Ultra-low potential electrochemiluminescence carboxylesterase activity detection biosensor and kit

Abstract

The present invention provides an ultra-low potential electrochemiluminescent carboxylesterase activity detection biosensor and a kit, relating to the field of electrochemiluminescent detection technology. The present invention provides a novel electrochemiluminescent material perylene imide derivatives (PTC derivatives), using the electrochemiluminescent material to detect carboxylesterase activity, with ultra-low potential (-0.4V) and high-efficiency electrochemiluminescence characteristics, while it also has excellent film-forming properties. The present invention modifies the electrode with the obtained electrochemiluminescent material to construct an electrochemiluminescent carboxylesterase activity detection biosensor, and the obtained "on-off" type carboxylesterase activity detection biosensor and kit have the significant advantages of ultra-low potential, high sensitivity, and label-free when performing carboxylesterase activity detection.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence detection, in particular to an ultra-low potential electrochemiluminescence carboxylesterase activity detection biosensor and a kit. Background Art

[0002] Carboxylesterase (CES) is an important serine hydrolase that plays a crucial role in biological processes such as the hydrolysis and metabolism of many endogenous esters and ester-based drugs. Studies have shown that abnormal expression of CES is closely associated with metabolic diseases such as hyperlipidemia, alcoholic liver damage, and obesity. Furthermore, compared to alpha-fetoprotein, a typical serological biomarker for hepatocellular carcinoma, CES as a biomarker can more effectively distinguish hepatocellular carcinoma, a highly lethal disease, from other forms of liver disease in early diagnosis (Sherman et al., 2011; Yin et al., 2023). Therefore, developing a rapid, convenient, low-cost, highly sensitive, and specific CES detection method is of great significance for the early diagnosis and prognostic assessment of related diseases.

[0003] Currently, commercial carboxylesterase activity detection kits are based on colorimetry, but colorimetry has low sensitivity and is susceptible to interference from complex matrices in serum, prone to false positive or false negative results. Methods for detecting carboxylesterase activity based on fluorescent probes have been reported (CN102788776B and CN107502652A), but are susceptible to the influence of autofluorescence and scattered light, and have the disadvantages of poor selectivity, high detection cost, complex sample pretreatment, expensive detection equipment, low detection sensitivity, and high background signal. Therefore, there is an urgent need to develop a highly sensitive, highly specific, low-cost, fast and convenient carboxylesterase detection method.

[0004] Electrochemiluminescence (ECL) provides an opportunity for the highly sensitive and specific detection of carboxylesterases due to its advantages such as cheap and readily available equipment, high sensitivity, low background signal, and good controllability. However, there is still a lack of reports on electrochemiluminescent carboxylesterase detection methods, and there is an urgent need to develop an electrochemiluminescent carboxylesterase detection kit. The successful development of a carboxylesterase kit requires overcoming the following two points: first, solving the problem of high potential and difficulty in immobilization of commercial luminescent systems; second, finding a suitable carboxylesterase catalytic substrate and cleverly constructing a label-free signal conversion switch to overcome the time-consuming and error-prone sensor caused by labeling and interface assembly. Ru(bpy)3 2+ Biochemical technologies related to the Ru(bpy)3 / tripropylamine luminescence system have been widely used in commercial in vitro clinical diagnosis. 2+The Ag / tripropylamine system is difficult to immobilize and has an excitation potential of approximately +1.2 V (vs Ag / AgCl). 1.2 V, close to the electrolysis potential of water, can cause bubbles to form on the electrode surface, resulting in damage to thinner electrodes due to electrode contamination and separation from the substrate (Peng et al., 2022). In addition, high trigger potentials may interfere with the stability of biomolecules (Ding et al., 2021). ECL systems with low trigger potentials have the advantages of less inherent electrochemical interference and improved long-term stability of the working electrode. Therefore, constructing ECL systems that are easy to form films and have low excitation potentials is a key driving force for the commercial application of ECL biochemical technology.

[0005] 3,4,9,10-Perylenetetracarboxylic dianhydride (PTCDA) is considered a promising ECL luminescent material due to its excellent film-forming properties, easy immobilization, large planar π-conjugation, large specific surface area, and excellent photoelectric properties. However, PTCDA's rigid coplanar configuration leads to strong intermolecular π-π stacking, resulting in aggregation-induced quenching (AIS), which significantly reduces its ECL performance. Professor Zhuo Ying of Southwest University has partially addressed the AIS problem in PTCDA-based electrochemiluminescent materials by condensing PTCDA with melamine at high temperature to form a porous covalent organic framework. However, the excitation potential of the luminescent material prepared by this method is too negative (-1.6 V, Zhuo et al., 2022). This excessively negative excitation potential exceeds the hydrogen potential of water decomposition, which can easily interfere with the electrode and affect its stability. Therefore, addressing the aggregation quenching and overly negative AIS caused by PTCDA's rigidity is of great significance for the construction of biosensors and test kits. Furthermore, specific target recognition is currently typically achieved through complementary base pairing of antigens / antibodies or nucleic acids, which involves labeling the probe with the recognition element. This is time-consuming and labor-intensive, and the number of labels is limited, impacting the practicality and performance of the sensor. Given this, screening for label-free carboxylesterase sensors and kits with catalytic substrates that respond to electrochemiluminescent carboxylesterases and trigger electrochemiluminescent signal conversion is both challenging and significant.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention aims to provide an ultra-low potential electrochemiluminescent biosensor and kit for detecting carboxylesterase activity. To address the shortcomings of existing carboxylesterase detection methods, which suffer from poor selectivity, high detection costs, complex sample pretreatment, expensive detection equipment, low detection sensitivity, and high background signals, the present invention proposes an ultra-low potential, highly sensitive, label-free electrochemiluminescent biosensor and kit for detecting carboxylesterase activity.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] One of the technical solutions of the present invention is to provide an electrochemiluminescent material having a structure shown in any one of Formulas I to III:

[0010]

[0011] The second technical solution of the present invention is to provide a method for preparing the electrochemiluminescent material, comprising the following steps:

[0012] hydrothermally reacting 3,4,9,10-perylenetetracarboxylic dianhydride with a nucleophilic reagent to obtain the electrochemiluminescent material;

[0013] The nucleophile is N,N-dimethylaminoethylenediamine, N,N-diethylethylenediamine or N,N-diisopropylethylenediamine. The preferred nucleophile is N,N-diisopropylethylenediamine.

[0014] 3,4,9,10-perylenetetracarboxylic dianhydride is hydrothermally reacted with N,N-dimethylaminoethylenediamine, N,N-diethylethylenediamine and N,N-diisopropylethylenediamine to obtain compounds of the structures represented by Formula I, Formula II and Formula III, respectively.

[0015] Furthermore, the temperature of the hydrothermal reaction is 50° C.-200° C.; the time of the hydrothermal reaction is 3-12 hours; and the molar ratio of the 3,4,9,10-perylenetetracarboxylic dianhydride to the nucleophilic reagent is 1:1-1:50.

[0016] The third technical solution of the present invention is to provide a biosensor comprising an electrode and the electrochemiluminescent material modified on the surface of the electrode.

[0017] A fourth technical solution of the present invention provides a method for preparing the above-mentioned biosensor, comprising the following steps:

[0018] The biosensor is obtained by modifying the electrochemiluminescent material (ECL probe) on the surface of the electrode.

[0019] The fifth technical solution of the present invention: provides the use of the above-mentioned electrochemiluminescent material or the above-mentioned biosensor in the detection of carboxylesterase activity.

[0020] Technical solution six of the present invention: provides a kit, comprising the above-mentioned biosensor, a catalytic substrate and a co-reaction reagent; the catalytic substrate is phenyl acetate, phenyl propionate, 1-naphthyl acetate, 1-naphthyl propionate, naphthol benzoate or 1,8-naphthyl dibenzoate; the preferred catalytic substrate is 1-naphthyl acetate.

[0021] The co-reactant is a salt containing persulfate.

[0022] The present invention provides a catalytic substrate that responds to carboxylesterase and can simultaneously trigger signal conversion. The molecular structure of the catalytic substrate is as follows:

[0023]

[0024] As a more preferred embodiment of the present invention, the kit further comprises a buffer, preferably a PBS buffer, having a pH of 7.0-8.0. This buffer at this pH ensures that the carboxylesterase on the biosensor efficiently catalyzes the substrate, thereby regulating the state of the biosensor's ECL signal.

[0025] Technical solution seven of the present invention: provides the use of the above-mentioned kit in the detection of carboxylesterase activity.

[0026] Furthermore, the application includes the following steps: incubating carboxylesterase in the presence of the catalytic substrate and the co-reactant, and monitoring the electrochemiluminescence response signal using the biosensor.

[0027] Furthermore, the concentration of the catalytic substrate is 2 mmol / L; the concentration of the persulfate ion in the co-reaction reagent is 15 mmol / L; and the incubation time is 30 min.

[0028] The present invention uses PTC-Dip produced by the nucleophilic substitution reaction of N,N-diisopropylethylenediamine and 3,4,9,10-perylenetetracarboxylic dianhydride to achieve the best effect.

[0029] The response mechanism of the biosensor of the present invention is as follows: in the absence of carboxylesterase, the electrochemiluminescent material exhibits extremely high ECL emission intensity. In the presence of carboxylesterase, the phenol or naphthol generated from the enzymatic reaction consumes the strong oxidative free radicals produced by the coreactant, thereby quenching the biosensor's ECL signal, resulting in a quenched ECL signal. This enables highly sensitive, label-free detection of carboxylesterase. In a preferred embodiment of the present invention, the biosensor's target molecule, carboxylesterase, catalyzes the substrate 1-naphthyl acetate to produce 1-naphthol, which quenches the electrochemiluminescence of PTC-Dip, resulting in a quenched sensor signal.

[0030] The present invention uses the π-conjugated molecule 3,4,9,10-perylenetetracarboxylic dianhydride as a matrix and uses the branched amino compounds N,N-dimethylaminoethylenediamine, N,N-diethylethylenediamine, and N,N-diisopropylethylenediamine as nucleophiles to synthesize a PTC-derivative ECL luminescent body with good film-forming properties, high luminous efficiency, and ultra-low potential under hydrothermal conditions. The structure of the luminescent body is as follows:

[0031]

[0032] The synthesis path of the above-mentioned PTC-derivative ECL luminophore of the present invention is as follows:

[0033]

[0034] The more specific synthesis steps of the PTC-derivative ECL luminophore of the present invention are as follows:

[0035] (1) The parent molecule 3,4,9,10-perylenetetracarboxylic dianhydride is dissolved in distilled water and ultrasonically shaken. A branched amino compound is then added as a nucleophile and ultrasonically shaken to uniformly disperse the two reactants in the distilled water. The uniformly dispersed mixed solution is then transferred to a hydrothermal reactor for a high-temperature hydrothermal reaction.

[0036] (2) After the hydrothermal reaction is completed, the reactor is cooled naturally to room temperature, and the product after the reaction is centrifuged and washed with a solvent for precipitation. The obtained precipitate is dried to obtain a solid powdered PTC-derivative ECL luminescent body.

[0037] The centrifugal condition is preferably 12000 r / min for 10 min; the washing solvent for centrifugation is distilled water; and the drying method of the solid precipitate is freeze drying.

[0038] The biosensor of the present invention does not require modification of the labeling probe; the carboxylesterase catalyzes the decomposition of the substrate to produce phenolic substances, which quench the electrochemical luminescence signal of the electrode, thereby realizing the detection of the carboxylesterase.

[0039] The present invention discloses the following technical effects:

[0040] The present invention provides a novel electrochemiluminescent material - perylene imide derivatives (PTC derivatives), which reacts with the coreaction reagent S2O8 2- Under the conditions of the presence of iodine, the carboxylesterase activity was detected with ultra-low potential (-0.4V), high electrochemiluminescence efficiency and excellent film-forming property.

[0041] The present invention modifies an electrode with the prepared electrochemiluminescent material to produce an electrochemiluminescent carboxylesterase activity detection biosensor. A kit is then constructed using the biosensor, a specific catalytic substrate, and a co-reactant. The on-off carboxylesterase activity detection biosensor and kit offer the significant advantages of ultra-low potential, high sensitivity, and label-free performance when performing carboxylesterase activity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 is a response mechanism diagram of the biosensor of the present invention;

[0044] Figure 2 This is a comparison chart of the ECL intensities of PTC-Dme, PTC-Det, and PTC-Dip of the present invention;

[0045] Figure 3 This is an optimization diagram of the amount of N,N-diisopropylethylenediamine and PTCDA substances of the present invention;

[0046] Figure 4 This is a temperature optimization diagram for the hydrothermal reaction of N,N-diisopropylethylenediamine and PTCDA of the present invention;

[0047] Figure 5 This is a reaction time optimization diagram for the hydrothermal reaction of N,N-diisopropylethylenediamine and PTCDA of the present invention;

[0048] Figure 6 : SEM images of PTCDA and PTC-Dip of the present invention; wherein A is the SEM image of PTCDA, and B is the SEM image of the reaction product PTC-Dip;

[0049] Figure 7 IR spectra of PTC-Dme, PTC-Det and PTC-Dip of the present invention;

[0050] Figure 8 The UV spectra of PTC-Dme, PTC-Det and PTC-Dip of the present invention are shown in FIG.

[0051] Figure 9 The fluorescence emission spectra of PTC-Dme, PTC-Det and PTC-Dip of the present invention (excitation light wavelength 496nm);

[0052] Figure 10 The ECL emission spectrum test results of PTC-Dip of the present invention;

[0053] Figure 11 The XPS spectra of PTCDA and PTC-Dip of the present invention are shown in Figure 1, wherein A is the XPS spectrum of PTCDA and B is the XPS spectrum of the reaction product PTC-Dip.

[0054] Figure 12CV graph (A) and corresponding voltage-light intensity ECL graph (B) of the PTC-Dip of the present invention;

[0055] Figure 13 Comparison of the ECL intensity of the PTC-Dip of the present invention (A) and the classic ECL intensity of ruthenium bipyridine (B);

[0056] Figure 14 This is a comparison chart of the signal responses produced by carboxylesterase catalyzing different substrates at the same concentration;

[0057] Figure 15 This is a graph showing the electrochemiluminescence response intensity of the biosensor of the present invention in different carboxylesterase activities;

[0058] Figure 16 is the electrochemiluminescence response working curve of the biosensor of the present invention in carboxylesterases with different activities;

[0059] Figure 17 is the specific response of the biosensor of the present invention. DETAILED DESCRIPTION

[0060] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0061] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0062] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0063] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0064] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0065] In the following examples, unless otherwise specified, all raw materials used are commercially available products.

[0066] Figure 1 Schematic diagram of the response mechanism of the biosensor of the present invention.

[0067] Example 1

[0068] Synthesis of electrochemiluminescent material PTC-Dip:

[0069] Accurately weigh 5 mg of 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and dissolve it in 8 mL of distilled water. Ultrasonicate for 5 minutes. Then, add twice the amount of N,N-diisopropylethylenediamine and ultrasonicate for 5 minutes to evenly disperse the reactants in the solvent. The resulting mixture is then transferred to a 30 mL reactor and reacted at 150°C for 8 hours. After the reaction, the reaction mixture is centrifuged at 12,000 rpm for 10 minutes. The precipitate is washed three times with distilled water and freeze-dried to obtain the luminescent solid powder PTC-Dip.

[0070] The previously freeze-dried phosphor powder PTC-Dip was weighed and prepared into a 1 mg / mL suspension with distilled water. Ultrasonic vibration was applied for 5 minutes to disperse the phosphor more evenly in the water. The suspension was then refrigerated at 4°C until use.

[0071] Example 2

[0072] Synthesis of electrochemiluminescent material PTC-Dme:

[0073] The only difference from Example 1 is that N,N-dimethylaminoethylenediamine is used to replace N,N-diisopropylethylenediamine in an equimolar amount to prepare PTC-Dme.

[0074] Example 3

[0075] Synthesis of electrochemiluminescent material PTC-Det:

[0076] The only difference from Example 1 is that N,N-diethylethylenediamine is used to replace N,N-diisopropylethylenediamine in an equimolar amount to prepare PTC-Det.

[0077] Figure 2 This is a solid-phase ECL intensity comparison chart of PTC-Dme, PTC-Det, and PTC-Dip at a concentration of 1 mg / mL in the present invention. The detection potential is 0-0.4 V, the photomultiplier tube high voltage is 600 V, the scan speed is 0.3 V / s, and the amplification level is 3.

[0078] Figure 3 This is an optimization diagram of the amount of N,N-diisopropylethylenediamine and PTCDA substances of the present invention; Figure 4 This is a temperature optimization diagram for the hydrothermal reaction of N,N-diisopropylethylenediamine and PTCDA of the present invention; Figure 5 This is a reaction time optimization diagram for the hydrothermal reaction of N,N-diisopropylethylenediamine and PTCDA of the present invention.

[0079] Figure 6 A is the SEM image of PTCDA; B is the SEM image of the reaction product PTC-Dip. Figure 6 It can be seen that the prepared PTC-Dip has a rectangular lamellar structure and has a certain degree of dispersion compared to PTCDA. The PTC-Dme, PTC-Det and PTC-Dip prepared in Examples 1-3 were subjected to Fourier transform infrared spectroscopy analysis to determine the composition of the luminophore. The analysis results are shown in FIG. Figure 7 As shown. At 3100cm -1 The stretching vibration absorption peak of CH on the aromatic ring appears at 1625cm -1 and 1450cm -1 The peaks with different intensities are the skeleton vibrations of the carbon-carbon double bonds in the aromatic rings, at 1750 cm -1 The strong peak at 2800 cm is the stretching vibration absorption peak of carbonyl. -1 The peak at is the stretching vibration absorption peak of CH on the terminal methyl group of the luminophore. Figure 7 The results of Fourier transform infrared spectroscopy showed that PTC-Dme, PTC-Det, and PTC-Dip were successfully prepared.

[0080] The UV-visible absorption spectra of PTC-Dme, PTC-Det and PTC-Dip are shown in Figure 2. Figure 8 As shown in the figure, the three electrochemiluminescent materials have strong absorption peaks around 500nm, which is attributed to their large π-conjugated structures.

[0081] The fluorescence emission spectra of PTC-Dme, PTC-Det and PTC-Dip are shown in Figure 2. Figure 9As shown. Figure 9 The maximum fluorescence emission wavelength of the three electrochemiluminescent materials is 547 nm. The fluorescence emission intensities of PTC-Dme, PTC-Det, and PTC-Dip are significantly enhanced compared to PTCDA, with PTC-Dip exhibiting the highest intensity. This is attributed to the significant steric hindrance of its side chains, which suppresses aggregation-induced quenching of the perylene ring.

[0082] The ECL emission spectrum test results of PTC-Dip are shown in Figure 10 .like Figure 10 As shown, PTC-Dip has obvious electrochemiluminescence at 571 nm.

[0083] Figure 11 The following are the X-ray photoelectron spectra of PTCDA and PTC-Dip. A is the X-ray photoelectron spectrum of PTCDA, and B is the X-ray photoelectron spectrum of PTC-Dip. The XPS spectrum of PTCDA shows that PTCDA contains C1s (285.08eV) and O1s (532.08eV), of which O1s mainly comes from the carboxyl oxygen of the PTCDA molecule, and C1s comes from the carbon in the PTCDA molecule; compared with the XPS spectrum of PTCDA, the XPS spectrum of PTC-Dip contains N1s (400.08eV) in addition to C1s and O1s. Since PTCDA does not contain nitrogen atoms, the N1s in the figure comes from the nitrogen of the imide in the luminophore molecule. By comparing the XPS spectra of the two substances, it can be seen that the amine is successfully embedded in the luminophore molecule through a hydrothermal reaction.

[0084] Example 4

[0085] Preparation of electrochemiluminescent PTC-Dip-based biosensor (solid-phase PTC-Dip nanomaterial-modified glassy carbon electrode):

[0086] A glassy carbon electrode with a diameter of 3 mm was polished using two different particle sizes of aluminum oxide powder, 3 μm and 0.05 μm, respectively. During the polishing process, the electrode was continuously rinsed with distilled water until the glassy carbon electrode was polished clean.

[0087] After the glassy carbon electrode was dried, 6 μL of the 1 mg / mL electrochemiluminescent suspension prepared in Example 1 was pipetted and drop-coated on the glassy carbon electrode, and dried at room temperature to obtain a biosensor.

[0088] Example 5

[0089] The electrochemiluminescence measurement of carboxylesterase activity using a biosensor is performed as follows:

[0090] Carboxylesterase was added to 3.0 mL of phosphate buffer (0.1 mol / L, pH 7.4) with a coreaction reagent, persulfate, at a concentration of 15 mmol / L and a catalytic substrate, 1-naphthyl acetate, at a concentration of 2 mmol / L. The reaction time was 30 min. The PTC-Dip biosensor prepared in Example 4 was tested with the enzymatic reaction solution using electrochemiluminescence detection.

[0091] In this example, a PTC-Dip-modified glassy carbon electrode was used as the working electrode, Ag / AgCl (saturated KCl) as the reference electrode, and a platinum wire as the auxiliary electrode. Cyclic voltammetry was used for electrochemical scanning, with a scanning potential of 0 V to 0.4 V and a photomultiplier tube (PMT) voltage set to 620 V. The ECL signal intensity of the prepared sensor was measured using an MPI-E II electrochemiluminescence analyzer.

[0092] The biosensor (PTC-Dip / GCE) prepared in Example 4 was tested for excitation potential and ECL response.

[0093] from Figure 12 The cyclic voltammogram (CV) of A shows that the excitation potential of PTC-Dip / GCE starts at -0.1V and reaches the maximum value at -0.27V. Its voltage-light intensity curve ( Figure 12 B) The graph shows that the trend of light intensity increasing with voltage is basically consistent with the voltage trend in the CV graph.

[0094] At 15mmol / L S2O8 2- Under the condition of co-reaction reagent, the electrochemiluminescence intensity of PTC-Dip and the classic luminescent material ruthenium bipyridine were tested. Figure 13 It can be seen that under a low voltage scan from 0 to -0.4V, the luminescence intensity of ruthenium bipyridine is 0, mainly because the low excitation potential of ruthenium bipyridine cannot be excited under this condition. Moreover, under this excitation potential, the electrochemiluminescence intensity of PTC-Dip can reach 17808a.u. Furthermore, this intensity is slightly higher than the electrochemiluminescence intensity of the classic luminophore ruthenium bipyridine scanned at -1.6V. This result shows that PTC-Dip can achieve ultra-low potential, high-efficiency electrochemiluminescence, and its ECL performance is better than that of traditional ruthenium bipyridine luminophores.

[0095] Using the biosensor (PTC-Dip / GCE) prepared in Example 4, the 2- The signal response comparison chart of the co-reactant and 1U / mL carboxylesterase system catalyzing 2mmol / L different substrates is shown in the figure. Figure 14 .

[0096] The biosensor was explored in terms of carboxylesterase activity detection and specific response:

[0097] First, the biosensor was tested. The electrode modified with PTC-Dip (PTC-Dip / GCE) was added with 1 U / mL and 1×10 -1 U / mL, 1×10 - 2 U / mL, 1×10 -3 U / mL, 1×10 -4 U / mL, 1×10 -5 U / mL, 1×10 -6 U / mL, 1×10 -7 U / mL, 1×10 -8 U / mL, 1×10 -9 The ECL response of the biosensor to carboxylesterase with different activities was studied by Figure 15 It can be seen that as the concentration of carboxylesterase in the system increases, the electrochemiluminescence intensity of the sensor gradually decreases. The change value of electrochemiluminescence intensity ( Δ I ECL =I blank -I) has a good linear relationship with the logarithm of the carboxylesterase concentration (such as Figure 16 As shown), the linear regression equation is ΔI ECL =1685.2lgc+18068.58(R 2 =0.986), and the detection limit was 2.56×10 -10 U / mL.

[0098] The biosensor prepared in Example 4 was used to investigate the ECL response of different interfering substances in the system. Acetylcholine oxidase (COD), bovine serum albumin (BSA), and glucose (GLU) were selected as interfering substances for the experiment. Interfering substances with a concentration 10 times that of carboxylesterase were added to the system, and the electrochemiluminescence response of the sensor to the interfering substances was measured under the same conditions. Figure 17 It can be observed that the electrochemiluminescence signal of the sensor with the interfering substance added is almost the same as the response without the interfering substance, and the signal is not quenched. However, the addition of carboxylesterase significantly reduces the sensor signal, indicating that the prepared carboxylesterase biosensor can produce a specific response to carboxylesterase.

[0099] The biosensor prepared in Example 4 was used to test the spike recovery experiment of carboxylesterase in serum to verify the ability of the sensor and kit to detect carboxylesterase in a complex matrix interference environment. Different concentrations of carboxylesterase were added to 50-fold diluted serum samples, and the recovery rate of CES was calculated according to the obtained regression equation. As shown in Table 1, the recovery rate of carboxylesterase was 95.30-102.1%, and the relative standard deviation was 3.7-4.6%. Therefore, the developed ECL sensor can detect carboxylesterase samples in complex biological matrices with acceptable interference, which may be attributed to the label-free and simple structure of the sensor, as well as the specific recognition of carboxylesterase for 1-naphthyl acetate, thereby triggering signal switching.

[0100] Table 1. Biosensor recovery results of CES spiked in healthy human serum

[0101]

[0102] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An application of an electrochemiluminescent material in the preparation of a carboxylesterase biosensor, characterized in that: The electrochemiluminescent material is used to detect carboxylesterase activity, and the electrochemiluminescent material has a structure shown in any one of Formula I to Formula III: 。

Images