CN114460159B - ALP activity detection kit based on photo-ATRP signal amplification strategy and its use method - Google Patents

Abstract

The invention discloses an ALP activity detection kit based on photo-ATRP signal amplification strategy and its use method. The kit includes the following raw materials: electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, Me ₆ TREN, FMMA, LiClO ₄ . The present invention uses the photo-ATRP reaction as a polymerization reaction signal amplification strategy to significantly improve the sensitivity of ALP activity detection, while avoiding the use of transition metal catalysts, and has the advantages of high efficiency, convenient operation, and environmental friendliness. Under optimal experimental conditions, the linear range of this method for ALP activity detection is 10-150mU/mL, and the limit of detection (LOD) is 2.12mU/mL, indicating that the method of the present invention has good sensitivity. Moreover, the method of the present invention has satisfactory selectivity, anti-interference, reproducibility and stability. The method has obtained good experimental results in clinical serum samples and inhibition rate experiments.

Description

ALP activity detection kit based on photo-ATRP signal amplification strategy and its use How to use

Technical field

The invention relates to an alkaline phosphatase (ALP) activity detection kit and a method of use based on a light-mediated atom transfer radical polymerization (photo-ATRP) signal amplification strategy, and belongs to the field of biological analysis technology.

Background technique

Alkaline phosphatase (ALP) is a homodimeric metalloprotease widely present in prokaryotes and eukaryotes. The zinc and magnesium atoms in its structure play a crucial role in the dephosphorylation of ALP. role. Under alkaline conditions, ALP can catalyze the hydrolysis of phosphate monoester structures in proteins, nucleic acids and small molecules. Therefore, ALP plays an important role in physiological functions such as cell division, osteogenesis, mineralization, and detoxification. Studies have shown that abnormal ALP activity is closely related to a variety of diseases. For example, elevated ALP activity in serum is often related to diseases such as biliary obstruction, osteoblastic bone tumors, osteomalacia, leukemia-like reaction or lymphoma; on the contrary, some metabolic diseases , such as Wilson's disease, chronic myelogenous leukemia, etc., will cause pathological inhibition of ALP activity, making its activity lower than the normal range. Therefore, ALP activity detection is of great significance for the diagnosis and treatment of related diseases. At present, the detection methods of ALP mainly include chromatography, surface-enhanced Raman scattering, chemiluminescence, fluorescence, colorimetry, electrochemiluminescence, etc. With the further understanding of the role of ALP in different body fluids or cellular locations, and the continuous expansion of sample types, higher requirements have been placed on the precision and sensitivity of ALP detection. Therefore, the development of sensitive, simple, and efficient ALP detection methods is an urgent problem that needs to be solved.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an ALP activity detection kit and its use method based on the photo-ATRP signal amplification strategy, which not only avoids the use of transition metal catalysts, but also significantly increases the electrochemical signal. output, with the advantages of high sensitivity, good selectivity, simple operation, and environmental friendliness.

In order to achieve the above objects, the technical solution of the present invention is:

An ALP activity detection kit based on photo-ATRP signal amplification strategy, including the following raw materials: electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, Me ₆ TREN, FMMA, LiClO ₄ .

When used, prepare some raw materials into solutions, in which the concentration of MPA solution is 10mM, the concentration of EDC in the EDC/NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, and the concentration of BIBB solution is 10mM, EY The solution concentration is 5mM, the Me ₆ TREN solution concentration is 10mM, the FMMA solution concentration is 10mM, and the LiClO ₄ solution concentration is 1M.

A method of using an ALP activity detection kit, including the following steps:

(1)MPA modification

Soak the gold electrode in the MPA solution and incubate at 25-37°C for 2-8 hours;

(2) Activation and modification of MPA carboxyl group phosphoethanolamine

Soak the electrode obtained in step (1) in the EDC/NHS mixed solution and incubate it at 37°C for 0.5 to 1 hour. Then soak the electrode in the O-phosphoethanolamine solution and incubate it at 37°C for 1 to 2 hours;

(3)ALP dephosphorylation and BIBB modification

Drop the solution to be detected on the surface of the electrode obtained in step (2), and incubate it at 37°C for 1 to 2 hours. Then soak the electrode in the BIBB solution and incubate it at 37°C for 0.5 to 2 hours;

(4) Light-mediated ATRP reaction

Soak the electrode obtained in step (3) in the photo-ATRP reaction solution, irradiate it with blue light, and react at 25-37°C for 3-6 hours;

(5)SWV detection

The electrode obtained in step (4) was immersed in the LiClO ₄ solution, and square wave voltammetry (SWV) electrochemical measurement was performed.

Further, the gold electrode is pretreated first. The pretreatment method is:

① Ultrasonically clean the gold electrode with absolute ethanol and water respectively, then polish the electrode with 0.3μm and 0.05μm alumina polishing powder respectively, and ultrasonically wash with absolute ethanol and ultrapure water respectively;

②After washing, soak the gold electrode in piranha acid solution, and then wash it ultrasonically with absolute ethanol and ultrapure water;

③ The electrode is soaked in 0.5M sulfuric acid solution, set the potential to -0.3~1.5V, the scanning rate is 0.1V/s, and the number of scanning segments is 40 until a repeatable cyclic voltammogram is obtained. Ultrasonic cleaning with ultrapure water and nitrogen blowing Dry and set aside.

Further, the photo-ATRP reaction solution was mixed with 5 mL ultrapure water, 3990 μL DMSO, 5 μL EY solution, 5 μL Me ₆ TREN solution, and 1 mL FMMA solution.

Furthermore, the scanning range of square wave voltammetry (SWV) electrochemical measurement is 0~0.6V potential.

Application of the above-mentioned kit in detecting ALP activity.

Application of the above-mentioned kit in screening ALP inhibitors.

A schematic diagram of the detection principle of the present invention is shown in Figure 1.

Beneficial effects of the present invention:

1. The present invention uses the photo-ATRP reaction as a polymerization reaction signal amplification strategy to significantly improve the sensitivity of ALP activity detection.

2. The present invention uses the photo-ATRP reaction as a signal amplification strategy to avoid the use of transition metal catalysts, and has the advantages of high efficiency, easy operation, and environmental friendliness.

3. The ALP activity detection method of the present invention based on the photo-ATRP signal amplification strategy first fixes 3-mercaptopropionic acid (MPA) on the surface of the gold electrode through self-assembly of gold-sulfur bonds, so that the ALP substrate O-phosphoethanolamine can pass through The amide bond is connected to the electrode surface; in the presence of ALP, the phosphate monoester structure in O-phosphoethanolamine is hydrolyzed into a hydroxyl group, which can be connected with the ATRP initiator BIBB; finally, under 470nm blue light irradiation, polymerization is carried out using EY as the photocatalyst The reaction causes ferrocenyl methyl methacrylate (FMMA) monomer to polymerize on the electrode surface. Grafting of polymers containing a large amount of ferrocene signal tags significantly enhanced the electrochemical signal output. Under optimal experimental conditions, the linear range of this method for ALP activity detection is 10-150mU/mL, and the limit of detection (LOD) is 2.12mU/mL, indicating that the method of the present invention has good sensitivity. Moreover, the method of the present invention has satisfactory selectivity, anti-interference, reproducibility and stability. The method has obtained good experimental results in clinical serum samples and inhibition rate experiments. Therefore, the photo-ATRP signal amplification strategy of the present invention has good application potential in clinical ALP-related disease diagnosis and inhibitor screening.

Description of drawings

Figure 1 is a schematic diagram of the detection principle of the present invention.

Figure 2 is a feasibility diagram of the method of the present invention used for ALP activity detection.

Figure 3 shows the electrochemical impedance spectroscopy (EIS) diagram of the gradually modified electrode.

Figure 4 shows the CV diagram of the modified electrode at different scan rates (inline diagram: represents the linear relationship between the reduction and oxidation current peaks and different scan rates).

Figure 5 is an atomic force microscope (AFM) image of the modified electrode before and after polymerization. A is before the photo-ATRP reaction, and B is after the photo-ATRP reaction.

Figure 6 is a water contact angle (WCA) diagram of the gradually modified electrode.

Figure 7 shows the optimization of BIBB reaction time.

Figure 8 shows the optimization of the concentration ratio of EY and Me ₆ TREN.

Figure 9 shows the linear relationship between ALP activity and electrical signal intensity.

Figure 10 shows the selectivity study of ALP detection.

Figure 11 is a study on the anti-interference property of the electrochemical detection of the present invention.

Figure 12 shows the study on the inhibition rate of sodium vanadate on ALP activity.

Detailed ways

The specific embodiments of the present invention will be further described in detail below with reference to examples.

Example 1: Kit

An ALP activity detection kit based on the photo-ATRP signal amplification strategy, including the following raw materials: electrode, 3-mercaptopropionic acid (MPA), 1-(3-dimethylaminopropyl)-3-ethylcarbodioxide Amine hydrochloride (EDC), N-hydroxysuccinimide (NHS), O-phosphoethanolamine, 2-bromoisobutyryl bromide (BIBB), dimethyl sulfoxide (DMSO), eosin Y (EY) , Tris-(N,N-dimethylaminoethyl)amine (Me ₆ TREN), ferrocenyl methyl methacrylate (FMMA), LiClO ₄ .

When used, prepare some raw materials into solutions, in which the concentration of MPA solution is 10mM, the concentration of EDC in the EDC/NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, and the concentration of BIBB solution is 10mM, EY The solution concentration is 5mM, the Me ₆ TREN solution concentration is 10mM, the FMMA solution concentration is 10mM, and the LiClO ₄ solution concentration is 1M.

Example 2: ALP activity detection method

(1)MPA modification

Soak the pretreated gold electrode in 400 μL 10mM MPA solution and incubate at 37°C for 2 hours;

(2) Activation and modification of MPA carboxyl group phosphoethanolamine

Soak the electrode obtained in step (1) in 400 μL EDC/NHS mixed solution (EDC concentration is 20mM, NHS concentration is 5mM), incubate at 37°C for 0.5h, and then soak the electrode in 400μL 10mM O-phosphoethanolamine solution, 37°C Incubate for 1h;

(3)ALP dephosphorylation and BIBB modification

Drop 10 μL of the solution to be detected (containing ALP) on the surface of the electrode obtained in step (2), and incubate it at 37°C for 1 hour. Then soak the electrode in 400 μL of 10mM BIBB solution and incubate it at 37°C for 1 hour;

(4) Light-mediated ATRP reaction

Soak the electrode obtained in step (3) in the photo-ATRP reaction solution (the reaction solution is obtained by adding 3990 μL DMSO, 5 μL 5mM EY solution, 5 μL 10mM Me ₆ TREN solution, and 1mL 10mM FMMA solution to 5mL of ultrapure water in sequence), Use 470nm blue light to illuminate and react at room temperature for 3 hours;

(5)SWV detection

The electrode obtained in step (4) was immersed in 1M LiClO ₄ solution, and square wave voltammetry (SWV) electrochemical measurement was performed under a scanning range of 0 to 0.6V potential.

The pretreatment method for gold electrodes is:

① Ultrasonically clean the gold electrode with absolute ethanol and water for 1 minute respectively, then polish the electrode with 0.3 μm and 0.05 μm alumina polishing powder for 3 to 5 minutes respectively, and ultrasonically wash it with absolute ethanol and ultrapure water for 1 minute;

② After washing, soak the gold electrode in the piranha acid solution for 15 to 20 minutes, and then ultrasonically wash it with absolute ethanol and ultrapure water for 1 minute; the preparation method of the piranha acid solution is: 98% H ₂ SO ₄ and H ₂ O ₂ is mixed in a volume ratio of 3:1;

③ The electrode is soaked in 0.5M sulfuric acid solution, set the potential to -0.3~1.5V, the scanning rate is 0.1V/s, and the number of scanning segments is 40 until a repeatable cyclic voltammogram is obtained. Ultrasonic cleaning with ultrapure water and nitrogen blowing Dry and set aside.

Example 3: Feasibility study

This study studied the feasibility of the method of the present invention in detecting alkaline phosphatase (ALP) activity through a series of blank control experiments. The SWV curves of various modified electrodes can be seen in Figure 2. Without adding MPA (curve b), O-phosphoethanolamine (curve c), ALP (curve d), BIBB (curve e), FMMA (curve f), EY (curve g), Me ₆ TREN (curve h) and In the absence of illumination (curve i), there is almost no electrochemical signal response except for a weak background signal. When the electrode is gradually modified according to the method of the present invention, an obvious oxidation current signal (curve a) can be observed, with a peak potential of about 0.28V, which is consistent with the potential range of ferrocene electroactive molecules in LiClO ₄ solution. The generation of oxidation current can be attributed to the electrochemical oxidation of ferrocene electroactive molecules, demonstrating that monomeric FMMA was successfully connected to the gold electrode through the photo-ATRP reaction. Therefore, this completely modified electrode is feasible for ALP activity detection.

Example 4: Electrochemical characterization and morphological characterization

1. Electrochemical characterization

Electrochemical impedance spectroscopy (EIS) can sensitively detect the transition of the solid-liquid interface and is used to characterize the gradual modification of the electrode surface. In the Nyquist diagram, the diameter of the semicircle in the high-frequency region is equal to the charge transfer resistance (R _ct ). The EIS diagram of the gradually modified electrode of the present invention is shown in Figure 3. Due to the good conductivity of the gold electrode, the _Rct of the bare gold electrode is the smallest, about 0.29kΩ(a). The self-assembly of MPA on the electrode surface hinders electron transfer, causing R _ct to rise to 0.44kΩ (b). After the carboxyl group of MPA was activated by EDC and NHS, the negatively charged carboxyl group was replaced by succinimide ester, and R _ct dropped to 0.23kΩ (c). Modification of O-phosphoethanolamine increased _Rct to 0.63kΩ(d). After the modified electrode was incubated with ALP, _Rct decreased slightly (e). After modifying the initiator BIBB, R _ct further increased to 1.02kΩ(f). Finally, R _ct increases significantly to 1.99 kΩ(g) due to polymer chains grafted on the electrode surface. These results demonstrate that the construction of fully modified electrodes was successful and smooth. It was demonstrated that the electroactive polymer was successfully grafted onto the gold electrode via the photo-ATRP strategy.

In order to prove the successful occurrence of the ATRP reaction, cyclic voltammetry (CV) was used to scan different modified electrodes. CV scanning of different modified electrodes was performed in LiClO ₄ solution with a potential range of 0 to 0.6V. As can be seen from Figure 4, as the scan rate increases from 0.01V/s to 1.0V/s, the redox peak current changes linearly. This proves that the redox process of ferrocene molecules is not through electrostatic adsorption, but is connected to the gold electrode surface through covalent bonds.

2. Morphological characterization

The morphology of the modified electrode was observed with atomic force microscopy (AFM). By comparing the change in surface height of the gold electrode before and after polymerization, it was verified that the monomer successfully formed a polymer chain through the targeted ATRP reaction (Figure 5). As can be seen from Figure 5, after modifying the initiator BIBB, the height of the gold electrode is 8.9nm (A), and the surface height increases to 29.7nm (B) after the photo-ATRP reaction. The increase in the surface height of the gold electrode proves that the monomers successfully formed polymers through the photo-ATRP reaction.

According to the different hydrophilicity of the surface groups of the modified electrode, the water contact angle (WCA) was used to characterize the stepwise modified electrode. The results are shown in Figure 6. Since the gold electrode has strong hydrophobicity, the WCA of the bare gold electrode is 90.6°. After MPA modification of the gold electrode, the introduction of carboxyl groups reduced the WCA (84.7°). Subsequently, the WCA increased (88.2°) due to the hydrophobic O-phosphoethanolamine modification of the carbon chain. After ALP catalyzes the hydrolysis of O-phosphoethanolamine, the generation of hydroxyl groups reduces the WCA to 86.6°. Next, modification of the initiator BIBB resulted in an increase in WCA (90.2°). Finally, the WCA increased significantly to 93.6° due to the high hydrophobicity of the polymer chain. The changes in WCA further indicate that the electrode modification was successful.

Example 5: Optimization of detection conditions

In order to achieve the best performance of the detection method, the present invention studied the reaction (incubation) time of BIBB and the molar concentration ratio of EY and ME ₆ TERN.

(1) Optimization of BIBB reaction time

In this study, SWV was used to record the signal response of BIBB modification time within 15 to 90 minutes. The results are shown in Figure 7. The SWV response gradually increases with the extension of reaction time. Subsequently, there was no significant change in current intensity after 60 min. Therefore, the optimal reaction time of BIBB in this method is 60 min and should be used in subsequent research.

(2) Optimization of the concentration ratio of EY and Me ₆ TREN

This study is based on the redox reaction of ferrocene molecules. The molar ratio of EY to ME ₆ TERN is key and must be optimized. Control the volumes of EY and ME ₆ TERN to both be 5 μL, the concentration of ME ₆ TERN to be 10mM, and change the EY concentration. The results are shown in Figure 8. The current signal increases as the EY concentration decreases, reaching a maximum value when the molar ratio of EY to ME ₆ TERN is 0.5:1. As the molar ratio of EY to ME ₆ TERN gradually becomes less than 0.5:1, the current signal decreases rapidly with the decrease of EY concentration. Therefore, the optimal molar ratio of EY to ME ₆ TERN is 0.5:1.

In summary, the optimal reaction time of BIBB is 60 min, and the optimal molar concentration ratio of EY and ME ₆ TERN is 0.5:1.

Example 6: Analyzing performance

Under the optimal experimental conditions obtained in Example 5, a series of ALPs with different activities were used to study the detection performance of the method of the present invention. The results are shown in Figure 9. In the range of ALP activity of 10 to 150 mU/mL, the current intensity increases with the increase of ALP activity, and shows a good linear relationship. The linear equation is I (μA) = 0.01933C _ALP ( mU/mL)+0.19188, R ² =0.998, and the calculated lowest detection limit LOD is 2.12mU/mL. Compared with reported ALP activity detection methods, this strategy has relatively high sensitivity. Therefore, the proposed method has good application potential for clinical detection of ALP activity.

Example 7: Selectivity, anti-interference ability, stability and reproducibility

In order to verify the selectivity of the strategy of the present invention for detecting ALP activity, under the optimal experimental conditions obtained in Example 5, pepsin (Pepsin), bovine serum albumin (BSA), glucose oxidase ( GOx) and ALP oxidation current intensity. The concentrations of ALP, Pepsin, GOx and BSA were 50mU/mL, 50mU/mL, 50mU/mL and 50μM respectively. The results are shown in Figure 10. An obvious current response was only observed in the ALP group. The results show that this strategy has high selectivity for the activity detection of ALP.

In order to evaluate the anti-interference ability of the constructed fully modified electrode, the electrochemical signals of different active ALPs in Tris-HCl buffer and 10% human serum were compared under the optimal experimental conditions obtained in Example 5. The results are shown in Figure 11. The current signal of 10% human serum was 97.5% (80mU/mL), 100.4% (100mU/mL) and 97.4% (120mU/mL) of Tris-HCl buffer. The results show that the method of the present invention has good anti-interference ability in serum matrix.

In order to verify the stability of the completely modified electrode, the modified electrode was stored in a water-saturated environment at 4°C for 3 weeks and then tested. It was found that the current intensity was 92.30% of the signal intensity of the newly prepared electrode. This indicates that the fully modified electrode has good stability. The reproducibility of the fully modified electrode was evaluated through intra-group and inter-group experiments (n=5), and the relative standard deviations within and between groups were 3.54% and 4.13%, respectively. The results show that the fully modified electrode has good reproducibility.

Example 8: Detection performance in real samples

Clinical human serum samples were used to verify the reliability of the method of the present invention. Five clinical human serum samples were obtained from the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine. The ALP activity in the serum was detected using the method of the present invention and compared with the provided clinical data (AMP buffer solution method detection) for comparison. The results are shown in Table 1. The relative error between the results obtained by the method of the present invention and the clinical data is less than 5%, indicating that the detection method of the present invention is reliable.

Table 1: Comparison of results between the AMP buffer method and the method of the present invention

Example 9: Inhibition rate experiment

In order to study the effectiveness of the method of the present invention in screening ALP activity inhibitors, an inhibition rate experiment was conducted using sodium vanadate Na ₃ VO ₄ as a model. Preactivated Na ₃ VO ₄ solutions of different concentrations were mixed with ALP samples, and the inhibition of ALP activity by Na ₃ VO ₄ was studied through changes in current intensity. The results are shown in Figure 12. As the concentration of Na ₃ VO ₄ increases, the inhibition efficiency also increases. It shows that the inhibition of ALP activity by Na ₃ VO ₄ is concentration-dependent. Therefore, it is proved that the completely modified electrode of the present invention is suitable for screening alkaline phosphatase inhibitors.

Claims (6)

1. ALP activity detection kit based on photo-ATRP signal amplification strategy, characterized by comprising the following raw materials: electrode, MPA, EDC, NHS, O-phosphoethanolamine, BIBB, DMSO, EY, me ₆ TREN、FMMA、LiClO ₄ The method comprises the steps of carrying out a first treatment on the surface of the The application method comprises the following steps:

(1) MPA modification of 3-mercaptopropionic acid

Immersing a gold electrode in an MPA solution, and incubating at 25-37 ℃ for 2-8 h;

(2) Activation of MPA carboxyl group and modified phosphoethanolamine

Immersing the electrode obtained in the step (1) in EDC/NHS mixed solution, incubating at 37 ℃ for 0.5-1 h, immersing the electrode in O-phosphoethanolamine solution, and incubating at 37 ℃ for 1-2 h;

(3) ALP dephosphorylation and BIBB modification

Dropping the solution to be detected on the surface of the electrode obtained in the step (2), incubating 1-2 h at 37 ℃, then soaking the electrode in BIBB solution, and incubating 0.5-2 h at 37 ℃;

(4) Light mediated ATRP reaction

Immersing the electrode obtained in the step (3) in a photo-ATRP reaction liquid, irradiating with blue light, and reacting at 25-37 ℃ for 3-6 h;

(5) SWV detection

Immersing the electrode obtained in the step (4) into LiClO ₄ In the solution, performing square wave voltammetry SWV electrochemical measurement;

the photo-ATRP reaction solution was prepared from 5mL ultrapure water, 3990. Mu.L DMSO, 5. Mu.L EY solution, and 5. Mu.L Me ₆ The TREN solution and 1mL of FMMA solution were mixed.

2. The ALP activity detection kit according to claim 1, whereinWhen in use, part of raw materials are prepared into a solution, wherein the concentration of MPA solution is 10mM, the concentration of EDC in EDC/NHS mixed solution is 20mM, the concentration of NHS is 5mM, the concentration of O-phosphoethanolamine solution is 10mM, the concentration of BIBB solution is 10mM, the concentration of EY solution is 5mM, and Me ₆ TREN solution concentration of 10mM, FMMA solution concentration of 10mM, liClO ₄ The solution concentration was 1M.

3. The ALP activity detection kit of claim 1, wherein the gold electrode is pretreated by:

(1) respectively ultrasonically cleaning a gold electrode by using absolute ethyl alcohol and water, polishing the electrode by using alumina polishing powder with the particle size of 0.3 mu m and alumina polishing powder with the particle size of 0.05 mu m, and respectively ultrasonically cleaning the electrode by using absolute ethyl alcohol and ultrapure water;

(2) after washing, the gold electrode is put into a water tiger fish acid solution to be soaked, and then ultrasonic washing is respectively carried out by absolute ethyl alcohol and ultrapure water;

(3) the electrode is soaked in 0.5M sulfuric acid solution, the potential is set to be-0.3-1.5V, the scanning speed is 0.1V/s, the scanning section number is 40, until a repeatable cyclic voltammogram is obtained, ultra-pure water is used for ultrasonic cleaning, and nitrogen is used for drying for standby.

4. The ALP activity detection kit of claim 1, wherein the square wave voltammetry SWV electrochemical measurement is scanned at a potential ranging from 0 to 0.6V.

5. Use of a kit according to claim 1 or 2 for detecting ALP activity.

6. Use of a kit according to claim 1 or 2 for screening an ALP inhibitor.