Gold electrode, non-enzymatic glucose sensor and construction method thereof

Abstract

The present invention provides a gold electrode, a non-enzymatic glucose sensor and a construction method thereof. The construction method of the gold electrode of the present invention is to use a pulse electrochemical method to etch the surface of the gold wire to form a specific microscopic electroactive structure, which can change the detection range of the glucose biosensor, achieve calibration-free, and facilitate clinical application in different environments.

Description

Technical Field

[0001] The present invention relates to the technical field of glucose detection, and in particular to a gold electrode, a non-enzymatic glucose sensor and a construction method thereof. Background Art

[0002] Diabetes is characterized by high blood sugar and is one of the chronic diseases that plague modern people. There is no cure yet. Only by continuously controlling blood sugar levels can we achieve state monitoring during prevention and treatment. At present, the main methods for detecting glucose include the following: spectrophotometry, rotation method, gas chromatography, high performance liquid chromatography, and electrochemical biosensor method.

[0003] Glucose biosensors have received a lot of attention from the academic community. However, glucose biosensors require calibration to correct for manufacturing differences between sensors and sensor drift. The need for calibration has proven to be one of the several significant barriers limiting the clinical use of biosensors.

[0004] In addition, the concentration range of glucose in different body fluid environments is different. The blood glucose concentration of normal people is 3.6-7.5mM, and the blood glucose concentration of diabetics is 1.1-20.8mM. The glucose concentration in other body fluid environments is related to blood glucose levels, such as 30-80μM in saliva, 20-600μM in sweat, and 100-600μM in tears.

[0005] Therefore, how to provide a calibration-free non-glucose sensor and improve the detection range in clinical applications is an important topic that needs to be studied urgently. Summary of the invention

[0006] Based on this, it is necessary to provide a gold electrode, a non-enzymatic glucose sensor and a construction method thereof. The non-enzymatic glucose sensor constructed using a gold electrode can be calibration-free, has adjustable sensitivity, and can adjust the blood glucose concentration detection range.

[0007] The present invention adopts the following technical solution:

[0008] The present invention provides a gold electrode, the surface of which is subjected to pulse electrochemical etching to form a granular nanostructure, a dendritic nanostructure or a cross-linked dendritic nanostructure, the roughness factor of which is not less than 2.8, and the electroactive area of ​​which is not less than 1000 μC / cm 2 .

[0009] The invention also provides a method for constructing a gold electrode, which uses a pulse electrochemical method to etch the gold surface in a concentrated sulfuric acid solution.

[0010] In some of the embodiments, the process steps of the pulse electrochemical etching method are: using a chronoamperometry method, repeatedly performing pulse scanning under the conditions of 2.0V and 0V, and each pulse lasts for 20ms.

[0011] In some of the embodiments, before the pulse electrochemical etching, the method further includes a step of cyclically cleaning the electrode in a NaOH solution at a voltage of 0 to -1.5 V and a potential of 0.1 V / s.

[0012] In some of the embodiments, after the pulse electrochemical etching, the step of testing whether a redox peak is generated on the surface at a potential of 0.1 V / s, 0 V and 1.65 V is further included.

[0013] The present invention also provides a non-enzymatic glucose sensor, which uses a gold electrode prepared by pulse electrochemical etching as a working electrode.

[0014] In some embodiments, when the gold electrode is subjected to pulse electrochemical etching for 50 to 200 times, the test frequency is 50 Hz; when the gold electrode for glucose detection is subjected to pulse electrochemical etching for 200 to 500 times, the test frequency is 10 to 50 Hz.

[0015] In some embodiments, the non-enzymatic glucose sensor further includes a counter electrode and a reference electrode, the counter electrode is a platinum wire, and the reference electrode is an Ag / AgCl electrode.

[0016] In some of the embodiments, when the surface of the gold electrode is a dendritic nanostructure, the glucose concentration detection range of the non-enzymatic glucose sensor is 20 to 1000 μM; when the surface of the gold electrode is a dendritic nanostructure, the glucose concentration detection range of the non-enzymatic glucose sensor is 6 to 1000 μM; when the surface of the gold electrode is a cross-linked dendritic nanostructure, the glucose concentration detection range of the non-enzymatic glucose sensor is 1.6 to 100 μM, which can be used directly or after diluting the sample for blood glucose concentration detection in saliva, sweat, and tears.

[0017] In some of the examples, square wave voltammetry was used to measure the glucose concentration in PBS buffer.

[0018] The beneficial effects of the present invention are:

[0019] Compared with the prior art, the present invention provides for the first time a gold electrode whose surface is etched by pulse electrochemical etching to form a granular nanostructure, a dendritic nanostructure or a cross-linked dendritic nanostructure. It is calibration-free during glucose detection, has high sensitivity, and can adjust the detection range of blood glucose concentration, thereby improving its clinical applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the gold electrode surface etching method and structural changes.

[0021] Figure 2 The microstructure of the gold electrode surface after different etching times.

[0022] Figure 3 is the surface area of ​​the gold electrode after different etching times.

[0023] Figure 4 This is the standard concentration curve of glucose sensors with different etching times and different test frequencies in PBS.

[0024] Figure 5 Glucose concentration curves of three electrodes etched at 500r.

[0025] Figure 6 Glucose concentration calibration curve for the three electrodes etched at 500r.

[0026] Figure 7 This is the glucose free calibration curve under 500r etching conditions.

[0027] Figure 8 Estimated concentration accuracy for the glucose sensor.

[0028] Fig. 9 This is the standard concentration curve of glucose sensors with different etching times in PBS.

[0029] Fig.10 To test the response and selectivity of glucose sensors in different body fluid environments. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0031] The following embodiments are only used to illustrate the present invention, but are not limited to the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0032] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0033] The present invention adopts gold wire as working electrode, platinum wire as counter electrode and Ag / AgCl reference electrode, and CHI 1040C electrochemical workstation adopts eight-channel constant potential instrument as inspection channel, and realizes accurate measurement of detection object under the condition of multiple working electrodes simultaneously. The eight working electrodes of the electrochemical workstation and a common reference electrode and a common counter electrode are in the same electrolytic cell for measurement.

[0034] Open the CHI 1040C software, check whether the instrument is connected properly, place the prepared working electrode in the electrolytic cell test system, and correctly connect the electrode to the workstation to ensure good contact and that the electrodes do not touch each other.

[0035] The electrochemical etching method of the present invention etches the surface of the gold wire by a pulse electrochemical method in a 0.5M sulfuric acid solution. Different etching times will result in different microstructures on the electrode surface.

[0036] The present invention specifically provides a method for electrochemically etching the surface of a gold wire, comprising the following steps:

[0037] S1, cleaning the electrode surface:

[0038] At 0 to -1.5V and 0.1V s -1 At a potential of 0.5 molL -1 The electrode was cleaned 10 times using the electrochemical method using NaOH solution. After this step, the NaOH solution on the electrode surface was rinsed off with ultrapure water.

[0039] S2, gold electrode surface etching:

[0040] like Figure 1 As shown, the chronoamperometry was used to perform repeated pulse scans at 2.0V and 0V, with each pulse lasting 20ms. Under 2.0V, the surface gold atoms on the original gold electrode dissolved and diffused to the vicinity of the electrode surface. Under 0V, the dissolved gold ions near the electrode were fixed to the surface of the gold electrode to form a micro-nano structure. By repeating these two steps, the gold atoms on the electrode surface repeatedly "dissolved" and "fixed", and the micro-nano structure gradually changed.

[0041] The morphology of the micro-nanostructure is affected by the number of cycles of electrochemical etching. In theory, the more cycles there are, the more complex the micro-nanostructure on the electrode surface. The results of electrochemical and SEM characterization are shown in Figure 2. Figure 2 and 3 .

[0042] This etching step is carried out at 0.5 molL -1The redox washing step is carried out in H2SO4 solution. The important purpose of this step is to form gold nanostructures of different morphologies on the electrode surface by electrochemical etching, increase the effective specific surface area of ​​the gold electrode, thereby increasing the electron conduction efficiency and matching the linear relationship between the surface gold nanomorphology and the body fluid system to be tested.

[0043] The difference from conventional technology is that the method has simple raw materials, does not need to add additional gold source (such as chloroauric acid), does not need to add complexing agent (such as perchlorate, hydrochloride), and is convenient and fast.

[0044] S3, area measurement of gold electrode using CV:

[0045] At 0.05 molL -1 The H2SO4 solution was used to test the –1 Potential, 0V and 1.65V voltage test, observe the redox peaks appearing on the upper curve. It can be determined that the electroactive area of ​​each gold electrode is not less than 1000μC cm -2 The surface area of ​​a freshly cleaned electrode is typically 0.03 (±30%) cm 2 .

[0046] This step is carried out at 0.05 molL -1 The gold electrode surface is completely cleaned in H2SO4 solution. During scanning, a gold oxide layer is generated on the surface, which is then reduced to produce a redox peak.

[0047] The specific tests and analysis are as follows:

[0048] like Figure 2 As shown in the SEM images, different nanostructures were generated on the surface of the gold electrode under different etching times. Electrochemical etching increases the surface area of ​​the electrode by promoting the formation of nanostructures. As can be seen from the figure, the initial surface of the gold electrode is relatively smooth and no nanostructure is generated. As the number of etchings increases, the nanostructure on the electrode surface also changes. When the number of etchings increases to 50 times, many nanoparticles appear on the surface of the gold electrode. When the number of etchings increases to 200 times, many dendritic nanostructures appear on the surface of the gold electrode. When the number of etchings reaches 500 times, the dendritic nanostructures on the surface of the gold electrode increase and form cross-links.

[0049] like Figure 3 As shown in the figure, the surface area of ​​the electrode after etching is significantly increased compared with the original smooth gold electrode. In addition, the surface area of ​​the electrode increases with the increase in the number of etching times. The roughness factor is one of the most important parameters for characterizing the surface. The roughness factor of the gold electrode is defined as the ratio of the effective area of ​​the gold electrode to the geometric area. Table 1 records the roughness factors of 8 gold electrodes after 0, 50, 200, and 500 times of rough processing.

[0050] Table 1 Roughness factor of gold electrode with different etching times

[0051]

[0052] The results show that when there is no roughness, the roughness factor of the gold electrode is close to 1. The roughness factor of the gold electrode increases with the increase of etching degree. Electrochemical etching significantly increases the electroactive area of ​​the gold electrode.

[0053] The glucose concentration in PBS buffer was determined by square wave voltammetry. Glucose titration tests were performed on glucose sensors with different gold electrode etching times at various frequencies (10 Hz, 50 Hz, 100 Hz and 200 Hz). Figure 4 Specifically:

[0054] (1) For the sensor with the gold electrode etched for 0r and 50r, the standard concentration curves under the test conditions of 200Hz, 100Hz, 50Hz and 10Hz are relatively similar, and the current and glucose concentration response.

[0055] (2) For the sensor with the gold electrode etched 200 times, under the test conditions of 100Hz, 50Hz and 10Hz, the standard concentration curves are similar, and the current and glucose concentration respond. However, under the test condition of 200Hz, the standard concentration curves are similar, and the current and glucose concentration do not respond.

[0056] (3) For the sensor with the gold electrode etched 500 times, the standard concentration curves under the test conditions of 50 Hz and 10 Hz are similar, and the current and glucose concentration respond. However, under the test conditions of 200 Hz and 100 Hz, the standard concentration curves are similar, and the current and glucose concentration do not respond.

[0057] In addition, the optimal test frequency of glucose sensors with different gold electrode etching times was further explored. The results showed that:

[0058] (1) For sensors with zero gold electrode etching times, the optimal test frequency is 200 Hz.

[0059] (2) For sensors with 50 gold electrode etching times, the best test frequency is 50 Hz.

[0060] (3) For sensors with 200 gold electrode etching times, the best test frequency is 50 Hz.

[0061] (4) For sensors with 500 gold electrode etching times, the optimal test frequency is 10 Hz.

[0062] like Figure 5As shown, for the sensor with 500 gold electrode etching times, the standard concentration curves of the three electrodes under the test conditions of 100 Hz, 50 Hz and 10 Hz are relatively close, while the standard concentration curves of the three electrodes under the test condition of 200 Hz are quite different.

[0063] The standard concentration curves of sensors in PBS with different gold electrode etching times under different frequency conditions were further investigated. The results showed that:

[0064] (1) Under the etching condition of 500r, the standard concentration curves in PBS under the test conditions of 10Hz and 50Hz are similar and respond to the concentration of glucose. The standard concentration curves in PBS under the test conditions of 10Hz and 50Hz are almost responsive to the concentration of glucose.

[0065] In order to eliminate the influence of test frequency on glucose test and realize calibration-free, the glucose test results under the conditions of 10Hz, 50Hz, 100Hz and 200Hz were processed. The glucose test currents of 100Hz and 200Hz with the same glucose concentration were divided by the glucose test currents of 10Hz and 50Hz respectively. The results are as follows: Figure 6 and Figure 7 ( Figure 7 The X-axis is the logarithm of the concentration), and a glucose test calibration-free curve was obtained.

[0066] The curve fitting results are shown in Table 2:

[0067] Table 2. Fitting results of glucose calibration curve etched at 500r

[0068]

[0069]

[0070] The fitting results of the calibration curve were applied to estimate the glucose concentration ( Figure 8 ), where X = 10^(-B2 / 4B1+0.5*(B2 2 / 4B1 2 -2B3 / 3B1+C) 0.5 +0.5*(B2 2 / 2B1 2 -4B3 / 3B1-C-(-B2 3 / B1 3 +4B2B3 / B1 2 -8B 4 / B1) / (4*(B2 2 / 4B1 2 -2B3 / 3B1) 0.5 +C),

[0071] In the above formula, C = 2 1 / 3 *C1 / (3B1*(C2+(-4C1 3 +C2 2 ) 0.5 ) 1 / 3 )+(C2+(-4C1 3 +C2 2 ) 0.5 ) 1 / 3 / (3*2 1 / 3 *B1), in this second formula C1=B3 2 -3B2B4-12AB1,C2=2B3 3 -9B2B3B4+27B1B4 2 -27B2 2 A+72B1B3A. ±30% concentration estimation was achieved in the range of 10 to 1000 μM.

[0072] After the gold electrode was etched, the standard concentration curve of the glucose sensor in PBS was as follows: Fig. 9 As shown in Figure 2, with the increase of etching times, the detection limit of the sensor gradually decreased from 50μM to 1.6μM. Specifically:

[0073] (1) For the sensor with 0 etching times and smooth gold electrode surface, the detection range is 50-1000 μM.

[0074] (2) For the sensor with 50 etching times and granular nanostructures on the gold electrode surface, the detection range is 20 to 1000 μM.

[0075] (3) For the sensor with 200 etching times and dendritic nanostructures on the gold electrode surface, the detection range is 6 to 1000 μM.

[0076] (4) For the sensor with 500 etching times and cross-linked dendritic nanostructures on the gold electrode surface, the detection range is 1.6 to 100 μM.

[0077] By adjusting the etching times, the electrochemical sensor can be applied to test in body fluid environments with different glucose levels, such as blood, urine, and sweat. Figure 7 shown.

[0078] like Figure 6 As shown in a, b, and c, the measured current is linearly related to the glucose concentration in the body fluid. The number of roughening times required for the gold electrode varies depending on the body fluid test:

[0079] (1) The concentration of glucose in the blood is the highest, and the gold electrode etched 50 times has the best effect.

[0080] (2) In urine and sweat tests with lower glucose concentrations, the etching times required for the gold electrode are 200 to 500 times, respectively.

[0081] In addition, the present invention also tests the effect of interfering compounds (such as uric acid, lactic acid, ascorbic acid, etc.) in human body fluids on electrode reactions. 0.1M of the above interfering compounds were added to different systems, and their reaction currents were tested and compared with the reaction currents of glucose at the same concentration. The results are shown in FIG. Fig.10 shown.

[0082] The results show that the glucose sensor with the etching conditions is highly selective for glucose testing in the presence of interferences such as uric acid, lactic acid and ascorbic acid.

[0083] The method of adjusting the test sensitivity and test range of the non-enzymatic glucose sensor by adjusting the number of gold electrode etching times can be applied to monitor glucose in blood, sweat and urine. This method has important application value in diabetes screening.

[0084] In addition, compared with the nano-gold structures prepared by other processes (for example, the need to add external gold sources such as chloroauric acid / HAuCl4·4H2O, deposition of gold-metal alloys and other additive methods), the present invention adopts an electrochemical etching method, which has the advantages of low addition, low consumption, low emissions, and low electrochemical residues. At the same time, the obtained gold nanostructure is controllable and has adjustable performance, and can efficiently detect glucose in a variety of body fluid environments and conditions.

[0085] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A non-enzymatic glucose sensor, characterized in that A gold electrode is used as a working electrode, and the surface of the gold electrode is subjected to pulse electrochemical etching to form a granular nanostructure, a dendritic nanostructure or a cross-linked dendritic nanostructure, and the roughness factor of the gold electrode is not less than 2.8, and the electroactive area is not less than 1000 μC / cm 2 ; The method for constructing the gold electrode comprises the following steps: The gold wire was cyclically cleaned in a NaOH solution at a voltage of 0~-1.5V and a potential of 0.1V / s; In sulfuric acid solution, the surface of the gold wire was etched by pulse electrochemical method: using chronoamperometry, repeated pulse scanning was performed under 2.0V and 0V conditions, each pulse lasted 20 ms, and the number of pulse electrochemical etching was 50-500 times; When the number of pulse electrochemical etching of the gold electrode is 50 to 200 times, the test frequency is 50 Hz; When the gold electrode is subjected to pulse electrochemical etching for 200 to 500 times, the test frequency is 10 to 50 Hz.

2. The non-enzymatic glucose sensor according to claim 1, characterized in that It comprises a counter electrode and a reference electrode, wherein the counter electrode is a platinum wire, and the reference electrode is an Ag / AgCl electrode.

3. The non-enzymatic glucose sensor according to claim 1 or 2, characterized in that: When the surface of the gold electrode is a granular nanostructure, the glucose concentration detection range of the non-enzymatic glucose sensor is 20-1000 μM; When the surface of the gold electrode is a dendritic nanostructure, the glucose concentration detection range of the non-enzymatic glucose sensor is 6 to 1000 μM; When the surface of the gold electrode is a cross-linked dendritic nanostructure, the glucose concentration detection range of the non-enzymatic glucose sensor is 1.6-100 μM.

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