrate, thus obtaining the deposited electrode AuNPs / RGO / GCE.

[0014] (3) Preparation of MIP / AuNPs / RGO / GCE

[0015] AuNPs / RGO / GCE were electropolymerized in an electrolyte polymerization solution containing the template substance cyclic adenosine monophosphate and the functional monomer o-phenylenediamine. The initial polymerization potential was 0.1-0.5V, the termination potential was 0.8-1.6V, and the polymerization was carried out for 20 cycles to obtain a dense and insulating polymer film.

[0016] The polymerized electrode was immersed in the elution solution and eluted using cyclic voltammetry for 22-26 cycles to obtain MIP / AuNPs / RGO / GCE.

[0017] As a limitation of the preparation method of the present invention, in step (1), the mass-to-volume ratio of the reduced graphene oxide to dimethylformamide is (0.5-2.5):1g / mL.

[0018] As another limitation of the preparation method of the present invention, in step (1), the mass-volume ratio of chitosan to acetic acid solution is (2.5-12.5):1g / mL.

[0019] As a third limitation of the preparation method of the present invention, in step (1), the volume ratio of A to B is 1:1.

[0020] As a fourth limitation of the preparation method of the present invention, in step (2), the mass concentration of chloroauric acid in the deposition solution is 0.2-1.0 g / L, the molar concentration of sulfuric acid is 0.034-0.167 mol / L, and the volume ratio between the two is 1:1.

[0021] As a fifth limitation of the preparation method of the present invention, in step (3), the molar ratio of the template material to the functional monomer is 1:(1-5).

[0022] As a sixth limitation of the preparation method of the present invention, in step (3), the electrolyte is PBS, and the molar ratio of the template substance to the electrolyte is 1:(2-6).

[0023] As a seventh limitation of the preparation method of the present invention, in step (3), the elution solution is H2SO4 with a concentration of 0.2-0.6 mol / L.

[0024] like Figure 1 The diagram shows the NBO charge structure of each atom, and the cyclic adenosine monophosphate (cAMP). Figure 1 In Figure A, the NBO charges of the two O atoms on the two hydroxyl groups, one oxygen atom, and the N atom on the amino group are O5 (-0.762), O6 (-0.946), O7 (-0.920), and N12 (-0.798), respectively. It is clear that all four atoms have strong electronegativity and readily accept H protons to form hydrogen bonds. Furthermore, the NBO charges of the four H atoms in the three groups of cyclic adenosine monophosphate are H29 (0.503), H31 (0.526), ​​H33 (0.433), and H34 (0.436), respectively. The more electronegative the H atom, the more readily it forms hydrogen bonds with the more electronegative atoms. The selected functional monomer, o-phenylenediamine (… Figure 1The NBO charges of the two amino groups on the N atom in Figure B are N4 (-0.874) and N5 (-0.874), and the NBO charges of the four H atoms are H10 (0.408), H11 (0.407), H13 (0.408), and H15 (0.407). The results indicate that o-phenylenediamine can act as both a proton donor and a proton acceptor to form hydrogen bonds with cyclic adenosine monophosphate (cAMP). The strength of the interaction between the template molecule and the functional monomer complex depends not only on the charge distribution of the functional groups but also on the spatial structure of the molecule and the steric hindrance of the binding site atoms. Binding sites with high steric hindrance, even with high charge density, are less likely to form hydrogen bonds due to the steric hindrance.

[0025] The above-mentioned technical solution of the present invention is a whole in which each step is closely related and mutually influential, and together they determine the morphological characteristics and performance of the product.

[0026] The above technical solution has the following advantages or beneficial effects:

[0027] 1. The preparation method of this invention is simple, the process is easy to control, and it is easy to promote and apply in industrial applications.

[0028] 2. The sensor prepared by this invention has specific recognition selectivity for cyclic adenosine monophosphate, high detection sensitivity, low detection limit, good reusability and stability.

[0029] This invention is applicable to the detection of cyclic adenosine monophosphate (cAMP) content in jujube peel and other samples. Attached Figure Description

[0030] Figure 1 The diagrams show the NBO charge structure of each atom in cyclic adenosine monophosphate (cAMP) and o-phenylenediamine, where: A is the NBO charge structure of each atom in cAMP, and B is the NBO charge structure of each atom in o-phenylenediamine.

[0031] Figure 2 The diagrams show the configurations of cyclic adenosine monophosphate (cAMP) complexes with different functional monomers in different proportions. Specifically: A is the configuration of the complex of cAMP and o-aminophenol; B is the configuration of the complex of cAMP and resorcinol; C is the configuration of the complex of cAMP and o-phenylenediamine; and D is the configuration of the complex of cAMP and acrylamide.

[0032] Figure 3 The figures show the electrochemical characterization of different electrodes using different electrochemical methods. In the figure, A is the CV characterization, B is the SWV characterization, and C is the EIS characterization.

[0033] Figure 4 This is a graph showing the selectivity analysis of the template molecules for the sensor.

[0034] Figure 5 The graphs show the adsorption of cyclic adenosine monophosphate (cAMP) standard solutions of different concentrations by the sensor. In the graphs, A is the peak current curve of adsorption of cAMP of different concentrations, and B is the linear relationship between the peak current value and cAMP of different concentrations.

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1

[0039] This embodiment prepares a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP) by following the steps in sequence:

[0040] (1) Preparation of RGO / GCE

[0041] 0.005 g of reduced graphene oxide was added to 10 mL of dimethylformamide. After stirring and sonication, the resulting light black dispersion was denoted as A. 0.025 g of chitosan was added to 10 mL of 1% acetic acid solution. After magnetic stirring and sonication, the resulting solution was denoted as B. Under stirring, 5 mL of B was added dropwise to 5 mL of A. After mixing, the reduced graphene oxide modifier was obtained.

[0042] 5 μL of reduced graphene oxide modifier was drop-coated onto the surface of a glassy carbon electrode and dried to obtain the modified electrode RGO / GCE.

[0043] (2) Preparation of AuNPs / RGO / GCE

[0044] RGO / GCE was placed in a 0.034 mol / L sulfuric acid deposition solution containing 0.2 g / L chloroauric acid (the volume ratio of chloroauric acid solution to sulfuric acid solution was 1:1), and gold nanoparticles were deposited on the surface of RGO / GCE for 300 s at a constant potential of -0.3 V to form a gold substrate, thus obtaining the deposited electrode AuNPs / RGO / GCE.

[0045] (3)MIP / AuNPs / RGO / GCE

[0046] AuNPs / RGO / GCE were electropolymerized in a PBS polymerization solution containing the template substance cyclic adenosine monophosphate and the functional monomer o-phenylenediamine (molar ratio 1:4) (molar ratio of cyclic adenosine monophosphate to PBS polymerization solution 1:2). The initial polymerization potential was 0.2V, the termination potential was 1.0V, and the polymerization was carried out for 20 cycles to obtain a dense and insulating polymer film.

[0047] The polymerized electrode was immersed in 0.4 mol / L H2SO4 and eluted using cyclic voltammetry for 24 cycles to obtain MIP / AuNPs / RGO / GCE.

[0048] The preparation method in this embodiment is simple and the process is easy to operate.

[0049] Examples 2-5

[0050] Examples 2-5 respectively prepared an electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP). The preparation process was the same as in Example 1, except that the corresponding technical parameters were different. The specific parameters are shown in the table below:

[0051]

[0052] The sensors prepared in Examples 2-5 are fast, sensitive, and accurate in detecting cyclic adenosine monophosphate (cAMP), with mild reaction conditions and simple preparation process.

[0053] Test case

[0054] Experimental Example 1: Selection of Functional Monomers and Determination of the Ratio of Template Molecule to Functional Monomer

[0055] This experimental example screens the selection of functional monomers and the ratio of template material to functional monomers during the polymerization process, and tests the ratio of cyclic adenosine monophosphate to four functional monomers (o-aminophenol, resorcinol, o-phenylenediamine, and acrylamide).

[0056] Based on the identified potential binding sites, complex configurations with different proportions were constructed. The B3LYP / 6-311+G(d,p) algorithm was used to optimize the complex models formed by cyclic adenosine monophosphate (cAMP) and four selected functional monomers in different proportions, and vibrational frequencies were calculated to obtain thermodynamic data. The spatial structure, bond lengths, interaction sites, and binding energies of the stable configurations of the complexes with different proportions were analyzed to investigate the interaction strength between the template molecule and the four functional monomers. Based on the principle that a higher number of hydrogen bonds and interaction sites with lower binding energies result in a more stable complex structure, the optimal functional monomers were screened, and the optimal ratio of the complexes was optimized.

[0057] like Figure 2As shown, the stable complex configurations of cyclic adenosine monophosphate (cAMP) with four functional monomers—o-aminophenol, resorcinol, o-phenylenediamine, and acrylamide—in ratios of (1:1 to 1:4) are illustrated, with the interaction sites and bond lengths marked in the figure. The figure shows that the template molecule cAMP and the four functional monomers are primarily linked through hydrogen bonds, and the hydrogen bond lengths all conform to the specified lengths. Figure 2 It can be seen that, by comparing the binding energies, the order of the binding energies (absolute values) of the four functional monomers o-aminophenol, resorcinol, o-phenylenediamine, and acrylamide with cyclic adenosine monophosphate (cAMP) in different proportions is o-phenylenediamine > resorcinol > o-aminophenol > acrylamide. Among them, o-phenylenediamine has the smallest binding energy with cAMP, but the largest absolute value, and the reaction releases the most heat. Therefore, when cAMP is bound to o-phenylenediamine for imprinting, the energy of the complex system is lower, and the interaction between the template molecule and the functional monomer is stronger. Thus, o-phenylenediamine is ultimately selected as the best functional monomer.

[0058] like Figure 2 As shown in Figure C, with the increasing proportion of the imprinted material, the amount of o-phenylenediamine in the complex also increases, leading to a corresponding increase in the released heat. The heat released during the formation of the cyclic adenosine monophosphate-4 o-phenylenediamine complex is 93.74 kJ·mol⁻¹ higher than that released during the formation of the cyclic adenosine monophosphate-3 o-phenylenediamine complex. -1 The heat released during the formation of the cyclic adenosine monophosphate-5 (cAMP-5) o-phenylenediamine complex increased by only 44.29 kJ·mol compared to the heat released during the formation of the cyclic adenosine monophosphate-4 (cAMP-4) o-phenylenediamine complex. -1 The increase is much smaller than before, indicating that at this point, the ability of o-phenylenediamine to bind to cyclic adenosine monophosphate (cAMP) has reached saturation. Both active sites are fully occupied, and the released heat reaches its maximum. Further increasing the amount of o-phenylenediamine would only lead to the formation of unnecessary non-specific interactions, reducing the effective binding sites. Therefore, the interaction between cAMP and o-phenylenediamine is strongest and the resulting complex exhibits optimal stability when the ratio is 1:4.

[0059] Experimental Example 2: Electrochemical Characterization of Different Electrodes

[0060] Three electrochemical methods, CV, SWV, and EIS, were used in a solution containing 0.1 mol·L⁻¹ -1 KCl and 5.0 mmol·L -1 0.2 mol·L⁻¹ of K₃[Fe(CN)₆] -1 The electrochemical behavior of different electrodes in PBS solution was characterized. For example... Figure 3As shown in Figure A, the electrochemical behavior of different electrodes was characterized using CV. The CV curve of the bare electrode shows symmetrical and reversible redox peaks (curve a). After modification with RGO (curve b), the redox peak current increases. This is because the large specific surface area and high electroactivity of RGO facilitate the diffusion of probe ions on the electrode surface, improving the catalytic activity of the electrode. Further deposition of AuNPs on the RGO / GCE surface (curve c) significantly increases the redox peak current, far exceeding the response peak current after RGO modification. This is because AuNP modification further improves the electrode's conductivity and surface area, promoting electron transfer; and the synergistic effect of RGO and AuNPs significantly enhances electrode sensitivity. After electropolymerization to form a polymer film (curve d), the redox peak current essentially disappears, indicating that the polymer film has been successfully prepared and hinders probe ion transfer. After the template molecules are eluted, a significant current response reappears (curve e). This is because a large number of imprinted holes are formed on the molecularly imprinted film after elution, allowing electron transfer to occur on the electrode surface. After re-adsorption of cyclic adenosine monophosphate (cAMP), the peak current of the probe ion decreases (curve f), indicating that the template molecule occupies part of the imprinted holes, thus hindering electron transfer. Furthermore, Figure 3 B represents the SWV characterization curves for different electrodes, from... Figure 3 As can be seen in B, the current changes in CV and SWV of different electrodes are consistent, further proving that the electrode modification effect and imprinting effect are good.

[0061] Electrochemical inductively coupled plasma (EIS) is an electrochemical method that monitors changes in interfacial properties during the assembly of modified electrodes by measuring electron transfer capabilities. At high frequencies, a larger semicircle diameter corresponds to a greater interfacial impedance. For example... Figure 3 As shown in Figure C, the interfacial impedance of GCE (curve a) further decreases after modification with RGO and AuNPs (curve b), indicating that GCE has poor conductivity, while RGO and AuNPs improve the electrode's conductivity. When a nearly insulating polymer film is formed on the AuNPs / RGO / GCE electrode surface through electropolymerization, its impedance increases sharply (curve c). After eluting the template molecules, a large number of imprinted holes are formed on the imprinted film, allowing probe ions to transfer electrons on the electrode surface, thus significantly reducing the impedance again (curve d). After re-adsorption of cyclic adenosine monophosphate (cAMP), the template molecules occupy some of the imprinted holes, hindering electron transfer, and the electron transfer impedance increases significantly again (curve e). The results of the three electrochemical characterization methods are consistent, indicating that a cAMP-molecularly imprinted electrochemical sensor has been successfully prepared.

[0062] Selectivity analysis of Experimental Example 3

[0063] This experimental example uses 5'-adenosine and 5'-adenosine triphosphate, which have structures similar to the template molecule cyclic adenosine monophosphate, as structural analogs, and selects miglitol, L-ascorbic acid, and oxytetracycline, which have significantly different structures and are water-soluble, as interfering substances to study the selectivity of the sensor. SWV characterization of the molecularly imprinted electrochemical sensor at the same concentration of 1.0 × 10⁻⁶... -4 mol·L -1 Under certain conditions, the changes in peak current before and after adsorption in standard solutions of cyclic adenosine monophosphate, 5'-adenosine monophosphate, 5'-adenosine triphosphate, miglitol, L-ascorbic acid, and oxytetracycline were investigated. For example... Figure 4 As shown in the figure, ΔI indicates that the molecularly imprinted electrochemical sensor has the strongest adsorption capacity for cyclic adenosine monophosphate (cAMP), but its adsorption capacity for structural analogs decreases, and its adsorption capacity for interfering substances is poor. The adsorption process is mainly driven by physical adsorption, and the results show that this sensor has good selectivity.

[0064] Experimental Example 4: Linearity and Detection Limit

[0065] This experimental example uses SWV to determine the adsorption of different concentrations of cyclic adenosine monophosphate standard solutions (1×10⁻⁶) by a molecularly imprinted electrochemical sensor. -8 mol·L -1 ~1×10 -4 mol·L -1 Peak current value () Figure 5 A), the corresponding peak current values ​​are respectively related to the cyclic adenosine monophosphate concentration at 1.0 × 10⁻⁶. -8 mol·L -1 ~1.0×10 -4 mol·L -1 Linear relationship within the range ( Figure 5 B) A standard curve was plotted with -lgC corresponding to different concentrations of cyclic adenosine monophosphate (cAMP) as the abscissa and the difference in peak current (ΔI) before and after adsorption as the ordinate. The detection limit of the template molecule was determined based on S / N = 3, and the results are as follows: Figure 5 As shown, the linear equations are ΔI=4.264lgC+36.574(R) 2 =0.9952), the detection limit is 2.46×10 -9 mol·L -1 (S / N = 3).

[0066] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP), characterized in that, Follow these steps in sequence: (1) Preparation of RGO / GCE Reduced graphene oxide was added to dimethylformamide, and after stirring and sonication, a light black dispersion was obtained, denoted as A. Chitosan was added to a 1% acetic acid solution, and after magnetic stirring and sonication, the resulting solution was denoted as B. Under stirring, B was added dropwise to A, and after the two were mixed, a reduced graphene oxide modifier was obtained. 5 μL of reduced graphene oxide modifier was drop-coated onto the surface of a glassy carbon electrode and dried to obtain the modified electrode RGO / GCE. (2) Preparation of AuNPs / RGO / GCE RGO / GCE was placed in a sulfuric acid deposition solution containing chloroauric acid, wherein the mass concentration of chloroauric acid in the deposition solution was 0.2-1.0 g / L, the molar concentration of sulfuric acid was 0.034-0.167 mol / L, and the volume ratio between the two was 1:

1. A constant potential of (-0.5)-(-0.1) V was used for deposition for 100-500 s to modify gold nanoparticles on the surface of RGO / GCE to form a gold substrate, thus obtaining the deposited electrode AuNPs / RGO / GCE. (3) Preparation of MIP / AuNPs / RGO / GCE AuNPs / RGO / GCE were electropolymerized in an electrolyte polymerization solution containing the template substance cyclic adenosine monophosphate and the functional monomer o-phenylenediamine. The molar ratio of the template substance to the functional monomer was 1:(1-5). The initial polymerization potential was 0.1-0.5V, the termination potential was 0.8-1.6V, and the polymerization was carried out for 20 cycles to obtain a dense and insulating polymer film. The polymerized electrode was immersed in an elution solution, namely H2SO4 with a concentration of 0.2-0.6 mol / L, and eluted using cyclic voltammetry for 22-26 cycles to obtain MIP / AuNPs / RGO / GCE.

2. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP) according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of the reduced graphene oxide to dimethylformamide is (0.5-2.5):1g / L.

3. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP) according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of chitosan to acetic acid solution is (2.5-12.5):1g / L.

4. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP) according to claim 1, characterized in that, In step (1), the volume ratio of A to B is 1:

1.

5. The method for preparing a molecularly imprinted electrochemical sensor for detecting cyclic adenosine monophosphate (cAMP) according to claim 1, characterized in that, In step (3), the electrolyte is PBS, and the molar ratio of the template substance to the electrolyte is 1:(2-6).

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