Electrochemical luminescence biological sensing electrode and construction method thereof and method for detecting gene

文档序号：133279 发布日期：2021-10-22 浏览：35次中文

阅读说明：本技术 一种电化学发光生物传感电极及其构建和用于检测基因的方法 (Electrochemical luminescence biological sensing electrode and construction method thereof and method for detecting gene ) 是由刘长金丁世家向加林闵迅董泽令袁建波于 2021-08-20 设计创作，主要内容包括：本方案公开了生物传感器技术领域的一种电化学发光生物传感电极,包括以下内容：(1)合成了PdCuBP介孔纳米酶,并发现其对溶解的O-(2)具有良好的催化活性。(2)首次合成PdCuBP@luminol介孔纳米球,在中性免H-(2)O-(2)介质中展现了优秀的电化学发光信号。(3)CRISPR/Cas12a系统作为检测触发器,特异性识别靶DNA,高效剪切界面发卡DNA-多巴胺(hpDNA-DA)猝灭剂。(4)联合PdCuBP@luminol和CRISPR/Cas12a构建电化学发光DNA生物传感器。该传感器可针对细胞色素c氧化酶亚基III基因检测,在临床诊断领域具有潜在应用价值。(The scheme discloses an electrochemiluminescence biosensor electrode in the technical field of biosensors, which comprises the following contents: (1) PdCuBP mesoporous nanoenzyme is synthesized and found to be used for dissolving O 2 Has good catalytic activity. (2) Firstly synthesizing PdCuBP @ lumineol mesoporous nanospheres, and avoiding H in neutrality 2 O 2 The medium shows excellent electrochemical luminescence signals. (3) The CRISPR/Cas12a system is used as a detection trigger, specifically recognizes target DNA, and efficiently cuts an interface hairpin DNA-dopamine (hpDNA-DA) quencher. (4) And (3) constructing an electrochemiluminescence DNA biosensor by combining PdCuBP @ lumineol and CRISPR/Cas12 a. The sensor can be used for detecting cytochrome c oxidase subunit III genes and has potential application value in the field of clinical diagnosis.)

1. A construction method of an electrochemiluminescence biosensing electrode is characterized by comprising the following steps:

(1) synthesizing PdCuBP mesoporous nanoenzyme: weighing DODAC, adding water to dissolve, and sequentially adding NH₄Solution F, H₃BO₃Solution, H₂PdCl₄Solution and CuCl₂A solution; stirring and heating for a certain time at a proper temperature, and then adding NH₃·H₂O, stirring to be colorless, and dripping NaH₂PO₂·H₂Gradually heating to 90-95 ℃, and keeping the temperature for a certain time; finally, adding DMAB, fully stirring, changing the color of the solution from white to dark brown, centrifuging, and washing to obtain PdCuBP mesoporous nanoenzyme;

(2) synthesis of PdCuBP @ luminol nanosphere

Adding a luminol dispersion liquid into the PdCuBP mesoporous nanoenzyme suspension obtained in the step (1), mixing, stirring for a certain time at room temperature in a dark place, centrifuging to remove the unloaded luminol, and obtaining and collecting PdCuBP @ luminol nanospheres;

(3) preparation of hpDNA-DA

Dissolving the hpDNA in EDC and NHS, standing, activating carboxyl of the hpDNA, then dripping DA solution, and fully stirring to obtain the hpDNA-DA;

(4) construction of an electrochemiluminescence biosensor electrode

And dripping the suspension of the PdCuBP @ luminol nanosphere on the surface of the cleaned gold electrode, drying to form a film, dripping the hpDNA-DA on the surface of the gold electrode, incubating for a certain time, and dripping MCH to block non-specific sites on the surface of the gold electrode to obtain the electrochemiluminescence biosensor electrode.

2. The method for constructing an electrochemiluminescence biosensing electrode according to claim 1, wherein the electrochemiluminescence biosensing electrode comprises the following steps: diluting the stock solution of the PdCuBP @ luminol nanosphere obtained in the step (2) by 5 times for later use, wherein the concentration of the hpDNA-DA in the step (4) is 3 mu M.

3. The method for constructing an electrochemiluminescence biosensing electrode according to claim 2, wherein the electrochemiluminescence biosensing electrode comprises the following steps: the cleaning treatment mode of the gold electrode is as follows: polishing a bare gold electrode by adopting alumina micro powder and a polishing pad, after ultra-pure water ultrasonic bath washing, etching for a certain time by using piranha solution, then cleaning by using deionized water, and drying and modifying under nitrogen; the piranha solution is H₂SO₄/H₂O₂Mixed solution of 3: 1.

4. The method for constructing an electrochemiluminescence biosensor electrode according to any one of claims 1 to 3, wherein the method comprises the following steps: stirring at 35 ℃ for 6 minutes in step (1), and adding NH₃·H₂O; dropping NaH₂PO₂·H₂Heating O to 95 deg.C, and keeping the temperature for 20 min.

5. The method for constructing an electrochemiluminescence biosensing electrode according to claim 4, wherein the electrochemiluminescence biosensing electrode comprises the following steps: the temperature for standing and stirring in the step (3) is 4 ℃.

6. The method for constructing an electrochemiluminescence biosensing electrode according to claim 5, wherein: the incubation temperature in the step (4) is 4 ℃, and the incubation time is 8 h; the obtained electrochemiluminescence biosensor electrode is washed by a washing solution, and then stored at 4 ℃ for later use, wherein the washing solution is 0.01M PBS (phosphate buffer solution), contains 0.05% (w/v) Tween-20 and has the pH value of 7.41.

7. An electrochemiluminescence biosensor electrode obtained by the method according to any one of claims 1 to 3, 5 and 6.

8. An electrochemiluminescence DNA sensor comprising the electrochemiluminescence biosensing electrode of claim 7.

9. The method for detecting genes using the electrochemiluminescence biosensor electrode according to claim 7, wherein: before detection, a Cas12a/crRNA complex is prepared in a reaction buffer solution, target DNA is added into the complex, the complex is incubated for a certain time at a proper temperature, a solution obtained after incubation is dripped on the surface of an electrochemiluminescence biosensing electrode, the complex is incubated for a certain time at a proper temperature, the surface of the electrode is washed and modified, scanning is carried out at a potential of-0.2-0.6V, the scanning rate is 0.1-0.15V/s, the pulse width is 50-100 ms, the photomultiplier is 800V, and electrochemiluminescence detection is carried out in PBS.

10. The method for detecting a gene according to claim 9, wherein: the Cas12a/crRNA complex was prepared as follows: adding 50nM crRNA and 50nM Cas12a into reaction buffer containing 1U RNase inhibitor and 1 XNEBuffer 2.1 to react; during gene detection, the concentration of the Cas12a/crRNA complex was 50 nM; the temperature of each incubation was 37 ℃ for 1 h.

Technical Field

The invention belongs to the technical field of biosensors, and particularly relates to an electrochemiluminescence biosensor electrode, a construction method thereof and a method for detecting genes.

Background

Currently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas) is of great interest to biological researchers as an efficient gene editing tool. The CRISPR/Cas system has different unique adjustable nuclease activities, including CRISPR/Cas9, CRISPR/Cas12a, CRISPR/Cas13a, CRISPR/Cas14 and the like, and is used for sequence-specific nucleic acid detection. The CRISPR/Cas12a belonging to the class II V-A CRISPR/Cas system activates high-efficiency DNase activity after specifically recognizing target double-stranded DNA (dsDNA), and is an ideal candidate molecule for next-generation DNA biosensing. The CRISPR/Cas12a is combined with fluorescence, lateral flow, colorimetry and other technologies, and a series of detection methods with high sensitivity and high specificity are rapidly developed. However, these methods have the disadvantages of expensive double-label, expensive optical equipment, low signal-to-noise ratio, poor color change resolution and the like, and thus the applicability of CRISPR/Cas12a bioanalysis is hindered.

Electrochemiluminescence (ECL), a commonly used biological analysis technique, has attracted much attention because of its advantages such as low background, high sensitivity, rapid analysis, miniaturization, and easy control. In view of these advantages, CRISPR/Cas12a binding to ECL may be an alternative technology to DNA biosensing. For example, Liang's group developed an electrochemiluminescence biosensor based on Cas12a, which showed better DNA detection performance in real samples. However, the method has the following disadvantages: volatile organic compounds (triethylamine, TEA) were added as exogenous co-reactants to the highly basic working solution (pH 11) to increase the intensity of ECL. The activity of CRISPR/Cas12a may be affected by the biotoxicity of overbased solutions and TEA, negatively affecting the sensitivity and specificity of the biosensor.

Among the numerous electrochemiluminescent reagents, luminol (also known as luminol) has the chemical name 3-aminoPhenylhydrazine) is widely used in electrochemiluminescence biosensors due to its excellent luminescence properties. Hydrogen peroxide (H)₂O₂) As an exogenous co-reactant, is used to enhance the emission signal of luminol. But H₂O₂The decomposability and instability of the luminol inhibit the application of the luminol in the field of biological assay. Furthermore, in order to enhance the luminol luminescence signal, luminol-based electrochemiluminescence biosensors need to work in biocompatible unfriendly alkaline solutions. Therefore, development of a neutral H-free catalyst₂O₂The luminol electrochemiluminescence biosensor is of great importance.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides an electrochemiluminescence biosensor electrode and a method for constructing a gene detection structure.

The construction method of the electrochemical luminescence biological sensing electrode in the scheme comprises the following steps:

(2) synthesis of PdCuBP @ luminol nanosphere

(3) preparation of hpDNA-DA

Dissolving the hpDNA in EDC and NHS, standing, activating carboxyl of the hpDNA, then dripping DA solution, and fully stirring to obtain the hpDNA-DA;

(4) construction of an electrochemiluminescence biosensor electrode

The working principle of the scheme is as follows: the PdCuBP mesoporous nanoenzyme and the PdCuBP @ luminol nanosphere in the scheme are multi-component metal-nonmetal synthetic substances, the PdCuBP mesoporous nanoenzyme has a huge surface area, and can carry abundant luminol (luminol) to form the PdCuBP @ luminol nanosphere which is used as an electrode modifier; the PdCuBP mesoporous nanoenzyme is a good ECL signal amplifier, has good oxide catalytic activity and accelerates the dissolution of O₂Converted to ROS, significantly enhancing the luminol ECL signal in neutral working solutions. The invention synthesizes PdCuBP @ lumineol mesoporous nanospheres for the first time, and H is avoided in neutrality₂O₂The medium shows excellent electrochemical luminescence signals.

Dopamine (DA) as a quencher was labeled on thiol (SH) hairpin dna (hpDNA) to form hpDNA-DA, which was then immobilized on the surface of Gold Electrode (GE) to quench ECL emission of PdCuBP @ luminol nanospheres.

The method comprises the steps that PdCuBP @ lumineol mesoporous nanospheres are loaded on the surface of a gold electrode, at the moment, because of an electrochemiluminescence signal of the PdCuBP @ lumineol mesoporous nanospheres, the signal is in an 'on' state, hairpin DNA-dopamine (hpDNA-DA) serving as a quencher is coupled on the surface of the gold electrode to quench the electrochemiluminescence signal of the PdCuBP @ lumineol mesoporous nanospheres, at the moment, the signal is in an 'off' state, when a system contains target DNA, the nuclease cutting activity of Cas12a is activated, and therefore the hpDNA-DA on the surface of the gold electrode is cut, and the signal is output.

The beneficial technical effect of this scheme is: the cytochrome c oxidase subunit III (COX III) gene is a novel biomarker of Acute Kidney Injury (AKI), and the CRISPR/Cas12a system of the prior art is specifically activated in the presence of the cytochrome c oxidase subunit III (COX III) gene and in the presence of the geneThe hpDNA-DA has non-specific trans-cutting capacity, so that an ECL signal is started, target DNA is specifically recognized, and an interface hpDNA-DA quencher is efficiently cut. The CRISPR/Cas12a system and the electrochemiluminescence biosensing electrode in the invention can construct a novel neutral-immunity H₂O₂The electrochemical luminescence DNA biosensor can show good biocompatibility, functional universality, room temperature operability and excellent detection capability in a neutral environment, does not need to add a high-alkalinity working solution and does not need to additionally add hydrogen peroxide (H)₂O₂) To enhance the electrochemiluminescence signal.

The electrochemical luminescence DNA biosensor which is constructed by using the electrochemical luminescence biosensor electrode and combining with the CRISPR/Cas12a system has good linear response between 1pM and 200nM, and the detection limit is 0.44 pM. Meanwhile, the prepared electrochemical luminescence DNA biosensor has good specificity and repeatability. In addition, the established method has been successfully evaluated in real urine, and the detection of cytochrome c oxidase subunit III gene by the established method is suggested to have potential application value in the field of clinical diagnosis.

Meanwhile, the invention solves the problems of high detection cost, complex operation, low sensitivity and the like in the prior art.

Further, the stock solution of the PdCuBP @ luminol nanosphere obtained in the step (2) is diluted by 5 times for standby, and the concentration of the hpDNA-DA in the step (4) is 3 mu M. The scheme improves the electrochemical luminous efficiency and reduces the development cost.

Further, the cleaning treatment mode of the gold electrode is as follows: polishing a bare gold electrode by adopting alumina micro powder and a polishing pad, after ultra-pure water ultrasonic bath washing, etching for a certain time by using piranha solution, then cleaning by using deionized water, and drying and modifying under nitrogen; the piranha solution is H₂SO₄/H₂O₂Mixed solution of 3: 1.

Further, in the step (1), NH was added after stirring at 35 ℃ for 6 minutes₃·H₂O; dropping NaH₂PO₂·H₂Heating O to 95 deg.C, and keeping the temperature for 20 min.

Further, the temperature of the standing and the stirring in the step (3) was 4 ℃.

Further, the incubation temperature in the step (4) is 4 ℃, and the incubation time is 8 h; the obtained electrochemiluminescence biosensor electrode is washed by a washing solution, and then stored at 4 ℃ for later use, wherein the washing solution is 0.01M PBS (phosphate buffer solution), contains 0.05% (w/v) Tween-20 and has the pH value of 7.41.

The electrochemiluminescence biosensor electrode constructed by the method can be combined with a CRISPR/Cas12a system to form an electrochemiluminescence DNA sensor for detecting genes.

The electrochemiluminescence biosensor electrode detection gene constructed by the method is characterized in that before detection, a Cas12a/crRNA complex is prepared in a reaction buffer solution, target DNA is added into the complex, the complex is incubated for a certain time at a proper temperature, a solution obtained after incubation is dripped on the surface of the electrochemiluminescence biosensor electrode, the surface of the electrode is washed and modified, potential scanning is carried out at-0.2-0.6V, the scanning speed is 0.1-0.15V/s, the pulse width is 50-100 ms, the photomultiplier is 800V, and electrochemiluminescence detection is carried out in PBS (0.01M, pH 7.4).

Wherein, the Cas12a/crRNA complex is prepared by: adding 50nM crRNA and 50nM Cas12a into reaction buffer containing 1U RNase inhibitor and 1 XNEBuffer 2.1 to react; during gene detection, the concentration of the Cas12a/crRNA complex was 50 nM; the temperature of each incubation was 37 ℃ for 1 h.

Drawings

FIG. 1 is a schematic diagram of the preparation process and detection principle of an electrochemiluminescence biosensor electrode according to the present invention;

FIG. 2 is the structure and composition analysis of PdCuBP mesoporous nanoenzyme and PdCuBP @ luminol mesoporous nanosphere;

wherein: (A) TEM and particle size distribution histogram of PdCuBP mesoporous nanoenzyme; (B-C) HRTEM image of PdCuBP mesoporous nanoenzyme; (D) HAADF-STEM diagram of PdCuBP @ luminol mesoporous nanospheres; (E-J) STEM-EDS element map of PdCuBP @ luminol;

FIG. 3 is a nitrogen adsorption-desorption isotherm of PdCuBP mesoporous nanoenzyme;

FIG. 4 is an XPS spectrum of PdCuBP @ luminol mesoporous nanospheres;

FIG. 5(A) electrochemiluminescence intensity of different modified electrodes: PdCuBP/GE (a), luminol/GE (b), PdCuBP @ luminol/GE (c), ECL intensity in PBS (pH 7.4), respectively; (B) electrochemiluminescence intensity of PdCuBP @ luminol/GE in alkaline (pH 9.0) (a) and neutral (pH 7.4) (b) PBS;

FIG. 6(A) PdCuBP @ luminol/GE at N₂Electrochemiluminescence signal in PBS saturated (a) and air saturated (b); (B) electrochemiluminescence response of PdCuBP @ luminol/GE in air saturated PBS containing 10mM DMSO (a) and 1mM p-benzoquinone (b);

fig. 7(a) feasibility analysis of Cas12 a-mediated cleavage; (B) feasible ECL analysis of developed biosensors: crRNA + target (a), Cas12a (b), Cas12a + crRNA (c), Cas12a + target (d), Cas12a + crRNA + target (e);

FIG. 8 electrochemical impedance Spectroscopy (A) and electrochemiluminescence (B) characterization: naked GE (a), PdCuBP @ luminal/GE (b), hpDNA-DA/PdCuBP @ luminal/GE (c), Cas12a-crRNA-target complex treated hpDNA-DA/PdCuBP @ luminal/GE (d);

FIG. 9 optimizes (A) dilution factor of PdCuBP @ luminol, (B) concentration of hpDNA-DA, (C) concentration of Cas12a/crRNA complex, (D) incubation time of Cas12a/crRNA/target complex;

FIG. 10(A) shows the electrochemiluminescence values of the biosensor designed at different target concentrations (a-i:1pM,5pM,50pM,100pM,1nM,10nM,50nM,100nM,200 nM). (B) Linear plot of electrochemiluminescence values versus log COX III concentration. (C) The designed biosensor electrochemiluminescence response was blank (no target DNA), 5nM COX I, 5nM COX II, 1nM COX III, and mixed (1nM COX III +5nM of each interfering substance). Error bars: SD, n is 3; (D) the electrochemiluminescence response of the biosensor was scanned for 10 cycles with a continuous cycling potential in the presence of 100pM COX III.

Detailed Description

The following is further detailed by way of specific embodiments:

firstly, constructing an electrochemiluminescence biosensor electrode, and detecting COX III gene, wherein the electrochemiluminescence biosensor electrode is shown in a combined figure 1.

1. Materials and methods

1.1 materials

Lba Cas12a (Cpf1) and 10 XNEBuffer 2.1(0.5M NaCl,0.1M Tris-HCl,0.1M MgCl2,1mg/mL BSA, pH 7.9) were purchased from New England Biolabs (Epstein, USA), HPLC purified CRISPR RNA (crRNA), RNase inhibitor, RNase-free water and DNA Marker from Takara Biotech (Dalian, China). GoldView I was purchased from Solambio Tech (Beijing, China). 6-mercapto-1-hexanol (MCH), tris (2-carboxy) -phosphate hydrochloride (TCEP), and polyethylene glycol sorbitan monolaurate (Tween-20) were purchased from Sigma-Aldrich (St. Louis, USA). All HPLC-purified DNA oligonucleotides were synthesized by Biotechnology Ltd (Shanghai, China). All oligonucleotide sequences are listed in table 1. Octacosyldimethylammonium chloride (DODAC, 96%) was purchased from Alfa Aesar (schischem, uk). Hypophosphorous acid (NaH)₂PO₂·H₂O, 98%), dopamine hydrochloride (99.9%), palladium (II) chloride (PdCl2, 99.9%) and anhydrous copper chloride (CuCl)₂99%) was purchased from Adamas-beta (shanghai, china). Borane dimethylamine (DMAB, 97%) complex was purchased from Acros Organics (Hell, Belgium). Ammonium fluoride (NH)₄F, 98%) and boric acid (H)₃BO₃99.5%) were purchased from great gent (shanghai, china). Anhydrous ethanol, hydrochloric acid (HCl) and ammonia (NH)₃·H₂O) was purchased from chemical ltd, chuan, china. Phosphate buffer (PBS,0.01M) (NaCl-Na)₂HPO₄-KH₂PO₄-KCl) as ECL working buffer, pH 7.4. All reagents were analytical grade, without any further purification procedures, and Milliq ultrapure water (. gtoreq.18 M.OMEGA.cm) was used throughout the work^-1,Millipore)。

TABLE 1 nucleic acid sequences used in this work

1.2 detection Instrument

ECL and electrochemical measurements were performed using an MPI-E multifunctional analyzer (west ampere, china) and a CHI 660E electrochemical workstation (shanghai, china). The three-electrode system consisted of a gold electrode (diameter 3 mm, GE, working electrode), a platinum wire (counter electrode) and an Ag/AgCl electrode (immersed in saturated KCl solution, reference electrode). Transmission Electron Microscopy (TEM) and High Resolution Transmission Electron Microscopy (HRTEM) were operated at 200kV using JEM-2100F. The elemental mapping analysis plots were collected on an Oxford X-MAX energy dispersive spectrometer. Brunauer-Emmett-Teller (BET) analysis was performed on powder samples using a Micromeritics TriStar II 3020 instrument. X-ray photoelectron spectroscopy (XPS) images were collected on an ESCALAB 250Xi spectrometer.

1.3 Synthesis of PdCuBP mesoporous nanoenzyme

180mg of DODAC was weighed out and dissolved in 60mL of ultrapure water by sonication. Then NH4F solution (6ml,0.337M), H were added in sequence₃BO₃Solution (6ml,0.101M), H₂PdCl₄Solution (3.84mL,10mM) and CuCl₂Solution (0.96mL,10 mM). Subsequently, the mixture was heated at 35 ℃ for 6 minutes with gentle stirring, and NH was rapidly added₃·H₂O (2.4mL,10 wt.%), and further stirred until colorless. Reacting NaH with₂PO₂·H₂O (6mL,0.034M) was added dropwise to the above solution, gradually heated to 35 ℃ to 95 ℃ and kept at 95 ℃ for 20min with an oil bath. Finally, freshly prepared DMAB (6ml,0.1M) was poured into the solution, stirred well and held at 95 ℃ for 30 minutes. After DMAB is reduced, the color is changed from white to dark brown, which shows that the PdCuBP mesoporous nano enzyme is synthesized. After centrifugation, the cells were washed 3 times with ethanol water and suspended in 6ml of deionized water.

1.4 Synthesis of PdCuBP @ luminol nanosphere

Firstly, 2mL of the synthesized PdCuBP mesoporous nano enzyme solution is mixed with 2mL of luminol dispersion liquid (solute is water, 5 mM). Then stirred at room temperature for 12h in the dark. After centrifugation to remove the unloaded luminol, the PdCuBP @ luminol nanospheres were collected and dispersed uniformly in 1ml of ultrapure water.

1.5 preparation of the hpDNA-DA Probe

First 500. mu.L of hpDNA (1. mu.M) was dissolved in 48mg of EDC and 7mg of NHS and left to stand at 4 ℃ for 1h to activate the carboxyl group of hpDNA. To the mixture was added dropwise DA solution (500. mu.L, 2. mu.M) and gently stirred at 4 ℃ for 12 hours to obtain hpDNA-DA.

1.6 construction of electrochemiluminescence biosensing electrode

The bare gold electrode (bare GE) was polished with alumina micropowder and a polishing pad. After rinsing with ultra pure water in an ultrasonic bath, piranha solution (H)₂SO₄/H₂O₂3:1) for 10 minutes, then rinsed with deionized water and dried under nitrogen for modification. Then, the PdCuBP @ luminol nano microsphere suspension is dripped on the surface of a clean gold electrode, and the gold electrode is dried at 37 ℃ to form a film. Then, 10. mu.L of hpDNA-DA was dropped on the treated gold electrode surface, incubated at 4 ℃ for 8h, and then a non-specific site on the gold electrode surface was blocked by dropping MCH (10. mu.L, 0.5 mM). Finally, the constructed electrochemiluminescence biosensor electrode was washed with a washing solution (0.01M PBS containing 0.05% (w/v) Tween-20, pH 7.4) and stored at 4 ℃ for further use.

1.7 electrochemiluminescence detection procedure

Prior to measurement, Cas12a/crRNA complex was prepared using 50nM crRNA,50nM Cas12a in a buffer containing 1U RNase inhibitor, 1 × NEBuffer 2.1. Subsequently, 4. mu.L of target DNA was added to 6. mu.L of reaction buffer and incubated at 37 ℃ for 10 minutes. Then, 10. mu.L of the above solution was dropped on the prepared electrode surface, and incubated at 37 ℃ for 1 hour. The surface of the modified electrode is lightly washed, potential scanning is carried out at-0.2-0.6V, the scanning speed is in the range of 0.1-0.15V/s, preferably 0.15V/s, the pulse width is in the range of 50-100 ms, preferably 50ms, the photomultiplier is 800V, and electrochemical luminescence detection is carried out in PBS (0.01M, pH 7.4).

Second, characterizing the PdCuBP mesoporous nanoenzyme and PdCuBP @ lumineol mesoporous nanosphere

In order to characterize the PdCuBP mesoporous nanoenzyme, technologies such as TEM, HRTEM, BET and the like are used. As shown in FIGS. 2A and B, the prepared PdCuBP mesoporous nanoenzyme is a high monodispersity nanosphere with the diameter of about 100 nm. As shown in fig. 1C, the PdCuBP mesoporous nanoenzyme is a dendritic dispersion-like spherical structure (fig. 2C). As shown in FIG. 3, the nitrogen adsorption-elution isotherm shows that the PdCuBP mesoporous nanoenzyme is a porous structure, and the surface area of the PdCuBP mesoporous nanoenzyme is about 41.17m by calculation²A/g and an average pore diameter of 5.02 nm.

To further explore the nanostructures and elements of PdCuBP @ luminol nanospheres, we performed High Angle Annular Dark Field (HAADF) -STEM and STEM-EDS elemental mapping analyses. As can be seen from fig. 2D, the PdCuBP mesoporous nanoenzyme still maintains the mesoporous nanocluster structure of the bulk after loading luminol. In fig. 2(E-J), the Pd, Cu, B, and P elements in the mesoporous nanoenzyme are uniformly distributed in the nanocluster, and the N of luminol is uniformly distributed on the nanoparticle. These results show that luminol is successfully loaded on PdCuBP mesoporous nanoenzyme to form PdCuBP @ luminol mesoporous nanospheres.

The surface electronic state and chemical bond configuration of the PdCuBP @ luminol mesoporous nanosphere are further researched by XPS. XPS spectra of N1s, Pd 3d, Cu 2P, B1 s and P2P in the PdCuBP @ luminol mesoporous nanosphere are shown in FIG. 4. As shown in FIG. 4A, the main peak of N1s appears at 399.3eV, which may be the-NH of luminol₂The N1s peak at 402eV may be due to the N-oxide. In addition, as shown in fig. 4B, Pd 3d (Pd) is present in the PdCuBP mesoporous nanoenzyme⁰And Pd²⁺). The valence change of Pd indicates that Pd-N bond interaction exists between PdCuBP and luminol.

Electrochemical luminescence property of PdCuBP @ luminol mesoporous nanosphere

In order to evaluate that the PdCuBP @ lumineol mesoporous nanospheres are free of H in neutrality₂O₂The electrochemical luminescence property in the working solution is compared with that of different modified electrodes. As shown in fig. 5A, no electrochemiluminescence signal was observed after the electrode was modified by PdCuBP mesoporous nanoenzyme (curve a). Furthermore, the luminol-modified electrode emits a very low electrochemiluminescence signal (curve b). After the electrode is modified by PdCuBP @ luminol, an extremely high electrochemical luminescence signal (curve c) is obtained, which shows that the PdCuBP mesoporous nanoenzyme has high contactable specific surface area, can adsorb a large amount of luminol, shows good biological catalytic activity, and has no H₂O₂The luminol is triggered to emit an electrochemiluminescence signal in the working solution. In addition, as shown in fig. 5B, there is no significant difference in the electrochemiluminescence signals of the PdCuBP @ luminol mesoporous nanospheres in the alkaline and neutral working solutions (curves a and B). The results show that the PdCuBP @ luminol mesoporous nanosphere can be neutral and has no H₂O₂High-intensity electrochemical luminescence signal output is realized in a working system.

Electrochemical luminescence mechanism of PdCuBP @ luminol mesoporous nanosphere

In order to understand the potential electrochemical luminescence mechanism of the PdCuBP @ luminol mesoporous nanospheres, ECL signals were detected in different solutions, and all experiments were performed in PBS (phosphate buffer solution) with pH 7.4. As shown in FIG. 6A, PdCuBP @ luminol nanospheres are shown in N₂There was almost no electrochemiluminescence signal in saturated PBS (curve a). Correspondingly, the PdCuBP @ luminol nanosphere modified electrode exhibited a significant electrochemiluminescence signal in air-saturated PBS (curve b). The result shows that the PdCuBP mesoporous nano enzyme can obviously improve the dissolution of luminol in O₂Light emission efficiency in (1).

Dissolved O is known₂Can be used as endogenous nuclear reactant to generate ROSs (such as O)₂ ^·-And OH^·) And reacted with an activated intermediate of oxidized luminol. Therefore, to further explore PdCuBP mesoporous nanoenzyme and dissolved O₂The mechanism of interaction-induced luminol ECL, we designed and performed the following experiments. As shown in FIG. 6B, addition of dimethyl sulfoxide (DMSO) scavenges Ross OH^·After that, we observed a slight signal change (curve a). In addition, p-benzoquinone is added to eliminate O2^·-After that, we observed a significant reduction of the electrochemiluminescence signal to 1266a.u (curve b). The results show that the PdCuBP mesoporous nanoenzyme promotes dissolved oxygen to generate O₂ ^·-Instead of OH^·Resulting in electrochemiluminescence of the luminol. PdCuBP @ luminol/O₂A possible ECL mechanism for the system may be described as follows:

luminol-2e^--2H⁺→luminol^·- (2)

luminol^·-+O₂ ^·-→3-AP^2-*+N₂ (3)

luminol^·-+O₂→O₂ ^·-+luminol (4)

3-AP^2-*→3-AP^2-+hv (5)

fifth, feasibility analysis of the biosensor

To confirm the trans-cleavage activity of Cas12a, the reaction products were subjected to PAGE electrophoretic analysis, as shown in fig. 7A. The electrophoresis positions (Lane 1-4) of the components of Cas12a/crRNA/target complex and single-stranded DNA (ssDNA) are labeled. Lane 5-7 shows that the nuclease ability of Cas12a cannot be activated due to imperfections in the Cas12a/crRNA/target complex structure. Lane 8 suggests that the Cas12a/crRNA complex is capable of specifically recognizing and degrading target DNA. As expected, Cas12a, crRNA, and target DNA can form a triple complex (Cas12a/crRNA/target complex) to degrade ssDNA (Lane 9), indicating that the nuclease activity of Cas12a can be fully activated by integrating the Cas12a/crRNA/target complex.

To validate the feasibility of the Cas12 a-based electrochemiluminescence platform for COX III DNA detection, we also performed electrochemiluminescence measurements to further study the nucleic acid cleavage performance on the modified electrode (hpDNA-DA/PdCuBP @ luminol nanosphere/GE). Also, as shown in fig. 7B, the triple structure of Cas12a/crRNA/target complex is incomplete and fails to activate Cas12a nuclease activity, thereby increasing the electrochemical signal of the hpDNA-DA quenching PdCuBP @ luminol (curves a-d). Only mixtures containing Cas12a, crRNA and target (50nM) assembled into a Cas12a/crRNA/target triplet, resulting in cleavage of hpDNA-DA and recovery of the ECL signal emitted by PdCuBP @ luminal (curve e). Both PAGE and ECL detection results indicate that an ECL biosensor based on Cas12a can be used for nucleic acid detection.

Sixth, characterization of electrochemical biosensor

The preparation process of the ECL biosensor is characterized by adopting electrochemical impedance spectroscopy and cyclic voltammetry, and the preparation process is carried out on 5mM Fe (CN) containing 0.1M KCl₆ ^3-/4-Is carried out in (1). In electrical impedance spectroscopy, the semi-circle diameter is equal to the electron transfer resistance (Ret). As shown in fig. 8A, the resistance value of the bare GE is small (curve a). Thereafter, with modifications of PdCuBP @ lu on GEThe impedance of the miniol nanospheres was slightly reduced (curve b), indicating that PdCUBP @ luminol has a certain conductivity. Subsequently, when hpDNA-DA was modified, the impedance increased significantly (curve c), due to hpDNA and DA blocking electron transfer between the electrode and the electrolyte solution. As expected, a significantly reduced impedance intensity (curve d) can be obtained after cleavage of hpDNA-DA by Cas12 a. The electrochemical impedance spectrum result shows that the designed ECL biosensor is successfully prepared.

To further verify the successful assembly of the electrochemiluminescence biosensor, the electrochemiluminescence values for each preparation process were recorded. As shown in fig. 8B, no electrochemiluminescence signal was observed by bare GE due to the absence of luminophores (curve a). Subsequently, due to the excellent electrochemiluminescence properties of the PdCuBP @ luminol nanospheres, very high signals were obtained (curve b). When the electrode was immobilized with hpDNA-DA, the electrochemiluminescence signal dropped significantly (curve c), demonstrating that DA in hpDNA can greatly quench the electrochemiluminescence emission of luminol. The electrochemiluminescence emission signal of PdCuBP @ luminol nanospheres can be restored after incubation with Cas12a/crRNA/target complex (curve d) because hpDNA is cleaved to release DA quencher. These results further demonstrate that the stepwise preparation of the manufactured biosensors was performed successfully as expected.

Seventhly, optimizing preparation and reaction conditions of biosensor

Several experimental conditions were systematically optimized for optimal analytical performance. The dilution times of the PdCuBP @ luminol nanosphere capable of improving the electrochemical luminescence efficiency and reducing the development cost are preliminarily researched. As shown in fig. 9A, the electrochemiluminescence biosensor shows the highest electrochemiluminescence intensity when the dilution factor of the PdCuBP @ luminol nanospheres is 5 times. Therefore, we selected five-fold dilution of PdCuBP @ luminol nanospheres as the optimal dilution factor for the modified electrode. Next, the concentration of hpDNA-DA, which is a factor in extinguishing the electrochemiluminescence efficiency of PdCuBP @ luminol nanospheres, was also optimized. As shown in fig. 9B, the electrochemiluminescence signal was nearly lowest after 3 μ M hpDNA-DA incubation on the modified electrode. Therefore, we selected a median concentration of 3 μ M as the optimal concentration in the next experiment. Furthermore, the concentration of Cas12a/crRNA complex in the working buffer is the basic condition for constructing biosensors due to specific recognition and trans-cleavage ability. Subsequently, we optimized a series of 20-60nM Cas12a/crRNA complexes. As shown in fig. 9C, the electrochemiluminescence signal of the biosensor increased with the change in Cas12a/crRNA complex concentration from 20nM to 50nM, and then remained constant with the further increase in Cas12a/crRNA complex concentration. Generally, the incubation time of Cas12a/crRNA/target complex on the modified electrode will affect the performance of the detection system. Figure 9D shows ECL values at different incubation times, with an optimal cleavage time of Cas12a/crRNA/target complex of 60 min.

Eighth, analytical Properties of electrochemiluminescence biosensor

Under the optimized condition, the developed electrochemiluminescence biosensor is used for quantitatively detecting COX III genes with different concentrations. FIG. 9A shows the quantitative determination of DNA of COX III gene signals for electrochemiluminescence in the concentration range from 1pM (curve a) to 200nM (curve i). FIG. 10B shows a good linear regression equation as: 2556lg C_COXIII+33490(R²0.9904). The calculated limit of detection (LOD) was 0.44pM (S/N — 3). This is mainly due to the high ECL emission efficiency of the PdCuBP @ luminol nanospheres and the high cleavage activity of the activated CRISPR/Cas12a on hpDNA-DA.

To demonstrate the specificity of the proposed electrochemiluminescence biosensor, we performed cross-reaction experiments on several interfering substances, including COX I, COX II, COX III and mixtures containing COX III. Compared to the blank, the change in electrochemiluminescence was negligible when non-specific interfering substances were present (FIG. 9C). Meanwhile, in the presence of the target substance, a significant electrochemiluminescence response is obtained as compared with each interfering substance. The results show that the developed biosensor has excellent specificity due to the typically good recognition ability of the Cas12a/crRNA complex.

The stability of the electrochemiluminescence biosensor is a key parameter to consider. The stability of the method was evaluated as Relative Standard Deviation (RSD). Under optimal conditions, the biosensor was scanned 10 times in succession. As can be seen from fig. 9D, the electrochemiluminescence intensity was relatively stable under continuous scanning, with an RSD of 2.27%, indicating that the stability of the biosensor was satisfactory.

FIG. 10(A) shows the electrochemiluminescence values of the biosensor designed at different target concentrations (a-i:1pM,5pM,50pM,100pM,1nM,10nM,50nM,100nM,200 nM). (B) Linear plot of electrochemiluminescence values versus log COX III concentration. (C) The designed biosensor electrochemiluminescence response was blank (no target DNA), 5nM COX I, 5nM COX II, 1nM COX III, and mixed (1nM COX III +5nM of each interfering substance). Error bars: SD, n is 3. (D) The electrochemiluminescence response of the biosensor was scanned for 10 cycles with a continuous cycling potential in the presence of 100pM COX III.

Ninthly, recovery test of electrochemical luminescence biosensor

In order to evaluate the potential clinical application value of the constructed electrochemiluminescence biosensor, quantitative analysis is carried out on target DNA (5pm, 500pm and 1000pm) added in a human urine sample, and a recovery test is carried out. As shown in table 2, the sample recovery rate was 97.81% to 103.73%, and RSD < 5% (n ═ 3). The results indicate a reliable and potential application of the proposed biosensor in clinical analysis.

TABLE 2 electrochemical luminescence biosensor developed for determining COX III content in normal urine

^*Recovery (%) is expressed as the ratio of calculated/spiked COX iii.

Sequence listing

<110> Zunyi medical university affiliated hospital

<120> an electrochemiluminescence biosensor electrode, its construction and method for detecting gene

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<170> SIPOSequenceListing 1.0

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<212> RNA