Seaweed residue-based electrochemical biosensor and preparation method thereof

Abstract

The present application relates to a seaweed residue-based electrochemical biosensor and a preparation method thereof, which uses seaweed residue as raw material and chitosan as crosslinking agent, and prepares carbon aerogel CAs through sol-gel, vacuum freeze-drying and high-temperature carbonization process, and uses the CAs modified electrode (CAs / GCE) for DA detection. The present application uses high-temperature pyrolysis of nitrogen in seaweed residue to increase the disorder of C in CAs, which can effectively increase the active sites for dopamine detection and improve the electrochemical detection activity of the sensor for DA. CAs do not contain substances harmful to organisms or polluting to the environment, which not only solves the problems of high cost and limited application range of other raw materials, but also solves the problems of high value-added development and utilization of waste seaweed residue.

Description

Technical Field

[0001] This invention relates to an electrochemical biosensor based on seaweed residue and its preparation method, belonging to the field of marine waste utilization technology. Background Technology

[0002] Dopamine (DA), or 3,4-dihydroxyphenylethylamine, is the most important catecholamine neurotransmitter in the mammalian nervous system, playing a crucial role in human metabolism, cardiovascular system, kidneys, central nervous system, and endocrine system. Therefore, in clinical medicine, serum DA levels have become an important parameter for diagnosing and preventing mental illnesses. Electrochemical analysis techniques for DA detection have been widely studied due to their advantages of high sensitivity, rapid response, simple operation, low cost, and real-time analysis. Therefore, the development of high-performance DA electrochemical biosensors and the search for active materials with good electrochemical properties are of great value.

[0003] Carbon nanomaterials have attracted widespread interest in the field of electrochemical detection due to their attractive electrical conductivity and structural properties (such as high electronic conductivity, large specific surface area, and high stability). Carbon aerogels (CAs), as an emerging type of carbon nanomaterial, can be prepared from biomass materials by adding cross-linking agents and then undergoing freeze-drying and carbonization processes. Compared with other materials, CAs have the advantages of being environmentally friendly, having low preparation costs, and being quick to prepare. The prepared CAs have numerous pores, low density, and large specific surface area, making them particularly prominent in applications as electrode materials. Currently, carbon aerogels (CAs) are widely used in biosensor research. For example, Zhang et al. (2016) modified Fe2O3 nanoparticles onto carbon aerogels. The resulting composite material exhibited a rapid amperometric response, a low detection limit, and good selectivity when used for the electrochemical detection of dopamine (DA). (See: Zhang, Y., et al., In SItu Growth of Fe2O3Nanoparticles on Highly PorousGraphene / Polyimide-Based Carbon Aerogel NanocomposItes for Effectively Selective Detection of Dopamine. Advanced Materials Interfaces, 2016.3(15)). Yang et al. (2015) used electropolymerization and molecular imprinting techniques to modify molecularly imprinted polypyrrole onto the surface of CAs, developing a novel electrochemical sensor with molecular recognition capabilities and high electrocatalytic activity for the detection of dopamine (DA). In addition, Chinese patent document CN115639257A discloses a method for preparing an electrochemical sensor for DA detection. This method combines nanotechnology, electrochemical analysis technology and sensor preparation technology, uses a platinum phthalocyanine layer as an electrode modification layer and dops it with Pt nanoparticles, and develops a novel material for the preparation of multi-DA electrochemical sensors.

[0004] However, the aforementioned existing technologies still suffer from high raw material costs, complex synthesis, and time-consuming preparation, making it difficult to meet the needs of industrial production. Therefore, developing a low-cost and rapidly prepared DA electrochemical biosensor remains a significant challenge. Summary of the Invention

[0005] To address the shortcomings of existing technologies, especially the high cost and complex preparation process of DA electrochemical biosensors, this invention proposes a method for preparing CAs using seaweed residue as raw material, and develops an electrochemical biosensor based on this method for DA detection.

[0006] Terminology Explanation:

[0007] DA: Dopamine.

[0008] CAs: Carbon aerogels.

[0009] GCE: Glassy carbon electrode.

[0010] Seaweed residue: The seaweed residue mentioned in this invention is the residue generated during the production of sodium alginate. Seaweed residue has a high organic content, mainly crude protein, crude fiber, and a small amount of seaweed polysaccharides. Processing techniques include, but are not limited to, rinsing, crushing, alkali dissolution, grinding, and foaming filtration to obtain seaweed filter residue. The seaweed used can be seaweed grown in various sea areas.

[0011] DMF: N,N-dimethylformamide.

[0012] Nafion reagent: Nafion reagent is a perfluorosulfonic acid polymer solution, typically composed of a fluorinated solvent containing Nafion polymer and some additives. Nafion polymer is a high molecular weight material with good thermal, chemical, and electrochemical stability. Nafion reagent is a commonly available commercial product.

[0013] The technical solution of the present invention is as follows:

[0014] A method for preparing CAs using seaweed residue as raw material includes the following steps:

[0015] (1) Dissolve seaweed residue in water, adjust the pH value to 2.5-5, add chitosan and stir until completely dissolved to obtain seaweed residue solution;

[0016] (2) After freezing the seaweed residue solution, vacuum dry it and then carbonize it at 700℃-1000℃ under inert gas to obtain CAs.

[0017] According to the present invention, preferably, in step (1) the concentration of seaweed residue in the seaweed residue solution is 0.01-0.07 g / mL, and the amount of chitosan added is 5%-20% of the mass of seaweed residue;

[0018] Preferably, acetic acid is used to adjust the pH, and preferably the pH is adjusted to 3.

[0019] According to the present invention, preferably, cellulose fiber powder is also added in step (1) to improve the mechanical strength of the prepared aerogel. The amount of powder added is 5%-20% of the mass of seaweed residue.

[0020] According to the present invention, preferably, the freezing temperature in step (2) is -50℃ to -80℃, the freezing time is 5-15h, and the freeze-drying time is 5-10h;

[0021] Preferably, the carbonization process is carried out in an Ar or N2 atmosphere;

[0022] Preferably, the heating rate of the carbonization process is 2-5℃ / min, and the carbonization time is 2-4h.

[0023] According to the present invention, CAs prepared by the above method are also provided.

[0024] According to the present invention, CAs prepared by the above method are also provided for use in the preparation of electrochemical biosensors;

[0025] Further preferred application is in the fabrication of electrochemical biosensors for DA detection.

[0026] According to the present invention, an electrochemical biosensor for DA detection is also provided, comprising CAs-modified GCE, i.e., CAs / GCE, wherein the CAs / GCE is obtained by casting a CAs suspension onto a GCE and drying it under an infrared lamp.

[0027] According to the present invention, the method for preparing the above-mentioned electrochemical biosensor for DA detection includes the following steps:

[0028] (1) After grinding CAs, add DMF solution and ultrasonically disperse evenly to obtain CAs suspension;

[0029] (2) After polishing and washing the GCE, the CAs suspension is cast onto the GCE and dried under an infrared lamp to obtain an electrochemical biosensor for DA detection.

[0030] According to the present invention, preferably, the concentration of CAs in the CAs suspension in step (1) is 3-10 mg / mL;

[0031] Preferably, Nafion reagent is also added, with a volume ratio of Nafion to DMF of 1:15-1:35.

[0032] According to the present invention, preferably, in step (1), the GCE is polished with 1μm, 0.3μm and / or 0.05μm alumina slurry and the GCE is ultrasonically cleaned with anhydrous ethanol and / or deionized water.

[0033] According to the present invention, preferably, the CAs / GCE prepared in step (2) has a diameter D = 0.2 mm and an effective working area S = 3.14 * 10⁻⁶. -2 cm 2 .

[0034] The principles and beneficial effects of this invention are as follows:

[0035] 1. This invention uses seaweed residue as raw material, which has the advantages of low cost, convenient acquisition, and environmental friendliness. At the same time, CAs do not contain substances that are harmful to organisms or pollute the environment, which solves the problems of high cost and limited application scope of other raw materials, and also solves the problem of high-value-added development and utilization of waste seaweed residue.

[0036] 2. In this invention, carbon aerogels (CAs) were prepared from seaweed residue using a sol-gel, vacuum freeze-drying, and high-temperature carbonization process. A CAs-modified electrode (CAs / GCE) was then used for dopamine (DA) detection. This invention utilizes the high-temperature pyrolysis of nitrogen in seaweed residue to increase the disorder of carbon (C) in CAs, effectively increasing the active sites for dopamine detection and enhancing the sensor's electrochemical detection activity for DA. CAs / GCE exhibits a low detection limit and a wide detection range in the electrochemical detection of DA. The detection limit reaches 0.165 μM, and the detection range is 0.5–150 μM.

[0037] 3. This invention provides an effective solution for the efficient utilization of biomass resources, which is conducive to maintaining the healthy development of human ecology. Attached Figure Description

[0038] Figure 1 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of Experiment Example 1.

[0039] Figure 2 The graph shows the cyclic voltammetry test results of CAs / GCE and bare electrode (GCE) in Experiment Example 2.

[0040] Figure 3 The It response curves of CAs / GCE obtained at different concentrations of DA in Experiment Example 3 are shown. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments. The description of the embodiments is only for the purpose of understanding the present invention and is not intended to limit the scope of protection of the present invention.

[0042] All raw materials used in the embodiments are commercially available. Among them, the seaweed residue used is obtained by using South American brown algae as raw material and undergoing processes such as rinsing, crushing, alkali dissolution and grinding, and foaming and filtration.

[0043] Example 1

[0044] Dissolve 3.0 g of seaweed residue in 100 ml of deionized water. Adjust the pH of the obtained seaweed residue solution to 3 with acetic acid, add 0.3 g of chitosan and dissolve, then add 0.3 g of cellulose fiber. During this process, continuously stir with a glass rod to ensure complete dissolution of the added chemicals and obtain a homogeneous seaweed residue solution. Place the homogeneous seaweed residue solution in a petri dish and freeze-dry at -80℃ for 12 h. Remove the frozen sample and vacuum-dry it in a freeze dryer for 12 h. The resulting seaweed aerogels are heated to 700℃ in a tube furnace at a rate of 5℃ / min and held for 2 h, then cooled to room temperature to obtain seaweed carbon aerogels (CAs-700).

[0045] Example 2

[0046] Dissolve 3.0 g of seaweed residue in 100 ml of deionized water. Adjust the pH of the seaweed residue solution to 3 with acetic acid, add 0.3 g of chitosan and dissolve, then add 0.3 g of cellulose fiber. During this process, continuously stir with a glass rod to ensure complete dissolution of the added chemicals and obtain a homogeneous seaweed residue solution. Place the homogeneous seaweed residue solution in a petri dish and freeze-dry at -80℃ for 12 h. Remove the frozen sample and vacuum-dry it in a freeze dryer for 12 h. The resulting seaweed aerogels are heated to 1000℃ in a tube furnace at a rate of 5℃ / min and held for 2 h, then cooled to room temperature to obtain seaweed carbon aerogels (CAs-1000).

[0047] Example 3

[0048] Dissolve 5.0 g of seaweed residue in 100 ml of deionized water. Adjust the pH of the obtained seaweed residue solution to 4 with acetic acid, and add 0.3 g of chitosan to dissolve it. During this process, continuously stir with a glass rod to ensure that the added chemical is fully dissolved and a homogeneous seaweed residue solution is obtained. Place the homogeneous seaweed residue solution in a petri dish and freeze it at -80℃ for 12 h. Remove the frozen sample and vacuum dry it in a freeze dryer for 12 h. The resulting seaweed aerogels are heated to 800℃ in a tube furnace at a rate of 3℃ / min and held for 2 h, then cooled to room temperature to obtain seaweed carbon aerogels (CAs-800).

[0049] Example 4

[0050] Dissolve 7.0 g of seaweed residue in 100 ml of deionized water. Adjust the pH of the obtained seaweed residue solution to 2.5 with acetic acid, and add 1 g of chitosan to dissolve it. During this process, continuously stir with a glass rod to ensure that the added chemical is fully dissolved and a homogeneous seaweed residue solution is obtained. Place the homogeneous seaweed residue solution in a petri dish and freeze it at -80℃ for 12 h. Remove the frozen sample and vacuum dry it in a freeze dryer for 12 h. The resulting seaweed aerogels are heated to 900℃ in a tube furnace at a rate of 4℃ / min and held for 2 h, then cooled to room temperature to obtain seaweed carbon aerogels (CAs-900).

[0051] Example 5

[0052] Dissolve 2.0 g of seaweed residue in 100 ml of deionized water. Adjust the pH of the obtained seaweed residue solution to 2.5 with acetic acid, and add 0.2 g of chitosan to dissolve it. During this process, continuously stir with a glass rod to ensure that the added chemical is fully dissolved and a homogeneous seaweed residue solution is obtained. Place the homogeneous seaweed residue solution in a petri dish and freeze it at -80℃ for 12 h. Remove the frozen sample and vacuum dry it in a freeze dryer for 12 h. The resulting seaweed aerogels are heated to 950℃ in a tube furnace at a rate of 5℃ / min and held for 2 h, then cooled to room temperature to obtain seaweed carbon aerogels (CAs-950).

[0053] Example 6

[0054] The CAs products obtained in Examples 1-5 were ground into powder, and then the powder was dispersed in DMF by ultrasonic treatment to obtain a uniform dark black suspension of 3-10 mg / mL; preferably, Nafion reagent was also added to the suspension, and the volume ratio of Nafion to DMF was 1:15-1:35.

[0055] Before electrode modification, the GCE was first polished on a microfiber polishing pad with alumina slurries of 1 μm, 0.3 μm, and 0.05 μm, respectively, and then rinsed with deionized water. Next, the electrode was ultrasonically cleaned for 3 minutes each in anhydrous ethanol and deionized water. Then, a CAs suspension was cast onto the GCE and dried under an infrared lamp to obtain a CAs-modified GCE (CAs / GCE), which is the electrochemical biosensor used for DA detection. The prepared CAs / GCE has a diameter D = 0.2 mm and an effective working area S = 3.14 × 10⁻⁶. -2 cm 2 .

[0056] Experimental Example 1

[0057] The elemental composition of CAs and seaweed residue aerogels obtained in Examples 1 or 2 was shown by X-ray photoelectron spectroscopy (XPS). XPS analysis determined the elemental composition of three different samples: seaweed residue aerogels, CAs-700, and CAs-1000. Figure 1 As shown. By Figure 1 It can be seen that with increasing carbonization and carbonization temperature, the oxygen (O) content gradually decreases while the carbon (C) content gradually increases. The C:O ratios are 2.8:1, 4.8:1, and 8:1, respectively. Furthermore, high-temperature carbonization significantly reduces the nitrogen (N) content, decreasing from 2.75% in the original seaweed residue to 0.88% in CAs-700, and not detected in CAs-1000. This indicates that the protein components in the raw seaweed residue are degraded by high temperature, and the amino groups in peptide bonds are broken, resulting in a significant decrease in nitrogen content. This also suggests that with increasing carbonization temperature, more vacancies appear in the material, generating more active sites. These defect sites are crucial for improving the electrochemical activity of the material.

[0058] Experimental Example 2

[0059] To evaluate the electrochemical activity of CAs / GCE, cyclic voltammetry (CV) tests were performed on CAs / GCE and the bare electrode (GCE). Figure 2 As shown. Wherein: Figure 2 (A) Demonstrates the CAs / GCE obtained from exposed GCE and CAs-700 and CAs-1000 in the presence of [Fe(CN)6]. 3- / 4- CV images of 0.1M KCl solution (1mM). (From...) Figure 2 (A) As can be seen, both electrodes exhibit distinct redox peaks. The peak current of bare GCE < 700℃ CAs / GCE < 1000℃ CAs / GCE. This indicates that 1000℃ CAs / GCE exhibits a higher electron transfer rate compared to bare GCE (Gu, Liu, & Zhou, 2019). The higher current density and sensitivity response of 1000℃ CAs / GCE can be attributed to the formation of more defect sites in the carbonized CAs.

[0060] like Figure 2 (B) illustrates the electrochemical response of the two working electrodes to DA. CV measurements were performed in 0.1 M phosphate-buffered saline (PBS) solution (pH = 7.0) at a scan rate of 50 mV·s. -1Neither electrode exhibited redox peaks in the absence of DA. However, when 30 μM DA was present in the solution, a pair of weak peaks were observed in the bare GCE, while a pair of distinct redox peaks were observed in the CAs / GCE. Furthermore, the peak current of the CAs / GCE was significantly higher than that of the bare GCE, reaching up to six times that of the bare GCE. This indicates that the CAs / GCE has a good electrocatalytic effect in the redox reaction of DA, and demonstrates a faster electron transfer rate and higher electrocatalytic activity than the bare GCE, making it suitable for DA detection.

[0061] Figure 2 (C) shows the scan rates (20–200 mV) at different scan rates. -1 In PBS solution (pH = 7.0), the CV response of CAs / GCE to DA (30 μM) was observed. With increasing scan rate, a positive shift in the oxidation peak potential (E-pa) and a negative shift in the reduction peak potential (E-pc) were observed, while the oxidation current (Ipa) and reduction current (Ipc) increased simultaneously.

[0062] Figure 2 (D) Explain the relationship between the redox peak current (Ip) and the square root of the scan rate (υ). 1 / 2 The linear relationship is good. The linear fitting equations are Ipa = 3.022υ. 1 / 2 -8.916(R 2 =0.993), Ipc = -3.102υ 1 / 2 +7.188(R 2 =0.992). Therefore, the redox of DA at CAs / GCE is diffusion-controlled.

[0063] Experimental Example 3

[0064] The It response curves of CAs / GCE at different concentrations of DA were tested, as shown in the figure. Figure 3 As shown.

[0065] Figure 3 (A) and (B) show the It response curves measured at 700℃ and 1000℃ with different concentrations of DA, respectively. The current response gradually increases as the DA concentration increases from 0.1 μM to 150 μM. Figure 3 As can be seen in (A), CAs / GCE at 700℃ will produce a significant current signal when the DA concentration is 1μM. Figure 3 (B) It shows that at 1000℃, CAs / GCE can produce a significant current signal at a DA concentration of 0.5 μM.

[0066] like Figure 3As shown in (C), through linear fitting, the regression equation for CAs / GCE at 700℃ is Ipa=0.0324C+0.139(R 2 =0.997), the regression equation for CAs / GCE at 1000℃ is: Ipa = 0.0356C + 0.218(R) 2 =0.993). Calculations show that the detection limit of CAs / GCE at 700℃ is approximately 0.33 μM, with a detection range of 1-150 μM. The detection limit of CAs / GCE at 1000℃ is 0.166 μM, with a detection range of 0.5-150 μM. Clearly, CAs / GCE at 1000℃ exhibits higher sensitivity in DA detection. Furthermore, the calculated current density of CAs / GCE at 700℃ and 1000℃ at a dopamine concentration of 50 μM is 59.39 μA / cm². 2 and 67.26 μA / cm 2 .

[0067] In electrode detection, compared to CAs / GCE at 700℃, CAs / GCE at 1000℃ exhibits more significant advantages, such as a wider detection range, a lower detection limit, and a higher current density. This is likely due to the higher number of defect sites and electron transfer rate present in 1000℃ CAs / GCE.

[0068] Measurements were taken using IT, under the same conditions, with the addition of 30 μM DDA and various interfering compounds (100 times the concentration of KNO3, glucose, and the same concentrations of AA and UA). Figure 3 As shown in (C) and (D), to test the selectivity of CAs / GCE at 700℃ and 1000℃, KNO3, glucose, ascorbic acid, and uric acid were added to the PBS solution. When these four substances were added, it was observed that the It curve did not fluctuate significantly, but after the addition of DA, the It curve fluctuated significantly, indicating that both electrodes have good selectivity.

[0069] To verify the stability of the modified electrode, three CAs / GCE samples were sealed and stored at -2℃ for 7 days at 1000℃, and the electrochemical analysis of DA was performed every other day. The DA concentration used was 30 μM. The test results are as follows: Figure 3 As shown in (F), after three days of continuous use, the response current of CAs / GCE to DA was still 96.8% of the initial value, and it could still reach 85.5% after seven days. This indicates that the electrochemical detection capability of CAs / GCE to DA did not change significantly, and the modified electrode has good stability and can be stored for long-term use.

[0070] Test Example 4

[0071] The effectiveness of CAs / GCE for real-world sample determination was verified by detecting DA in human urine. Urine samples were centrifuged at 8000 rpm for 10 minutes. The urine samples were then diluted 100-fold with 0.1 M PBS solution (pH = 7.0). DA was determined using the standard addition method, with each sample measured in triplicate. Table 1 lists the recovery results in human urine.

[0072] Table 1

[0073]

[0074] As shown in Table 1, the recovery rate is 99.02%–104.4%, and the RSD is less than 5%. Therefore, this method is reliable for the detection of DA in real samples.

Claims

1. A seaweed residue-based electrochemical biosensor for dopamine detection, characterized in that, The seaweed residue-based electrochemical biosensor is a glassy carbon electrode modified by carbon aerogel prepared by using seaweed residue as raw material; the preparation steps of the carbon aerogel are as follows: (1) the seaweed residue is dissolved in water, the pH value is adjusted to 2.5-5, chitosan is added and stirred until dissolved, then cellulose fiber powder is added, the addition amount is 5%-20% of the mass of the seaweed residue, to obtain a seaweed residue solution; (2) the seaweed residue solution is frozen and vacuum dried, and then carbonized at 700-1000℃ under the introduction of inert gas to obtain carbon aerogel.

2. The seaweed residue-based electrochemical biosensor for dopamine detection according to claim 1, wherein, In step (1), the concentration of seaweed residue in the seaweed residue solution is 0.01-0.07 g / mL, and the addition amount of chitosan is 5%-20% of the mass of the seaweed residue.

3. The seaweed residue-based electrochemical biosensor for dopamine detection according to claim 1, wherein, In step (1), the pH value is adjusted to 3 by acetic acid.

4. The seaweed-slag-based electrochemical biosensor for dopamine detection according to claim 1, wherein, In step (2), the freezing temperature is-50℃ ~ -80℃, and the freezing time is 5-15h; the freeze-drying time is 5-10h.

5. The seaweed residue-based electrochemical biosensor for dopamine detection according to claim 1, wherein, In step (2), the carbonization treatment is carried out in Ar or N2 atmosphere.

6. The seaweed waste-based electrochemical biosensor for dopamine detection according to claim 1, wherein, In step (2), the heating rate of carbonization treatment is 2-5℃ / min, and the carbonization time is 2-4h.

7. The seaweed waste-based electrochemical biosensor for dopamine detection according to claim 1, wherein, The seaweed residue-based electrochemical biosensor is prepared by the following method: (i) the carbon aerogel is ground and added to DMF solution, and then ultrasonically dispersed to obtain a carbon aerogel suspension; (ii) the glassy carbon electrode is polished and washed, then the carbon aerogel suspension is cast onto the glassy carbon electrode, and dried under infrared lamp to obtain the seaweed residue-based electrochemical biosensor.

8. The seaweed waste-based electrochemical biosensor for dopamine detection according to claim 7, wherein, In step (i), the concentration of carbon aerogel in the carbon aerogel suspension is 3-10 mg / mL.

9. The seaweed waste-based electrochemical biosensor for dopamine detection according to claim 7, wherein, In step (i), Nafion reagent is also added after the carbon aerogel is added to the DMF solution, and the volume ratio of Nafion to DMF is 1:15-1:

35.

10. The seaweed residue-based electrochemical biosensor according to any one of claims 1-9 for use in dopamine detection.

Images