An ultramicroelectrode for detecting lipopolysaccharide and a preparation method thereof

Abstract

The application belongs to the technical field of electrode preparation and detection application, and discloses an ultramicro electrode for detecting lipopolysaccharide and a preparation method thereof. Based on UATRP, the application constructs an ultramicro electrode for signal amplification through polymer branch connection, introduces electrically active nanosilver particles into a polymer chain through a silver mirror-like reaction by using a UATRP method, combines a self-made ultramicro construction system, and generates a very high detection signal for sensitive determination of lipopolysaccharide. The linear detection range of the concentration of lipopolysaccharide is 10 ‑3 -10 9 pg / mL, and the detection limit is 2.72*10 ‑ 4 pg / mL (S / N=3).

Description

Technical Field

[0001] This invention belongs to the field of electrode preparation and detection application technology. This invention relates to an ultramicroelectrode for detecting lipopolysaccharides and its preparation method; specifically, this invention relates to an ultramicroelectrode for detecting lipopolysaccharides based on ultrafast atom transfer radical polymerization. Background Technology

[0002] Lipopolysaccharide (LPS) is an important structure of the outer membrane of most Gram-negative bacteria, playing a crucial role in bacterial protection against environmental stress, drug resistance, pathogenicity, and symbiosis. Even trace amounts of LPS can induce hyperthermia and a range of harmful symptoms, such as septic shock, allergic asthma, lung cancer, and colorectal cancer (CRC). Therefore, rapid, micro-volume, and ultrasensitive determination of LPS is of great significance for disease diagnosis and treatment. Currently, amoeboid cell lysate (LAL)-based analytical techniques have become the gold standard for traditional LPS analysis due to their extremely high sensitivity (or low detection limit) and selectivity for LPS. However, they suffer from low reproducibility, are relatively expensive and time-consuming, especially when used in the low LPS concentration range. With the increasing demand in the medical field for accurate, simple, economical, efficient, and sensitive biomarker detection systems, electrochemical biosensors for LPS detection have emerged. However, the basic principle of most LPS sensors is that they work by binding to proteins, peptides, and artificial affinity recognition molecules. However, the designed biomolecules or synthetic probes have poor stability and may cross-bind and coexist with other species during LPS detection. Therefore, there are many challenges in LPS ultrasensitive detection that require further improvement, and research on LPS signal amplification in micro-volumes is also rare.

[0003] Traditional atom transfer radical polymerization (ATRP) requires transition metal complexes as catalysts. Transition metal catalysts are sensitive to air, often requiring anaerobic conditions for polymerization, are toxic to biomolecules, and tend to remain in the polymer, making them difficult to remove and affecting the performance of optoelectronic materials. Common ATRP methods involve lengthy polymerization processes, high polymer dispersion, and currently, relatively low conversion rates. Therefore, relying solely on ATRP detection methods has limited detection range. To overcome the detection range limitations of single-method ATRP detection, signal amplification strategies combining ATRP with other methods are common. However, while the detection range has improved, polymerization time, polymer dispersion, and conversion rates still require further improvement.

[0004] To prevent / reduce chemical waste, minimize pollution, rationally allocate and utilize chemical resources, and better comply with "green analytical chemistry" standards and sustainability principles, the green concept of ensuring chemical processes have a positive environmental impact has always attracted attention, but many challenges remain to be overcome. For example, further simplification of the application of precious or high-performance materials and solutions to the problem of excessively high experimental budgets are needed. Furthermore, while ensuring the rational utilization of chemical resources, it is easier to achieve rapid synthesis and high throughput. The positive utilization of analytical polymerization from mL to μL and micro-volumes is also receiving widespread attention in the biomedical field. Due to the extremely low concentration of LPS in serum, the sensitivity of LPS electrochemical detection needs further improvement to achieve trace detection of LPS. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides an ultramicroelectrode for detecting lipopolysaccharide and its preparation method. Based on carbon quantum dot-catalyzed ultrafast visible light-induced atom transfer radical polymerization, a large number of electroactive silver nanoparticles are introduced on the electrode surface, and an electrochemical method is used to detect LPS with high sensitivity.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] An ultramicroelectrode includes a gold disk microelectrode, hydrochloric acid, chloroauric acid, sodium chloride, an aptamer (Apt), and bovine serum albumin (BSA).

[0008] There are no special restrictions on the gold disk microelectrode, but the commercially available model CHI105 (Φ=12.5μm, purchased from Shanghai Chenhua Instrument Co., Ltd.) is preferred.

[0009] The specific steps of the above-mentioned preparation method of the ultramicro electrode are as follows:

[0010] S1. A clean gold microelectrode was placed in an electrolyte consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. The gold microelectrode was modified by cyclic voltammetry with a potential range of 0.4 to -0.5 V and a scan rate of 20 mV / s for 2 cycles to obtain AuNCs / Au UME.

[0011] S2. Drop 5 μL of aptamer (purchased from Shanghai Sangon Biotech Co., Ltd.) onto AuNCs / AuUME and incubate for 0.5-8 hours to obtain Apt / AuNCs / Au UME.

[0012] S3. Immerse Apt / AuNCs / Au UME in 2 mg / mL bovine serum albumin for 15 min to avoid non-specific binding of analytes, and obtain the ultramicroelectrode BSA / Apt / AuNCs / Au UME.

[0013] The application of the ultramicroelectrode prepared by the above method in the detection of lipopolysaccharides is as follows:

[0014] The microelectrode BSA / Apt / AuNCs / Au UME was incubated in a lipopolysaccharide-containing sample for 0.25–4 hours to obtain LPS / Apt / AuNCs / Au UME. The LPS / Apt / AuNCs / Au UME was then incubated for 15–75 minutes in a 1–9 mM ZrOCl2 solution prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME. An initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, bringing Zr... 4+ / LPS / Apt / AuNPs / Au UME is immersed in a 0.25-4 mM α-bromophenylacetic acid (BPAA) solution prepared with 40% (v / v) 99.7% anhydrous ethanol and 60% water and incubated for 0.25-4 hours to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / AuUME. BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME is immersed in a carbon quantum dot-catalyzed ultrafast visible light-induced atom transfer radical polymerization (UATRP) solution. 1 mL of the polymerization solution includes 5-80 mM copper bromide, 2.7-43.2 mM methacrolein (MLA), 4.3-68.8 μM tris(2-pyridinemethyl)amine (TPMA), 0.25-4 mg carbon quantum dots (CQDs), and 0.36 mL deionized water. The polymerization is carried out at room temperature and under LED light in a BPAA / Zr... 4+ The surface of / LPS / Apt / AuNPs / Au UME was activated for 0.5-4.5 min to obtain PMLA / LPS / Apt / AuNPs / Au UME. Finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 5-25 mmol / L Ag(NH3)2OH solution for 10-160 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME. At this point, AgNPs / PMLA / LPS / Apt / AuNPs / Au UME was used as the working electrode, platinum wire as the counter electrode, and a platinum-hydrogen micro-reference electrode (PME) as the reference electrode. The signal was amplified by SWV in 5 μL of 0.1 M KCl solution. The linear regression equation was expressed as I(μA) = 35.3595logc(10... -3 pg / mL) +108.7291, R 2 =0.9971; the test result is obtained.

[0015] The specific method for preparing PME is as follows:

[0016] Polyvinylpyrrolidone (PVP) and polyethersulfone (PES) were dissolved in dimethylformamide (DMF) at a mass ratio of 3:2 at room temperature. The solution was stirred at 40°C for 24-32 hours, and then concentrated by distillation on a rotary evaporator to prepare a concentrated liquid film. A cleaned platinum wire was immersed in a 0.01 mol / L palladium chloride solution at a flow rate of 0.02 A / cm. 2 Electrodeposition was performed at a current density for 30 min. Finally, the palladium-plated reference electrode was immersed in a PVP-PES liquid film until a uniform layer of liquid film was coated on the electrode surface, and then placed in an 80℃ oven to allow the DMF to completely evaporate. This process was repeated several times until a uniform PVP-PES polymer layer was covered on the surface of the palladium-plated electrode, yielding PME. The prepared PME was then placed in a 2 mol / L H2SO4 solution for later use.

[0017] The specific methods for preparing carbon quantum dots are as follows:

[0018] Dissolve 1 g of o-phenylenediamine in 100 mL of deionized water, add the solution to a reaction vessel, and heat at 200 °C for 8 h. After the reaction vessel has cooled naturally to room temperature, filter through a 0.22 μm membrane and freeze-dry to obtain CQDs.

[0019] The advantages of this invention compared to the prior art are:

[0020] Compared with existing methods, the LPS microelectrode preparation method of this invention is ultra-micro, ultra-fast, highly sensitive, and highly operable. Furthermore, this method features low background signal, high selectivity, avoids false positive results, is relatively simple to operate, and is cost-effective, showing great application potential in LPS detection and analysis.

[0021] This invention, based on UATRP, constructs a type of ultramicroelectrode that amplifies signals through polymer grafting. Using the UATRP method, electroactive silver nanoparticles (AgNPs) are introduced into the polymer chain via a silver mirror-like reaction. Combined with a self-made ultramicrostructure system, this generates a very high detection signal for the sensitive determination of lipopolysaccharide (LPS). The linear detection range for LPS concentration is 10⁻⁶. -3 -10 9 pg / mL, detection limit is 2.72×10 pg / mL. -4 pg / mL (S / N = 3). Attached Figure Description

[0022] Figure 1The Au UME(a), AuNCs / Au UME(b), Apt / AuNCs / Au UME(c), BSA / Apt / AuNCs / Au UME(d), LPS / Apt / AuNCs / Au UME(e), and Zr are from Embodiment 1 of the present invention. 4+ / LPS / Apt / AuNCs / Au UME(f)、BPAA / Zr 4+ Electrochemical impedance spectroscopy of / LPS / Apt / AuNCs / Au UME(g), PMLA / LPS / Apt / AuNPs / Au UME(h), and AgNPs / PMLA / LPS / Apt / AuNPs / Au UME(i).

[0023] Figure 2 The electrodes (a), (b), (c), and Zr) of AuNCs / Au UME and incubated with BSA according to Embodiment 1 of the present invention are: AuNCs / Au UME. 4+ / LPS / Apt / AuNCs / Au UME(d)、BPAA / Zr 4+ Square wave voltammetry curves of / LPS / Apt / AuNCs / Au UME(e), PMLA / LPS / Apt / AuNPs / Au UME(f), and AgNPs / PMLA / LPS / Apt / AuNPs / Au UME(g) in 0.1 mol / L KCl.

[0024] Figure 3 This is a selectivity diagram of AgNPs / PMLA / LPS / Apt / AuNPs / Au UME prepared in Example 1 of the present invention for LPS, glucose (Glu), dopamine (DA), ascorbic acid (AA), bovine serum albumin (BSA), citrate, cholesterol (TC), adrenaline (AD), uric acid (UA), and sodium 1,2-dipalmitoylphosphatidyl(1-propanetriol) (DPPG).

[0025] Figure 4 This invention, under the conditions of Example 1, uses different concentrations of lipopolysaccharide (10... -3 -10 9 Square wave voltammetry curves and working curves of AgNPs / PMLA / LPS / Apt / AuNPs / Au UME modified with polymer nanoparticles prepared from pg / mL solution in 0.1 mol / L KCl solution. Detailed Implementation

[0026] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used are all commercially available. Items not specifically described in the embodiments are all currently available commercially and are not subject to special limitations.

[0027] Example 1

[0028] 1. Fabrication of ultramicroelectrodes:

[0029] A clean gold microelectrode was placed in an electrolyte solution consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. Cyclic voltammetry was used to modify the gold microelectrode within a potential range of 0.4 to -0.5 V at a scan rate of 20 mV / s for two cycles, yielding AuNCs / Au UME. 5 μL of aptamer was drop-cast onto AuNCs / Au UME and incubated for 2 hours to obtain Apt / AuNCs / Au UME. Apt / AuNCs / Au UME was then immersed in 2 mg / mL bovine serum albumin for 15 min to avoid non-specific binding of the analyte, resulting in the BSA / Apt / AuNCs / Au UME microelectrode.

[0030] 2. Application of the ultramicroelectrode prepared by the above method in the detection of lipopolysaccharides

[0031] BSA / Apt / AuNCs / Au UME was incubated in a lipopolysaccharide-containing sample for 1 hour to obtain LPS / Apt / AuNCs / Au UME. Then, LPS / Apt / AuNCs / Au UME was incubated in 5 mM ZrOCl2 prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water for 45 minutes to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME. An initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, bringing Zr... 4+ / LPS / Apt / AuNPs / Au UME was immersed in a solution of 1 mM BPAA prepared with 40% V / V 99.7% anhydrous ethanol and 60% water and incubated for 1 hour to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME. Then, BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME were immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 20 mM copper bromide, 10.8 mM MLA, 17.2 μM TPMA, 1 mg CQDs, and 0.36 mL deionized water. The polymerization was carried out under LED light at room temperature on BPAA / Zr... 4+The surface of / LPS / Apt / AuNPs / Au UME was activated for 2.5 min to obtain PMLA / LPS / Apt / AuNPs / Au UME. Finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 15 mmol / L Ag(NH3)2OH solution for 40 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME. At this point, AgNPs / PMLA / LPS / Apt / AuNPs / Au UME was used as the working electrode, platinum wire as the counter electrode, and PME as the reference electrode, placed in 5 μL of 0.1 M KCl solution for signal amplification via SWV. The linear regression equation is expressed as I(μA) = 35.3595logc(10) / μA / AuNPs / AuUME. -3 pg / mL) +108.7291, R 2 =0.9971; the test result is obtained.

[0032] Figure 1 EIS plots show the stepwise construction process of the ultramicroelectrode. Curve (a) is the EIS curve of Au UME, showing a small semi-circular region, indicating the presence of [Fe(CN)6] on the electrode surface. 3- / 4- Electron transfer resistance (R) of probe ions ct The value is relatively small. Curve (b) is the EIS curve of AuNCs / Au UME, R. ct The smaller size is likely due to the formation of clustered nanoflower structures on the Au UME surface, which significantly increases the effective area of ​​the microelectrode and substantially improves the measured limiting diffusion steady-state current. When Apt is assembled on the surface of the microelectrode, Apt / AuNCs / Au UME, R ct The value increases (curve c), because Apt is an electrochemically inert molecule, thus weakening electron transfer. Curve (d) is the EIS curve of BSA / Apt / AuNCs / Au UME with BSA blocking non-specific recognition sites. This may be because BSA is a biomacromolecule and exhibits electrochemical inertness, increasing the system resistance. Curve (e) is the EIS curve of LPS / Apt / AuNCs / AuUME, and R... ct The value continuously increases. This may be because LPS is an electrochemically inert molecule with a large volume, which is unfavorable for electron conduction, thus increasing the system resistance. Curve (f) represents Zr. 4+ EIS curve of / LPS / Apt / AuNCs / Au UME, R ct The increase in value is likely due to the interaction between the positively charged Zr(IV) and the negatively charged [Fe(CN)6]. 3- / 4- Electrostatic interactions between the molecules lead to adsorption. The adsorbed [Fe(CN)6] 3- / 4-It exhibits electrostatic repulsion against electron transfer, leading to a further increase in system impedance. BPAA / Zr 4+ / LPS / Apt / AuNCs / Au UME(curve g), R ct As the value continues to increase, BPAA, being an electrochemically inert substance, is detrimental to electron transfer. After UATRP polymerization (curve h), PMLA / LPS / Apt / AuNPs / Au UME is obtained, R ct The addition of [something] demonstrates that after UATRP polymerization, a large amount of methacrolein covers the electrode surface, forming an inert polymer molecular layer on the ultramicroelectrode surface, which hinders electron transfer by the electrochemical probe. After the silver mirror reaction, electroactive AgNPs are formed in situ on the AgNPs / PMLA / LPS / Apt / AuNPs / Au UME surface, which is beneficial for electron transfer. Therefore, the R [something] of the obtained electrode [something]. ct The values ​​decreased significantly (curve i), indicating that the method based on UATRP polymerization and AgNP deposition is effective.

[0033] By analyzing the SWV curves of different modified ultramicroelectrodes in 0.1 mol / L KCl solution after incubation in silver ammonia solution, the signal amplification behavior of the electrodes during the modification process was ruled out. The results are as follows... Figure 2 As shown, AuNPs / Au UME were incubated with BSA (a), Apt / AuNPs / Au UME (b), LPS / Apt / AuNPs / Au UME (c), and Zr. 4+ / LPS / Apt / AuNPs / Au UME(d)、BPAA / Zr 4+ No current peak was observed on the SWV curves for / LPS / Apt / AuNPs / Au UME(e) and PMLA / LPS / Apt / AuNPs / Au UME(f). This phenomenon can be explained by the fact that none of the processes involved chemical interaction with the silver ammonia solution, and there was no SWV signal response in the entire process without the introduction of AgNPs. When AgNPs / PMLA / LPS / Apt / AuNPs / Au UME (curve g), a strong signal peak was observed at 0.068V. This peak potential may be due to the oxidation of AgNPs to Ag. + The potential of the microelectrode was further demonstrated, proving that the microelectrode was based on the AgNPs signaling method.

[0034] To evaluate the selectivity of the prepared LPS sensor, Glu, DA, AA, BSA, Citrate, TC, AD, UA, and DPPG were used as interfering substances for relevant tests. The results are as follows: Figure 3As shown in the figure, the SWV peak current values ​​of the AgNPs / PMLA / LPS / Apt / AuNPs / Au UME electrodes with the same concentration of interfering substances are collected. It can be seen from the figure that although interfering substances are present, the resulting electrochemical signal is weak and has no significant impact on detection. This may be due to the specific binding between LPS and Apt; the interfering substances cannot generate specific binding sites, meaning they cannot effectively introduce signal molecules. Figure 3 This indicates that the microelectrode has good selectivity and anti-interference ability for LPS.

[0035] Figure 4 As shown, 1-13 are 10 respectively. 9 pg / mL, 10 8 pg / mL, 10 7 pg / mL, 10 6 pg / mL, 10 5 pg / mL, 10 4 pg / mL, 10 3 pg / mL, 10 2 pg / mL, 10 1 pg / mL, 1 pg / mL, 10 -1 pg / mL, 10 -2 pg / mL, 10 -3 Square wave voltammetry curves and working curves of AgNPs / PMLA / LPS / Apt / AuNPs / Au UME prepared with pg / mL LPS in 0.1 mol / L KCl solution. The oxidation peak current of AgNPs increases with increasing LPS concentration. When the LPS concentration is 10 pg / mL... -3 ~10 pg / mL and 10~10 9 When the concentrations were between pg / mL, a strong linear correlation was found between the logarithm of the concentration in the ultrafine system and the peak current magnitude in the normal system. The linear regression equation in the ultrafine system was expressed as I(μA)=35.3595logc(10 -3 pg / mL) +108.7291, R 2 =0.9971; the linear regression equation under normal conditions is expressed as I(μA) = 46.2862logc(10 - 3 pg / mL) + 99.6321, R 2 =0.9966. Based on 3σ / slope (σ represents standard deviation), the detection limit obtained by this method is 2.72 × 10⁻⁶. -4 Both pg / mL values ​​are superior to those reported in commonly available literature.

[0036] Example 2:

[0037] 1. Fabrication of ultramicroelectrodes:

[0038] A clean gold microelectrode was placed in an electrolyte solution consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. Cyclic voltammetry was used to modify the gold microelectrode within a potential range of 0.4 to -0.5 V at a scan rate of 20 mV / s for two cycles, yielding AuNCs / Au UME. 5 μL of aptamer was drop-cast onto AuNCs / Au UME and incubated for 0.5 h to obtain Apt / AuNCs / Au UME. Apt / AuNCs / Au UME was then immersed in 2 mg / mL bovine serum albumin for 15 min to avoid non-specific binding of the analyte, resulting in the BSA / Apt / AuNCs / Au UME microelectrode.

[0039] 2. Application of the ultramicroelectrode prepared by the above method in the detection of lipopolysaccharides

[0040] BSA / Apt / AuNCs / Au UME was incubated in a lipopolysaccharide-containing sample for 0.25 hours to obtain LPS / Apt / AuNCs / Au UME. Then, LPS / Apt / AuNCs / Au UME was incubated for 15 minutes in a 1 mM ZrOCl2 solution prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME. An initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, bringing Zr... 4+ / LPS / Apt / AuNPs / Au UME was immersed in a 0.25 mM BPAA solution prepared with 40% (v / v) 99.7% anhydrous ethanol and 60% water and incubated for 0.25 hours to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME. Then, BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME were immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 5 mM copper bromide, 2.7 mM MLA, 4.3 μM TPMA, 0.25 mg CQDs, and 0.36 mL deionized water. The polymerization was carried out under LED light at room temperature on BPAA / Zr... 4+The surface of / LPS / Apt / AuNPs / Au UME was activated for 0.5 min to obtain PMLA / LPS / Apt / AuNPs / Au UME. Finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 5 mmol / L Ag(NH3)2OH solution for 10 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME. At this point, AgNPs / PMLA / LPS / Apt / AuNPs / AuUME was used as the working electrode, platinum wire as the counter electrode, and PME as the reference electrode, placed in 5 μL of 0.1 M KCl solution for signal amplification via SWV. The linear regression equation is expressed as I(μA)=35.3595logc(10) -3 pg / mL) +108.7291, R 2 =0.9971; the test result is obtained.

[0041] Example 3:

[0042] 1. Fabrication of ultramicroelectrodes:

[0043] A clean gold microelectrode was placed in an electrolyte solution consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. Cyclic voltammetry was used to modify the gold microelectrode within a potential range of 0.4 to -0.5 V at a scan rate of 20 mV / s for two cycles, yielding AuNCs / Au UME. 5 μL of aptamer was drop-cast onto AuNCs / Au UME and incubated for 1 hour to obtain Apt / AuNCs / Au UME. Apt / AuNCs / Au UME was then immersed in 2 mg / mL bovine serum albumin for 15 min to avoid non-specific binding of the analyte, resulting in the BSA / Apt / AuNCs / Au UME microelectrode.

[0044] 2. Application of the ultramicroelectrode prepared by the above method in the detection of lipopolysaccharides

[0045] BSA / Apt / AuNCs / Au UME was incubated in a lipopolysaccharide-containing sample for 0.5 hours to obtain LPS / Apt / AuNCs / Au UME. Then, LPS / Apt / AuNCs / Au UME was incubated for 30 minutes in a 3 mM ZrOCl2 solution prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME. An initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, bringing Zr... 4+ / LPS / Apt / AuNPs / Au UME was immersed in a 0.5 mM BPAA solution prepared with 40% (v / v) 99.7% anhydrous ethanol and 60% water and incubated for 0.5 hours to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME. Then, BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME were immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 10 mM copper bromide, 5.4 mM MLA, 8.6 μM TPMA, 0.5 mg CQDs, and 0.36 mL deionized water. The polymerization was carried out under LED light at room temperature on BPAA / Zr... 4+ The surface of / LPS / Apt / AuNPs / Au UME was activated for 1.5 min to obtain PMLA / LPS / Apt / AuNPs / Au UME. Finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 10 mmol / L Ag(NH3)2OH solution for 20 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME. AgNPs / PMLA / LPS / Apt / AuNPs / AuUME was then used as the working electrode, platinum wire as the counter electrode, and PME as the reference electrode, placed in 5 μL of 0.1 M KCl solution for signal amplification via SWV. The linear regression equation is expressed as I(μA) = 35.3595logc(10... -3 pg / mL) +108.7291, R 2 =0.9971; the test result is obtained.

[0046] Example 4:

[0047] 1. Fabrication of ultramicroelectrodes:

[0048] A clean gold microelectrode was placed in an electrolyte solution consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. Cyclic voltammetry was used to modify the gold microelectrode within a potential range of 0.4 to -0.5 V at a scan rate of 20 mV / s for two cycles, yielding AuNCs / Au UME. 5 μL of aptamer was drop-cast onto AuNCs / Au UME and incubated for 4 hours to obtain Apt / AuNCs / Au UME. Apt / AuNCs / Au UME was then immersed in 2 mg / mL bovine serum albumin for 15 min to avoid non-specific binding of the analyte, resulting in the BSA / Apt / AuNCs / Au UME microelectrode.

[0049] 2. Application of the ultramicroelectrode prepared by the above method in the detection of lipopolysaccharides

[0050] BSA / Apt / AuNCs / Au UME was incubated in a lipopolysaccharide-containing sample for 2 hours to obtain LPS / Apt / AuNCs / Au UME. Then, LPS / Apt / AuNCs / Au UME was incubated for 60 minutes in a 7 mM ZrOCl2 solution prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME. An initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, bringing Zr... 4+ / LPS / Apt / AuNPs / Au UME was immersed in a 2 mM BPAA solution prepared with 40% (v / v) 99.7% anhydrous ethanol and 60% water and incubated for 2 hours to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME. Then, BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME were immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 40 mM copper bromide, 21.6 mM MLA, 34.4 μM TPMA, 2 mg CQDs, and 0.36 mL deionized water. The polymerization was carried out under LED light at room temperature on BPAA / Zr... 4+ The surface of / LPS / Apt / AuNPs / Au UME was activated for 3.5 min to obtain PMLA / LPS / Apt / AuNPs / AuUME. Finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 20 mmol / L Ag(NH3)2OH solution for 80 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME. At this point, AgNPs / PMLA / LPS / Apt / AuNPs / Au UME was used as the working electrode, platinum wire as the counter electrode, and PME as the reference electrode, placed in 5 μL of 0.1 M KCl solution for signal amplification via SWV. The linear regression equation is expressed as I(μA)=35.3595logc(10) -3 pg / mL) +108.7291, R 2 =0.9971; the test result is obtained.

[0051] Example 5:

[0052] 1. Fabrication of ultramicroelectrodes:

[0053] A clean gold microelectrode was placed in an electrolyte solution consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. Cyclic voltammetry was used to modify the gold microelectrode within a potential range of 0.4 to -0.5 V at a scan rate of 20 mV / s for two cycles, yielding AuNCs / Au UME. 5 μL of aptamer was drop-cast onto AuNCs / Au UME and incubated for 8 hours to obtain Apt / AuNCs / Au UME. Apt / AuNCs / Au UME was then immersed in 2 mg / mL bovine serum albumin for 15 min to avoid non-specific binding of the analyte, resulting in the BSA / Apt / AuNCs / Au UME microelectrode.

[0054] 2. Application of the ultramicroelectrode prepared by the above method in the detection of lipopolysaccharides

[0055] BSA / Apt / AuNCs / Au UME was incubated in a lipopolysaccharide-containing sample for 4 hours to obtain LPS / Apt / AuNCs / Au UME. Then, LPS / Apt / AuNCs / Au UME was incubated for 75 minutes in a 9 mM ZrOCl2 solution prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME. An initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, bringing Zr... 4+ / LPS / Apt / AuNPs / Au UME was immersed in a 4 mM BPAA solution prepared with 40% (v / v) 99.7% anhydrous ethanol and 60% water and incubated for 4 hours to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME. Then, BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME were immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 80 mM copper bromide, 43.2 mM MLA, 68.8 μM TPMA, 4 mg CQDs, and 0.36 mL deionized water. The polymerization was carried out under LED light at room temperature on BPAA / Zr... 4+The surface of / LPS / Apt / AuNPs / Au UME was activated for 4.5 min to obtain PMLA / LPS / Apt / AuNPs / Au UME. Finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 40 mmol / L Ag(NH3)2OH solution for 160 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME. At this point, AgNPs / PMLA / LPS / Apt / AuNPs / AuUME was used as the working electrode, platinum wire as the counter electrode, and PME as the reference electrode, placed in 5 μL of 0.1 M KCl solution for signal amplification via SWV. The linear regression equation is expressed as I(μA)=35.3595logc(10) -3 pg / mL) +108.7291, R 2 =0.9971; the test result is obtained.

[0056] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. An application of an ultramicroelectrode in the detection of lipopolysaccharides, characterized in that, The microelectrode BSA / Apt / AuNCs / Au UME was incubated in samples containing lipopolysaccharides for 0.25–4 hours to obtain LPS / Apt / AuNCs / Au UME; LPS / Apt / AuNCs / Au UME was incubated in a 1–9 mM ZrOCl2 solution prepared with 20% (v / v) 99.7% anhydrous ethanol and 80% water for 15–75 minutes to obtain Zr. 4+ / LPS / Apt / AuNPs / Au UME; Initiator is introduced via phosphate-ZrOCl2-carboxylate complexation, Zr 4+ / LPS / Apt / AuNPs / Au UME is immersed in a 0.25–4 mM α-bromophenylacetic acid solution prepared with 40% (v / v) 99.7% anhydrous ethanol and 60% water and incubated for 0.25–4 hours to obtain BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME; change BPAA / Zr 4+ / LPS / Apt / AuNPs / Au UME were immersed in a carbon quantum dot-catalyzed ultrafast visible light-induced atom transfer radical polymerization solution. 1 mL of the polymerization solution contained 5-80 mM copper bromide, 2.7-43.2 mM methacrolein, 4.3-68.8 μM tris(2-pyridinemethyl)amine, 0.25-4 mg carbon quantum dots, and 0.36 mL deionized water. The polymerization was carried out at room temperature and under LED light in a BPAA / Zr... 4+ The surface of / LPS / Apt / AuNPs / Au UME was activated for 0.5-4.5 min to obtain PMLA / LPS / Apt / AuNPs / Au UME; finally, PMLA / LPS / Apt / AuNPs / Au UME was immersed in 5 mL of 5-25 mmol / L Ag(NH3)2OH solution for 10-160 min to obtain AgNPs / PMLA / LPS / Apt / AuNPs / Au UME; AgNPs / PMLA / LPS / Apt / AuNPs / Au UME was used as the working electrode, platinum wire as the counter electrode, and platinum-hydrogen micro-reference electrode as the reference electrode, and the signal was amplified by SWV in 5 μL of 0.1 M KCl solution; and the result was expressed as a linear regression equation. I( μA) = 35.3595log c +108.7291, R 2 =0.9971; the test result is obtained; I The unit is μA; c This refers to the concentration of lipopolysaccharide, expressed in units of 10. -3 pg / mL; The specific steps for fabricating the ultramicroelectrode BSA / Apt / AuNCs / Au UME are as follows: S1. A clean gold microelectrode was placed in an electrolyte consisting of 0.366 wt% HCl, 0.293 mg / mL HAuCl4, and 0.0244 mol / L NaCl. The gold microelectrode was modified using cyclic voltammetry with a potential range of 0.4 ~ -0.5 V and a scan rate of 20 mV / s for 2 cycles to obtain AuNCs / Au UME. S2. Drop casting 5 μL of aptamer onto AuNCs / Au UME and incubating for 0.5–8 hours yields Apt / AuNCs / Au UME; S3. Soak Apt / AuNCs / Au UME in 2 mg / mL bovine serum albumin for 15 min to obtain the microelectrode BSA / Apt / AuNCs / Au UME.

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