METHOD AND SENSOR FOR DETECTING L-ARGININE

Abstract

A method and a sensor for detecting L-arginine are provided. The method includes synthesizing ferrocene-functionalized hexadecapeptide dithiocyclopentane (FC-P16 Peptide), preparing a polypeptide composite membrane-modified electrode (FC-P16 Peptide/AuE), detecting L-Arg and other steps. The results show that the polypeptide composite membrane-modified electrode (FC-P16 Peptide/AuE) exhibits excellent electrochemical response properties to L-Arg. In 10 mmol/L phosphate-buffered saline (PBS, pH=7.4), the DPV response peak current of the polypeptide composite membrane-modified electrode has an excellent linear relationship with the L-Arg concentration of 1.0×10.sup.−13 mol/L to 1.0×10.sup.−7 mol/L, with a detection limit of 1.0×10.sup.−13 mol/L. With prominent reproducibility, repeatability and selectivity, the modified electrode has potential application in life science and nutritional health.

Claims

1. A method for detecting L-arginine (L-Arg), comprising the following steps: (1) synthesis of ferrocene-functionalized hexadecapeptide dithiocyclopentane, wherein a structural formula of the ferrocene-functionalized hexadecapeptide dithiocyclopentane is shown as formula (I): ##STR00002## (2) preparation of a polypeptide composite membrane-modified electrode (FC-P16 Peptide/AuE): soaking a gold electrode in a Piranha solution to obtain a first soaked gold electrode, and then cleaning and polishing the first soaked gold electrode to obtain a polished gold electrode, cleaning and air drying the polished gold electrode with N.sub.2 to obtain a blow-dried gold electrode; soaking the blow-dried gold electrode in a ferrocene-functionalized hexadecapeptide dithiocyclopentane solution and phosphate-buffered saline (PBS) for 20 h to 30 h to obtain a second soaked gold electrode, wherein concentration of the ferrocene-functionalized hexadecapeptide dithiocyclopentane solution is 30 μmol/L to 80 μmol/L and a concentration of tris(2-carboxyethyl)phosphine (TCEP) in the PBS is 10 μmol/L to 80 μmol/L; then soaking the second soaked gold electrode in a 6-mercapto-1-hexanol (MCH) solution with a MCH concentration of 0.5 mmol/L to 2.0 mmol/L for 2 min to 30 min to obtain a third soaked gold electrode; rinsing the third soaked gold electrode with the PBS along a surface of the third soaked gold electrode to remove nonspecifically-adsorbed substances to obtain a cleaned gold electrode; and air drying the cleaned gold electrode with N.sub.2 to obtain the polypeptide composite membrane-modified electrode (FC-P16 Peptide/AuE); and (3) constructing a three-electrode system with the polypeptide composite membrane-modified electrode (FC-P16 Peptide/AuE) as a working electrode, a silver/silver chloride electrode as a reference electrode, and a platinum wire electrode as a counter electrode; then investigating electrochemical behaviors of differently-modified electrodes using cyclic voltammetry (CV) and differential pulse voltammetry (DPV); using the DPV to test L-Arg at different concentrations, and plotting a working standard curve; and then detecting L-Arg in test samples by a standard addition method.

2. The method for detecting the L-arginine (L-Arg) according to claim 1, wherein, in step (2), a surface of the gold electrode is polished with alumina powders of 1.0 μm, 0.3 μm, and 0.05 μm separately.

3. The method for detecting the L-arginine (L-Arg) according to claim 1, wherein, in step (2), the gold electrode has a diameter of 3 mm.

4. The method for detecting the L-arginine (L-Arg) according to claim 1, wherein, in step (3), the electrochemical behaviors of differently-modified electrodes are investigated in 10 mmol/L PBS, 2.0 mmol/L [Fe(CN).sub.6].sup.4−3−−10 mmol/L PBS, and 10 mmol/L PBS with 1.0×10.sup.−5 mol/L L-Arg using the CV and the DPV; the DPV is adopted to test a relationship between a current response and a concentration of the L-Arg, the DPV is adopted with the following parameters: amplitude: 0.05 V, pulse interval: 0.5 s, sampling width: 0.02, and pulse width: 0.2 s; and the CV is adopted with the following parameters: sampling interval: 0.001 V, and scan rate: 100 mV/s.

5. A sensor for detecting L-Arg, comprising a polypeptide composite membrane-modified electrode as a working electrode; wherein the polypeptide composite membrane-modified electrode comprises a gold matrix; the gold matrix has a surface modified with a polypeptide composite membrane layer; the polypeptide composite membrane layer comprises polypeptide molecules; and the polypeptide molecules are ferrocene-functionalized hexadecapeptide dithiocyclopentane molecules.

6. The sensor for detecting the L-Arg according to claim 5, wherein the polypeptide composite membrane layer further comprises MCH molecules.

7. The sensor for detecting the L-Arg according to claim 5, wherein, the gold matrix has a thickness of 1.0 mm to 5.0 mm, and the polypeptide composite membrane layer has a thickness of 2 nm to 20 nm.

8. The sensor for detecting the L-Arg according to claim 5, wherein the sensor has linear relationship with a concentration of the L-Arg, a linear detection range of the sensor is 1.0×10.sup.−13 to 1.0×10.sup.−7 mol/L, and a detection limit of the sensor is 1.0×10.sup.−13 mol/L.

9. A method of using ferrocene-functionalized hexadecapeptide dithiocyclopentane in a preparation of a biosensor, wherein the ferrocene-functionalized hexadecapeptide dithiocyclopentane has a structural formula shown as formula (I): ##STR00003##

10. The sensor according to claim 6, wherein the sensor has a linear relationship with a concentration of the L-Arg, a linear detection range of the sensor is 1.0×10.sup.−13 to 1.0×10.sup.−7 mol/L, and a detection limit of the sensor is 1.0×10.sup.−13 mol/L.

11. The sensor according to claim 7, wherein the sensor has a linear relationship with a concentration of the L-Arg, a linear detection range of the sensor is 1.0×10.sup.−13 to 1.0×10.sup.−7 mol/L, and a detection limit of the sensor is 1.0×10.sup.−13 mol/L.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a working structural diagram of an L-Arg detection sensor based on a polypeptide composite membrane-modified electrode;

[0023] FIG. 2 shows the sequence of ferrocene-functionalized hexadecapeptide dithiocyclopentane (FC-P16-C4-DTCP, FC-P16 Peptide);

[0024] FIG. 3 shows the cyclic voltammogram of differently-modified electrodes in 2.0 mmol/L [Fe(CN).sub.6].sup.3−/[Fe(CN).sub.6].sup.4− (at a concentration ratio of 1:1)-PBS (10 mmol/L, pH=7.4), with (a) representing AuE, and (b) representing FC-P16 Peptide/AuE;

[0025] FIG. 4 shows the cyclic voltammogram of AuE (curve a) and FC-P16 Peptide/AuE (curve b) electrodes in PBS (10 mmol/L, pH=7.4), with (a) representing AuE, and (b) representing FC-P16 Peptide/AuE;

[0026] FIG. 5A shows the differential pulse voltammogram of AuE (curve a) and FC-P16 Peptide/AuE (curve b) electrodes in PBS with 1.0×10.sup.−5 mol/L-Arg, with (a) representing AuE, and (b) representing FC-P16 Peptide/AuE;

[0027] FIG. 5B shows the differential pulse voltammogram of AuE (curve a) and FC-P16 Peptide/AuE (curve b) electrodes in PBS without L-Arg, with (a) representing AuE, and (b) representing FC-P16 Peptide/AuE;

[0028] FIG. 6A shows the differential pulse voltammogram of FC-P16 Peptide/AuE responding to different concentrations of L-Arg;

[0029] FIG. 6B shows the response relation curve of peak current of FC-P16 Peptide/AuE vs. logarithmic concentration L-Arg;

[0030] FIG. 6C shows the piecewise linear relationship curve of peak current of FC-P16 Peptide/AuE vs. logarithmic concentration L-Arg;

[0031] FIG. 6D shows the piecewise linear relationship curve of peak current of FC-P16 Peptide/AuE vs. logarithmic concentration L-Arg.

[0032] In FIG. 1: 1: silver/silver chloride electrode; 2: platinum wire electrode; 3: surface-modified gold electrode; 4: solution to be tested; 5: gold matrix; 6: polypeptide composite membrane layer; 7: polypeptide molecule; 8: MCH molecule; and 9: L-Arg.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] In examples, a polypeptide stock solution (1.0 mmol/L) was prepared with ultrapure water (UPW). The polypeptide stock solution, when used, was diluted with PBS (10 mmol/L, pH=7.4) to a desired concentration. 0.20 mol/L NaOH and 0.20 mol/L HCl were used to adjust the pH of PBS. The reagents used were all analytical reagents (AR), and the experimental water was UPW (resistivity≥18.3 MΩ.Math.cm). In the following description, amino acids are described with abbreviations.

I. Experimental Process

[0034] 1. Preparation of a Polypeptide Composite Membrane-Modified Electrode (FC-P16 Peptide/AuE)

[0035] A gold electrode (with a diameter of 3 mm) was soaked in Piranha solution for 5 min, and then washed with UPW. The gold electrode was polished with A1203 powders of 1.0 μm, 0.3 μm and 0.05 μm in sequence to have a mirror surface, then subjected to sonication in UPW, absolute ethanol and UPW separately for 5 min, cleaned, and air dried with N.sub.2. At room temperature, the blow-dried gold electrode was soaked in PBS (10 mmol/L, pH=7.4) with 40 μmol/L ferrocene-functionalized hexadecapeptide dithiocyclopentane (FC-P16-C4-DTCP, FC-P16 Peptide) and 50 μmol/L TCEP for 24 h. In order to block the remaining active sites on the surface of the gold and allow the polypeptide to be perpendicular to the surface, the modified electrode was soaked in 1.0 mmol/L MCH solution for 5 min, then rinsed with PBS along the surface of the gold to remove other nonspecifically-adsorbed substances, and air dried with N.sub.2 to obtain a polypeptide composite membrane-modified electrode (FC-P16 Peptide/AuE), which was stored at 4° C. for later use.

[0036] 2. Electrochemical Detection for L-Arg

[0037] Referring to FIG. 1, electrochemical detection was conducted for L-Arg 9 using an electrochemical workstation, a three-electrode system with a surface-modified gold electrode as a working electrode, a silver/silver chloride (saturated potassium chloride) electrode 1 as a reference electrode and a platinum wire electrode 2 as a counter electrode, and 10 mmol/L PBS as a buffer solution. The electrochemical behaviors of differently-modified electrodes were investigated in 10 mmol/L PBS (pH=7.4), 2.0 mmol/L [Fe(CN).sub.6].sup.4−3−−10 mmol/L PBS, and PBS (10 mmol/L, pH=7.4) with 1.0×10.sup.−5 mol/L L-Arg using CV and DPV. The DPV was adopted to test the relationship between the current response and the concentration for L-Arg at different concentrations, and a working standard curve was plotted. The DPV was adopted with the following parameters: amplitude: 0.05 V, pulse interval: 0.5 s, sampling width: 0.02 and pulse width: 0.2 s. The CV was adopted with the following parameters: sampling interval: 0.001 V and scan rate: 100 mV/s.

[0038] The surface-modified gold electrode 3 was the polypeptide composite membrane-modified electrode in the sensor for detecting L-Arg of the present invention. The polypeptide composite membrane-modified electrode included a gold matrix 5. The gold matrix 5 had a surface modified with a polypeptide composite membrane layer 6. The polypeptide composite membrane layer 6 included polypeptide molecules 7 and MCH molecules 8. And the polypeptide molecules 7 were ferrocene-functionalized hexadecapeptide dithiocyclopentane molecules (FC-P16 Peptide), namely, ferrocene-functionalized hexadecapeptide-3-n-butyl-1,2-dithiocyclopentane (FC-P16-C4-DTCP), which had an amino acid sequence shown as GGGGFGHIHEGYGGGG with -GGGG- at two termini as linkers. The gold matrix 5 had a thickness of 1.0 mm to 5.0 mm, and the polypeptide composite membrane layer 6 had a thickness of 2 nm to 20 nm.

[0039] 3. Treatment and Determination for Samples

[0040] The standard addition method was adopted to detect L-Arg in pig serum samples (solution 4 to be tested). Pig serum samples (derived from five healthy Duroc×Landrace×Yorkshire piglets, each weighing 7 kg to 15 kg) were provided by the Institute of Subtropical Agriculture, Chinese Academy of Sciences (Changsha). 50.00 μL of each of the five different pig serum samples was diluted 100 times with PBS (4.950 mL) of pH=7.4, and then L-Arg solutions with different concentrations were added to the pig serum solutions. DPV was adopted for determination.

II. Experimental Results and Analysis

[0041] 1. The Electrochemical Behaviors of L-Arg on the Surface of the Electrode

[0042] The CV and AC impedance method were adopted to observe the assembly of the polypeptide composite membrane-modified electrode. As shown in FIG. 3, it can be found that, in the 2.0 mmol/L [Fe(CN).sub.6].sup.3−/[Fe(CN).sub.6].sup.4− (1:1)−10 mmol/L PBS solution, a pair of extremely-distinct oxidation/reduction peaks are observed at the bare gold electrode (AuE, curve a), indicating that there is rapid electron transfer on the bare gold electrode. Compared with the bare gold electrode (AuE), no distinct oxidation/reduction peak is prone to be observed at the FC-P16 Peptide/AuE electrode (curve b). Obviously, after the ferrocene-functionalized hexadecapeptide dithiocyclopentane (FC-P16-C4-DTCP, FC-P16 Peptide) was assembled on the gold electrode, the formed polypeptide composite membrane blocked the electron transfer of the electrode surface to a solution, thereby resulting in a significant decrease in the current value.

[0043] Furthermore, the CV behaviors of AuE and FC-P16 Peptide/AuE electrodes were investigated in 10 mmol/L PBS with pH=7.4 (FIG. 4). As shown in FIG. 4, there is no distinct oxidation/reduction peak for AuE (curve a), and the FC-P16 Peptide/AuE (curve b) electrode has a relatively-distinct oxidation/reduction peak at 0.45 V to 0.55 V, indicating that the ferrocene linked to the head of the polypeptide has undergone oxidation-reduction reaction at the interface.

[0044] The DPV behaviors of AuE and FC-P16 Peptide/AuE electrodes were investigated in 10 mmol/L PBS with 1.0×10.sup.−5 mol/L-Arg (FIG. 5A) and in PBS without Arg (FIG. 5B). It can be seen from FIG. 5A that the reduction peak of L-Arg on the AuE electrode is not distinct, indicating that L-Arg is difficult to be reduced on the surface of AuE (curve a). Compared with AuE, the FC-P16 Peptide/AuE electrode exhibits a stronger response to L-Arg (curve b), the response peak current is increased, and the peak potential is at E.sub.p=0.32 V, indicating that the polypeptide molecules on the FC-P16 Peptide/AuE electrode can well bind L-Arg. In contrast, as shown in FIG. 5B, AuE has no distinct reduction peak in PBS without Arg (curve a), while the FC-P16 Peptide/AuE electrode has a relatively-distinct reduction peak near 0.32 V (curve b), indicating that the ferrocene linked to the head of the polypeptide has undergone reduction reaction at the interface. Therefore, it can be shown that the ferrocene group is linked to the polypeptide molecule.

[0045] 2. Linear Range and Detection Limit

[0046] Under the optimized experimental conditions, the DPV was adopted to detect L-Arg at different concentrations with the FC-P16 Peptide/AuE electrode (FIG. 6A). It can be seen from the figure that, during the process where the concentration of L-Arg is increasing from 1.0×10.sup.−13 mol/L to 1.0×10.sup.−4 mol/L, the response peak current decreases steadily (FIG. 6B), indicating that, as more and more L-Arg molecules are bound to the polypeptide, the steric hindrance between the surface of the gold electrode and the ferrocene is increased, and the electron transfer efficiency is reduced. It can be seen from FIG. 6C that the reduction peak current of L-Arg has an excellent linear relationship with the concentration of L-Arg in the range of 1.0×10.sup.−13 mol/L to 1.0×10.sup.−7 mol/L, with a linear equation of I=0.0065371gc-0.4054, a correlation coefficient of R=0.9967, a lower detection limit of 1.0×10.sup.−13 mol/L. Moreover, it can be seen from FIG. 6D that the reduction peak current of L-Arg has a certain linear correlation with the concentration of L-Arg in the range of 1.0×10.sup.−7 mol/L to 1.0×10.sup.−4 mol/L, with a linear equation of I=0.024081gc-0.2841, and a correlation coefficient of R=0.9656. It can be found that, compared with the L-Arg electrodes reported in other literatures (see Table 1), and especially compared with the enzyme-free electrode, the FC-P16 Peptide/AuE electrode prepared in the present invention has better performance.

TABLE-US-00001 TABLE 1 Comparison of performance with different modified electrodes Linear range LOD Refer- Modified electrode Analyte (μmol/L) (μmol/L) ence ADI/PANi/Nafion/Pt-SPE L-Arg  3-200  1 [1] U/A/PANi-Nafion/PtE L-Arg  70-600 38 [2] U-yeast cells/ L-Arg  0-600 85 [3] PANi-Nafion/PtE EA#14.3 aptamer/AuE L-Arg   0-0.86 1.6 × 10.sup.−6 [4] Urease-ISFET L-Arg  100-2000 50 [5] FC-P16 Peptide/AuE L-Arg 1.0 × 10.sup.−7 to 0.1 1.0 × 10.sup.−7 This work Note: ADI: arginine deiminase; PANi: Polyaniline; SPE: screen-printed electrode; PtE: Pt electrode; U/A: urease and arginase I; EA#14.3 aptamer: 96 unit thiolated G-quadruples DNA; ISFET: ion-selective field effect transistor.

References

[0047] [1] Zhybak M T, Fayura L Y, Boretsky Y R, Gonchar M V, Sibirny A A, Dempsey E, Turner A P F, Korpan Y I. Amperometric L-arginine biosensor based on a novel recombinant arginine deiminase[J]. Microchimica Acta, 2017, 184: 2679-2686.

[0048] [2] Stasyuk N, Smutok O, Gayda Vus B, Koval'chuk Y, Gonchar M. Bi-enzyme 1-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode[J]. Biosensors & Bioelectronics, 2012, 37(1): 46-52.

[0049] [3] Stasyuk N Y, Gayda G Z, Gonchar M V. L-Arginine-selective microbial amperometric sensor based on recombinant yeast cells over-producing human liver arginase I[J]. Sensors and Actuators B: Chemical, 2014, 204: 515-521.

[0050] [4] Carter Z A, Kataky R. A G-quadruplex aptamer based impedimetric sensor for free lysine and arginine[J]. Sensors and Actuators B: Chemical, 2017, 243: 904-909.

[0051] [5] Sheliakina M, Arkhypova V, Soldatkin O, Saiapina O, Akata B, Dzyadevych S. Urease-based ISFET biosensor for arginine determination[J]. Talanta, 2014, 121: 18-23.

[0052] 4. Reproducibility and Repeatability of Electrodes

[0053] Six polypeptide composite membrane-modified electrodes prepared in the same batch under the same conditions were used to detect 1.0×10.sup.−8 mol/L L-Arg, and the relative standard deviation was 2.1%, indicating that the polypeptide-modified electrodes had excellent reproducibility. The same electrode was used to continuously detect 1.0×10.sup.−8 mol/L L-Arg 3 times, and the relative standard deviation was 0.56%, indicating that the electrode had excellent repeatability.

[0054] 5. Anti-Interference Test

[0055] In a three-electrode system with PBS (10 mmol/L, pH=7.4) as the base solution, the effect of common amino acid substances on the detection of L-Arg by the FC-P16 Peptide/AuE-modified electrode was investigated. In the presence of L-Arg (1.0×10.sup.−8 mol/L), 50-fold concentration of interfering components were added. The results showed that, after methionine (Met), tyrosine (Tyr), isoleucine (Ile), aspartic acid (Asp), glutamine (Gln), leucine (Leu), valine (Val), threonine (Thr), alanine (Ala), phenylalanine (Phe), proline (Pro), histidine (His), glutamate (Glu), glycine (Gly), lysine (Lys) and tryptophan (Trp) were added, the peak current of the modified electrode hardly changed significantly, indicating the FC-P16 Peptide/AuE-modified electrode had excellent selectivity for L-Arg.

[0056] In the present invention, an amino acid sequence that can specifically bind L-Arg is designed, a ferrocene probe is introduced, and then a modified electrode based on a ferrocene-functionalized hexadecapeptide composite membrane (FC-P16 Peptide/AuE) is constructed. The polypeptide composite membrane-modified electrode has prominent selectivity, repeatability, reproducibility and low detection limit for L-Arg, and can be applied to the determination of L-Arg in pig serum samples. Therefore, the polypeptide composite membrane-modified electrode has important application prospects in life science and nutritional health.