CN111896596B

CN111896596B - Preparation method and application of electrochemical luminescence sensor

Info

Publication number: CN111896596B
Application number: CN202010546543.9A
Authority: CN
Inventors: 梁汝萍; 曹姝萍; 邱建丁
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2020-06-15
Filing date: 2020-06-15
Publication date: 2021-06-22
Anticipated expiration: 2040-06-15
Also published as: CN111896596A

Abstract

The invention discloses a preparation method and application of an electrochemiluminescence sensor, and belongs to the technical field of electrochemiluminescence sensing. Dropping a mixed solution of covalent organic frameworks Tp-Bpy COF and chitosan on the surface of a glassy carbon electrode, and reacting (AC) through an amide reaction₂₉Assembled to the electrode surface and hybridized by DNA complementation (GT)₂₉Made by bonding to electrodes (GT)₂₉/(AC)₂₉Tp-Bpy COF modified electrode in Ir (ppy)₃With a strong cathodic ECL signal. When Ru (bpy)₃ ²⁺By electrostatic interaction into (GT)₂₉/(AC)₂₉Double stranded, Ru (bpy)₃ ²⁺ECL enhancement of (D) Ir (ppy)₃The ECL of (a) is reduced. When As (III) is present, Ir (ppy)₃ECL Signal enhancement of Ru (bpy)₃ ²⁺ECL Signal decrease of (d), Ir (ppy)₃And Ru (bpy)₃ ²⁺The ratio of ECL signal(s) is linear with As (III) concentration, thus constructing a Tp-Bpy COF-based co-reactant effect and Ru (Bpy)₃ ²⁺And Ir (ppy)₃And for the ultrasensitive detection of As (III).

Description

Preparation method and application of electrochemical luminescence sensor

Technical Field

The invention relates to a preparation method and application of an electrochemiluminescence sensor, and belongs to the technical field of electrochemiluminescence sensing.

Background

Arsenic is widely distributed in the natural world, wherein inorganic arsenic is a carcinogenic and highly toxic substance and can cause lung cancer and skin cancer. Arsenic poisoning mainly comes from polluted drinking water, so that the development of a detection method for arsenic in a water sample in a complex environment has important significance. In recent years, the detection means of arsenite as (iii) are various, including inductively coupled plasma mass spectrometry, fluorescence, raman scattering, electrochemical methods, colorimetric methods, Electrochemiluminescence (ECL), and the like, wherein ECL has the characteristics of high sensitivity, wide linear range, low background signal, low cost, and the like, and has outstanding advantages in analysis and detection applications. The dual wavelength ratio ECL further reduces background signal and improves selectivity and sensitivity of detection by using ECL emitters with distinguishable emission wavelengths. However, the ECL sensing method has been poorly studied for detecting arsenic. Therefore, the development of the As (III) detection method of the dual-wavelength ratio ECL sensor has great significance for detecting the As (III) pollution in the environmental water sample.

Terpyridyl ruthenium (Ru (bpy)₃ ²⁺) And iridium terphenyl pyridine (Ir (ppy)₃) The common ECL luminescent reagent is common ECL luminescent reagent, common coreactants of the ECL luminescent reagent mainly comprise tripropylamine, triethylamine, triethanolamine and the like, but the amine coreactants are easy to volatilize and have high toxicity and harm to human health. Ru (bpy)₃ ²⁺Ru (bpy) produced by reduction when used as a cathode electrochemiluminescence light-emitting reagent₃ ⁺Unstable in aqueous solution, and Ru (bpy)₃ ^2+/+Has a reduction potential comparable to that of hydrogen evolution, resulting in Ru (bpy)₃ ²⁺Has limited research on ECL, and therefore, research on Ru (bpy)₃ ²⁺The novel co-reactants of (a) are of great significance for cathodic ECL applications. Xing et al developed a boron nitride quantum dot as Ru (bpy)₃ ²⁺The ECL method of anodic co-reactants was used to detect dopamine (Xing, H.; ZHai, Q.; Zhang, X.; Li, J.; Wang E. boron nitride sodium dots as an effective coreactant for enhanced electrochemiluminescence of ruthenium (II) tris (2, 2' -bispyridyl), anal. chem.,2018,90, 2141-. Raju et al have studied glutathione as Ru (bpy)₃ ²⁺Glutathione was detected by ECL method with a cathode co-reactant (Raju, C.V.; Kumar, S.S.G. sensitive novel electrochemiluminescence of tris (2, 2' -dipyridine) glutathione (II) using glutathione as a co-reactant, chem.Commun.,2017,53, 6593-. At present, Ir (ppy)₃The study of the cathode ECL co-reactant of (a).

The Covalent Organic Framework (COF) is a crystalline polymeric material, and has excellent application prospects in the aspects of separation and recovery of energy storage metal ions, catalysis, gas adsorption and the like. COF materials have significant advantages over Metal Organic Frameworks (MOFs) and other polymeric materials, such as regular pore structure, flexible topological connectivity, excellent tunable functionality, etc. Recently, the fluorescent properties of COFs have been used to detect small molecules and ions. However, no study report of COF as an ECL co-reactant has been found.

Disclosure of Invention

The invention aims to provide a preparation method of an electrochemical luminescence sensor taking a covalent organic framework as a co-reactant and application of the electrochemical luminescence sensor in arsenite detection, and the electrochemical luminescence sensor has the advantages of high detection sensitivity, low detection limit and good selectivity.

The invention is realized by the following technical scheme:

an electrochemical luminous method using covalent organic frame as co-reactant is characterized by that firstly, the trialdehyde phloroglucinol and 5,5 '-diamino-2, 2' -dipyridine are reacted by Schiff base to synthesize covalent organic frame (Tp-Bpy COF) containing pyridine, the mixed solution of Tp-Bpy COF and chitosan is dropped on the surface of glassy carbon electrode, and (AC) is reacted by amide₂₉Single-stranded DNA is assembled on the surface of the electrode and then hybridized by DNA complementation (GT)₂₉The single-stranded DNA is attached to an electrode (GT)₂₉/(AC)₂₉Tp-Bpy COF modified glassy carbon electrode with (GT)₂₉/(AC)₂₉The Tp-Bpy COF modified glassy carbon electrode is taken as a working electrode, and the working electrode, the reference electrode and a counter electrode are placed in Ir (ppy)₃In the acetonitrile solution of (2), the composition takes Tp-Bpy COF as Ir (ppy)₃Tp-Bpy COF-Ir (ppy) of a cathodic electrochemiluminescence coreactant of₃The system adopts an MPI-B type electrochemical luminescence test system to test the ECL signal of the Tp-Bpy COF modified glassy carbon electrode in the potential range of-2.5 to + 0.2V; (GT)₂₉/(AC)₂₉Electrode modified with/Tp-Bpy COF in Ir (ppy)₃The acetonitrile solution has strong cathode electrochemiluminescence signals; general (GT)₂₉/(AC)₂₉the/Tp-Bpy COF modified electrode is arranged on Ru (Bpy)₃ ²⁺After reaction with phosphate buffer solution of (3), Ru (bpy)₃ ²⁺By electrostatic interaction into (GT)₂₉/(AC)₂₉In the double strand, the composition is represented by Tp-Bpy COF as Ru (Bpy)₃ ²⁺Tp-Bpy COF-Ru (Bpy) of a cathodic electrochemiluminescence coreactant of₃ ²⁺System to produce strong Ru (bpy)₃ ²⁺A cathodic electrochemiluminescence signal of (a); furthermore, Ir (ppy)₃Electrochemiluminescence emission spectrum of (2) and Ru (bpy)₃ ²⁺Overlap of ultraviolet absorption spectra of Ir (ppy)₃Ru (bpy) as an energy donor₃ ²⁺Ru (bpy) as an energy receptor₃ ²⁺And Ir (ppy)₃The electrochemiluminescence resonance energy transfer occurs between the two, so that Ru (bpy)₃ ²⁺Ir (ppy)₃The electrochemiluminescence signal of (a) is reduced.

(GT)₂₉/(AC)₂₉The preparation method of the Tp-Bpy COF modified glassy carbon electrode comprises the following steps:

s1, preparation of Tp-Bpy COF: adding trialdehyde phloroglucinol and 5,5 '-diamino-2, 2' -bipyridyl into a glass tube, adding 1, 4-dioxane, 1.0mL mesitylene and 6M acetic acid solution, quickly freezing the mixed solution in a liquid nitrogen bath, vacuumizing the glass tube and sealing flame at the same time, placing the sealed glass tube in an oven at 120 ℃ for reaction for 3 days, washing the product with anhydrous tetrahydrofuran, and drying the obtained brownish red product at 100 ℃ for 24 hours to prepare a covalent organic framework Tp-Bpy COF containing pyridine;

s2, pretreating a glassy carbon electrode: polishing a glassy carbon electrode by using 1.0, 0.3 and 0.05um alumina suspension in sequence, then ultrasonically cleaning the glassy carbon electrode in 0.1M nitric acid, absolute ethyl alcohol and ultrapure water for 1 minute in sequence, and drying the surface of the electrode by using nitrogen;

S3、(GT)₂₉/(AC)₂₉preparation of Tp-Bpy COF modified glassy carbon electrode: the mixed solution of Tp-Bpy COF and chitosan prepared in step S1 is dripped on the surface of the glassy carbon electrode cleaned in step S2, dried at room temperature, and then carboxyl modified (AC) is reacted by amide reaction₂₉Modifying single-stranded DNA to the surface of glassy carbon electrode, and performing DNA complementary hybridization reaction to obtain (GT)₂₉The single-stranded DNA is ligated to an electrode to prepare (GT)₂₉/(AC)₂₉The Tp-Bpy COF modifies the glassy carbon electrode.

Preferably, threeThe mass of the aldehyde phloroglucinol and the 5,5 '-diamino-2, 2' -bipyridyl is 0.3mmol and 0.2mmol respectively, and the ratio of the mass of the aldehyde phloroglucinol to the mass of the 5,5 '-diamino-2, 2' -bipyridyl is 3: 2; the concentration of the phosphate buffer solution is 20mM, and the pH value is 7.4; the concentration of Tp-Bpy COF dripped on the surface of the glassy carbon electrode is 0.5mg mL^-1The mass percentage concentration of the chitosan is 0.002%; ir (ppy)₃The concentration of (2) is 5. mu.M; (AC)₂₉And (GT)₂₉Is 100 nM; (AC)₂₉The reaction condition of the single-chain modification to the electrode surface is reaction for 12h (GT) at room temperature₂₉And (AC)₂₉Reacting at 37 ℃ for 30min to hybridize and form double chains; ru (bpy)₃ ²⁺In a concentration of 50mM, electrode and Ru (bpy)₃ ²⁺The reaction time of the solution is 30 min; ir (ppy)₃The electrochemiluminescence of (a) is positioned at 505nm, Ru (bpy)₃ ²⁺The electrochemiluminescence of (a) is at 620 nm.

The electrochemical luminescence method using covalent organic frame as co-reactant is applied to arsenite radical detection, firstly (GT)₂₉/(AC)₂₉Soaking the Tp-Bpy COF modified glassy carbon electrode in phosphate buffer solution containing As (III) with different concentrations for 12h, cleaning the surface of the electrode with ultrapure water, and placing the electrode in 50uL50mM Ru (Bpy)₃ ²⁺The surface of the electrode was washed with ultrapure water for 30min, the electrode was used as a working electrode, and the working electrode, a reference electrode and a counter electrode were placed together in 5uM Ir (ppy)₃In the acetonitrile solution, an MPI-B type electrochemical luminescence test system is adopted to test an electrochemical luminescence signal of an electrode in a potential range of-2.5 to +0.2V and under the condition of 505nm and 620nm filters, according to Ir (ppy) at 505nm₃And Ru (bpy) at 620nm₃ ²⁺Ratio of electrochemiluminescence signals of (ECL)₅₀₅/ECL₆₂₀) The relation between the concentration of As (III) and the concentration of As (III) realizes the detection of As (III), the linear range of the detection of As (III) is 0.1ppt-20ppb, and the detection limit is as low as 0.05 ppt.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention takes Tp-Bpy COF as Ru (Bpy)₃ ²⁺And Ir (ppy)₃Cathode EC ofL co-reactant, establishes Tp-Bpy COF-Ir (ppy)₃And Tp-Bpy COF-Ru (Bpy)₃ ²⁺The new system of ECL of (1);

(2) the invention constructs a new method based on Ir (ppy)₃The ECL method of the two-wavelength ratio of the ECL-RET effect to the Tp-Bpy COF reveals the mechanism of the method and is used for sensitively detecting As (III);

(3) with Tp-Bpy COF as Ru (Bpy)₃ ²⁺And Ir (ppy)₃The cathode ECL coreactant replaces the traditional amine coreactant with large toxicity, and is beneficial to the sustainable development of the ecological environment;

(4) realizes the sensitivity and the selectivity detection of As (III) in an actual water sample, and has good application prospect.

Drawings

FIG. 1 is (A) a PXRD pattern for Tp-BpyCOF; (B) tp, Bpy and Tp-BpyCOF.

FIG. 2 is a schematic diagram of the construction process of the two wavelength ratio ECL method and the detection of As (III).

FIG. 3 is an ECL-potential curve. (A) GCE (a), Tp-Bpy COF/GCE (b), Tp/GCE (c) and Bpy/GCE (d) in Ir (ppy)₃In solution; (B) GCE (a), Tp-Bpy COF/GCE (b), Tp/GCE (c) and Bpy/GCE (d) in Ru (Bpy)₃ ²⁺In solution.

Fig. 4 is CV and EIS characterization of the electrode assembly process: gce (a); Tp-Bpy COF/GCE (b); (AC)₂₉/Tp-Bpy COF/GCE (c)；(GT)₂₉/(AC)₂₉/Tp-Bpy COF/GCE(d)；Ru(bpy)₃ ²⁺/(GT)₂₉/(AC)₂₉/Tp-Bpy COF/GCE(e)；Ru(bpy)₃ ²⁺/As(III)/(GT)₂₉/(AC)₂₉The concentration of/Tp-Bpy COF/GCE (f), As (III) is 10 ppb.

FIG. 5 is an ECL-time curve at 505nm and 620nm for different electrodes. Tp-Bpy COF/GCE (a), (AC)₂₉/Tp-Bpy COF/GCE(b)，(GT)₂₉/(AC)₂₉/Tp-Bpy COF/GCE(c)，Ru(bpy)₃ ²⁺/(GT)₂₉/(AC)₂₉/Tp-Bpy COF/GCE (d)，Ru(bpy)₃ ²⁺/As(III)/(GT)₂₉/(AC)₂₉Tp-Bpy COF/GCE (e). As (III) at a concentration of 10 ppb; inner partThe inset is Ru (bpy) at 620nm₃ ²⁺ECL signal magnification of (a).

FIG. 6 is (A) the response of ECL signals at 505nm and 620nm of a dual wavelength ECL sensor to different concentrations of As (III); (B) lg (ECL)₅₀₅/ECL₆₂₀)-lnC_As(III)Curve line.

FIG. 7 is (A) the effect of different interfering substances on ECL intensity of the cathode and anode, respectively; (B) interference of substance pair ECL₅₀₅/ECL₆₂₀The influence of (c). As (III) concentration is 10ppb, and the concentration of interfering substances is 100 ppb.

Detailed Description

The invention will be further elucidated with reference to the drawings and the embodiments without being limited thereto;

example 1

Preparation and characterization of Tp-Bpy COF

Preparation of Tp-Bpy COF: adding trialdehyde phloroglucinol (Tp) and 5,5 '-diamino-2, 2' -bipyridine (Bpy) into a glass tube, adding 1, 4-dioxane, 1.0mL mesitylene and 6M acetic acid solution, quickly freezing the mixed solution in a liquid nitrogen bath, vacuumizing the glass tube and sealing a flame at the same time, placing the sealed glass tube in an oven at 120 ℃ for reaction for 3 days, washing the product with anhydrous tetrahydrofuran, and drying the obtained brownish red product at 100 ℃ for 24 hours to prepare the covalent organic framework Tp-Bpy COF containing pyridine.

FIG. 1A is a powder X-ray diffraction Pattern (PXRD) of Tp-Bpy COF having a strong diffraction peak at 3.6 deg. due to reflection of the (100) crystal plane and diffraction peaks at 7.2 deg. and 26.9 deg. due to the (200) and (002) crystal planes of Tp-Bpy COF, respectively, indicating that Tp-Bpy COF has a high degree of crystallinity. In addition, the experimentally measured PXRD pattern substantially corresponds to the simulated PXRD pattern (fig. 1A inset). By N at 77K₂The specific surface area of Tp-Bpy COF is 755m measured by adsorption and desorption isotherms²g^-1. FIG. 1B is an IR spectrum of Tp, Bpy and Tp-Bpy COF, wherein Tp is 2895cm^-1(ii) a peak sum of stretching vibration of-CHO at Bpy of 3280cm^-1Of (a) NH of (b)₂The characteristic expansion vibration peak of (2) disappears at 1287cm^-1A new stretching vibration peak appears due to the stretching vibration of-C-N. The above results show that the method of the present invention successfully produces Tp-Bpy COF with high crystallinity. The results of thermogravimetric experiments show that Tp-Bpy COF is stable at the temperature of 340 ℃, which shows that the Tp-Bpy COF has good thermal stability.

Example 2

Co-reactant Effect of Tp-BpyCOF

Polishing the glassy carbon electrode by using alumina suspension liquid with the particle sizes of 1.0, 0.3 and 0.05 mu M in sequence, then ultrasonically cleaning the glassy carbon electrode for 1 minute in nitric acid with the particle size of 0.1M, absolute ethyl alcohol and ultrapure water in sequence, and drying the surface of the glassy carbon electrode by using nitrogen. 10uL 0.5mgmL^-1A mixed solution of Tp-Bpy COF and 0.002% Chitosan (CS) was applied dropwise to a cleaned GCE surface, dried at room temperature, and then the electrode was placed in a container containing 100nM carboxyl group-modified (AC)₂₉The single-stranded DNA of (1) was reacted in a PBS (20mM pH 7.4) buffer solution for 12 hours, and (AC)₂₉Modifying the surface of the electrode by amide reaction, washing the electrode with ultrapure water, and immersing the resulting electrode in a solution containing 100nM (GT)₂₉The single-stranded DNA of (4) was reacted at 37 ℃ for 30min in a PBS (20mM pH 7.4) buffer solution, (GT)₂₉Connecting to an electrode by DNA complementary hybridization to produce (GT)₂₉/(AC)₂₉The schematic diagram of the electrode modification process and the detection of As (III) of the electrode modification process of/Tp-Bpy COF/GCE is shown in FIG. 2.

As can be seen from FIG. 3A, in Ir (ppy)₃In the acetonitrile solution of (1), GCE only has weak cathode ECL (curve a), Tp-Bpy COF/GCE shows strong ECL signal (curve b) at-2.4V, and Tp/GCE (curve c) and Bpy/GCE (curve d) have slightly enhanced cathode signals, but the ECL signal is obviously worse than that of Tp-Bpy COF/GCE, so that Tp-Bpy COF/GCE can be used as Ir (ppy)₃Cathode ECL co-reactant enhancement of Ir (ppy)₃The cathode ECL signal of (a). In Ru (bpy)₃ ²⁺Tp-Bpy COF/GCE also showed an enhancement of cathodic ECL in phosphate buffered solutions (FIG. 3B). Thus, Tp-Bpy COF can be used as Ir (ppy)₃The cathode ECL co-reactant of (a) may also be used as Ru (bpy)₃ ²⁺The cathode ECL co-reactant of (a).

Effect mechanism explanation of co-reactant of Tp-Bpy COFThe following were used: with Tp-Bpy COF-Ru (Bpy)₃ ²⁺Example of the System, Ru (bpy)₃ ²⁺Loss of electrons at cathodic potential is reduced to Ru (bpy)₃ ⁺Meanwhile, pyridine N on the Tp-Bpy COF also loses electrons and undergoes protonation reaction, and a more stable Tp-Bpy COF is generated after electron transfer^·A free radical; Tp-Bpy COF^·And Ru (bpy)₃ ²⁺Reaction to yield Ru (bpy)₃ ³⁺And Tp-Bpy COF; ru (bpy) formed by the reaction₃ ³⁺Further with Ru (bpy)₃ ⁺Reaction to yield excited Ru (bpy)₃ ^2+*(ii) a Simultaneous Tp-Bpy COF^·Or with Ru (bpy)₃ ⁺Redox reaction to Ru (bpy)₃ ^2+*(ii) a Resulting Ru (bpy)₃ ^2+*The released energy returns to the ground state, producing a strong cathodic ECL signal. For Tp-Bpy COF-Ir (ppy)₃Systems, the reaction mechanism of which is in accordance with Tp-Bpy COF/GCE-Ru (Bpy)₃ ²⁺The system is the same.

Example 3

Construction and characterization of ECL sensors

The construction process of the ECL sensor is characterized by adopting Cyclic Voltammetry (CV) and alternating current impedance spectroscopy (EIS). As can be seen from FIG. 4A, in [ Fe (CN)₆]^3-/4-In the solution, GCE has a distinct redox peak (curve a); the peak current on Tp-Bpy COF/GCE is significantly reduced (curve b); since (AC)₂₉Single-stranded DNA having a negatively charged phosphate backbone, (AC)₂₉The current on/Tp-Bpy COF/GCE is further reduced (curve c); when (GT)₂₉The peak current was further reduced when connected to the electrode surface by DNA hybridization (curve d); (GT)₂₉/(AC)₂₉/Tp-Bpy COF/GCE in Ru (Bpy)₃ ²⁺After 30min reaction in solution, since Ru (bpy)₃ ²⁺Positively charged, the peak current of the electrode increases slightly (curve e); the peak current of the electrode is significantly enhanced (curve f) in the presence of 10ppb As (III), due to As (III) and (GT)₂₉Specifically bind to, such that (GT)₂₉And the DNA cannot be connected to the electrode by hybridization. FIG. 4B is EIS spectrum of electrode assembly process with high impedanceThe small corresponds to the cyclic voltammetry results, which indicate successful construction of ECL sensors.

FIG. 5 is an ECL-time curve at 505nm and 620nm for different electrodes. In Ir (ppy)₃In acetonitrile solution of (2), Tp-Bpy COF/GCE has a strong cathodic ECL signal at 505nm (curve a) due to Tp-Bpy COF as Ir (ppy)₃Can enhance Ir (ppy)₃The cathode ECL signal of (a). When (AC)₂₉Attachment to the electrode by amide reaction (curve b), (GT)₂₉By DNA hybridization to the electrode surface (curve c), the ECL signal is gradually reduced due to the electron transport hindered by the negative charge on the single-stranded and double-stranded phosphate backbone of the DNA. (GT)₂₉/(AC)₂₉/Tp-Bpy COF/GCE in Ru (Bpy)₃ ²⁺After 30min reaction in solution, Ru (bpy)₃ ²⁺Intercalation into DNA duplexes, producing strong Ru (bpy) at 620nm₃ ²⁺The cathodic ECL signal of (a); furthermore, since Ir (ppy)₃And Ru (bpy)₃ ²⁺The resonance transfer effect of energy between and Ru (bpy)₃ ²⁺Introducing into the electrode surface and reacting with Ir (ppy)₃Co-reactant enhancement competing for Tp-Bpy COF, Ir (ppy) at 505nm₃The ECL signal of (2) is significantly reduced due to the double quenching effect, Ru (bpy) at 620nm₃ ²⁺The ECL signal of (c) is significantly enhanced due to the double amplification effect (curve d). When As (III) is present, As (III) and (GT)₂₉Specific binding, resulting in (GT)₂₉Inability to attach to electrode surface by DNA complementary hybridization, Ru (bpy)₃ ²⁺Nor can it be embedded on the electrode surface by electrostatic interaction, so that Ir (ppy) at 505nm₃The ECL signal of (2) is recovered, and Ru (bpy) at 620nm₃ ²⁺ECL of (d) (curve e). The above results indicate that the sensor can be used to detect As (III).

Example 4

Detection of As (III) by a dual wavelength ratio ECL sensor

Firstly (GT)₂₉/(AC)₂₉Soaking the electrode in phosphate buffer solution containing different concentrations of As (III) and As (Tp-Bpy COF/GCE) for 12h, washing the surface of the electrode with ultrapure water, and removing the electrodePlaced in 50uL50mM Ru (bpy)₃ ²⁺The surface of the electrode was washed with ultrapure water for 30min, the electrode was used as a working electrode, and the working electrode, a reference electrode and a counter electrode were placed together in 5uM Ir (ppy)₃In the acetonitrile solution, an MPI-B type electrochemical luminescence test system is adopted to test an electrochemical luminescence signal of an electrode in a potential range of-2.5 to +0.2V and under the condition of 505nm and 620nm filters, according to Ir (ppy) at 505nm₃And Ru (bpy) at 620nm₃ ²⁺Ratio of electrochemiluminescence signals of (ECL)₅₀₅/ECL₆₂₀) And the concentration of As (III) to realize the detection of As (III),

FIG. 6A is a graph of the response of the ECL signal at 505nm and 620nm of a dual wavelength ECL sensor to different concentrations of As (III). As (III) concentration increases, As (III) and (GT)₂₉The more combinations, the more (GT)₂₉Inability to modify to the electrode by DNA hybridization results in Ru (bpy)₃ ²⁺Cannot be modified to the surface of the electrode, so that 505nm of Ir (ppy)₃The ECL signal of (2) gradually recovers, and Ru (bpy) at 620nm₃ ²⁺Gradually decreases. FIG. 6B shows lg (ECL)₅₀₅/ECL₆₂₀)-lnC_As(III)The linear range of the detection for As (III) is 0.1ppt-20ppb, the detection limit is 0.05ppt (S/N is 3), and the detection limit is far lower than the maximum limit (10ppb) of the concentration of As (III) in drinking water specified by the environmental protection agency. The limit of detection of As (III) by the method of the present invention is lower than that of fluorescence sensors constructed by Wang et al based on the binding of Arsenic with small molecule compounds, which detect Arsenic by 1.32ppb (Tian, X.; Chen, L.; Li, Y.; Yang, C.; Nie, Y.; Zhou, C.; Wang, Y.; Design and synthesis of a nanoparticle with aggregation-induced emission and analysis effects in the detection of Arsenic by Bimetallic nanoparticle anodic voltammetry, which detects Arsenic by 1.2ppb (Moghimi, N.; Mohapapapa, M.; Leung, K.T., biological for organic emission, 5587, 52).

To evaluate the selectivity of ECL sensors constructed by the method of the present invention, ECL sensors were examined for other ECL sensors containing 100ppbECL response of the ions, the results are shown in FIG. 7A, Ir (ppy) at 505nm₃ECL signal of (2) and Ru (bpy) at 620nm₃ ²⁺In Hg of the ECL signal²⁺And Ag⁺Slightly changed when present, respectively, due to Hg²⁺And Ag⁺Influence by binding to T and C bases, respectively (GT)₂₉And (AC)₂₉Thereby affecting Ru (bpy)₃ ²⁺Efficiency of intercalation into the duplex. However, the ratiometric ECL sensor responded well to as (iii) and hardly responded to other ions at concentrations ten times as (iii) (fig. 7B), indicating that the dual wavelength ratiometric ECL method constructed by the present invention has good selectivity for as (iii) detection.

In order to verify the practicability of the method in environmental sample analysis, the method performs a standard addition recovery experiment on tap water, lake water and Ganjiang water, and adds As (III) with different concentrations into an actual water sample respectively, wherein the standard addition recovery rate of the method to As (III) is 96.2% -110%, which shows that the method has good reliability on As (III) detection.

Claims

1. The preparation method of the electrochemical luminescence sensor taking the covalent organic framework as the co-reactant is characterized in that:

synthesizing a covalent organic framework containing pyridine by reacting trialdehyde phloroglucinol with 5,5 '-diamino-2, 2' -bipyridine through Schiff base;

dropping a mixed solution of a covalent organic framework containing pyridine and chitosan on the surface of a glassy carbon electrode, and reacting (AC) through an amide reaction₂₉Single-stranded DNA is assembled on the surface of the electrode and then hybridized by DNA complementation (GT)₂₉The single-stranded DNA is attached to an electrode (GT)₂₉/(AC)₂₉The Tp-BpyCOF modifies the glassy carbon electrode;

in order (GT)₂₉/(AC)₂₉The Tp-BpyCOF modified glassy carbon electrode is taken as a working electrode, and the working electrode, the reference electrode and a counter electrode are placed in Ir (ppy)₃In the acetonitrile solution of (2), the composition is that Tp-BpyCOF is Ir (ppy)₃Tp-BpyCOF-Ir (ppy) of a cathodic electrochemiluminescence coreactant of₃The system adopts MPI-B type electrochemical luminescence test systemTesting an ECL signal of the Tp-BpyCOF modified glassy carbon electrode in a potential range of-2.5 to + 0.2V;

(GT)₂₉/(AC)₂₉electrode modified with/Tp-BpyCOF in Ir (ppy)₃Has a strong cathode electrochemical luminescence signal in acetonitrile solution when (GT)₂₉/(AC)₂₉the/Tp-BpyCOF modified electrode is arranged on Ru (bpy)₃ ²⁺After reaction with phosphate buffer solution of (3), Ru (bpy)₃ ²⁺By electrostatic interaction into (GT)₂₉/(AC)₂₉In the double strand, the composition is Tp-BpyCOF Ru (bpy)₃ ²⁺Tp-BpyCOF-Ru (bpy) of a cathodic electrochemiluminescence coreactant of₃ ²⁺System to produce strong Ru (bpy)₃ ²⁺A cathodic electrochemiluminescence signal of (a);

Ir(ppy)₃electrochemiluminescence emission spectrum of (2) and Ru (bpy)₃ ²⁺Overlap of ultraviolet absorption spectra of Ir (ppy)₃Ru (bpy) as an energy donor₃ ²⁺Ru (bpy) as an energy receptor₃ ²⁺And Ir (ppy)₃The electrochemiluminescence resonance energy transfer occurs between the two, so that Ru (bpy)₃ ²⁺Ir (ppy)₃The electrochemiluminescence signal of (a) is reduced.

2. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

(GT)₂₉/(AC)₂₉the preparation method of the Tp-BpyCOF modified glassy carbon electrode comprises the following steps:

s1, preparation of Tp-BpyCOF: adding trialdehyde phloroglucinol and 5,5 '-diamino-2, 2' -bipyridyl into a glass tube, adding 1, 4-dioxane, 1.0mL mesitylene and 6M acetic acid solution, quickly freezing the mixed solution in a liquid nitrogen bath, vacuumizing the glass tube and sealing flame at the same time, placing the sealed glass tube in a 120-DEG oven for reaction for 3 days, washing a product with anhydrous tetrahydrofuran, and drying the obtained brownish red product for 24 hours at 100-DEG to prepare a covalent organic framework Tp-BpyCOF containing pyridine;

s2, pretreating a glassy carbon electrode: polishing a glassy carbon electrode by using alumina suspension liquid with the particle sizes of 1.0, 0.3 and 0.05 mu M in sequence, then ultrasonically cleaning the glassy carbon electrode for 1 minute in nitric acid with the particle size of 0.1M, absolute ethyl alcohol and ultrapure water in sequence, and drying the surface of the electrode by using nitrogen;

S3、(GT)₂₉/(AC)₂₉preparation of Tp-BpyCOF modified glassy carbon electrode: the mixed solution of Tp-BpyCOF and chitosan prepared in step S1 is dripped on the surface of the glassy carbon electrode cleaned in step S2, dried at room temperature, and then carboxyl modified (AC) is reacted by amide reaction₂₉Modifying single-stranded DNA to the surface of glassy carbon electrode, and performing DNA complementary hybridization reaction to obtain (GT)₂₉The single-stranded DNA is ligated to an electrode to prepare (GT)₂₉/(AC)₂₉The Tp-BpyCOF modifies the glassy carbon electrode.

3. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

the amount of the trialdehyde phloroglucinol and the 5,5 '-diamino-2, 2' -bipyridyl is 0.3mmol and 0.2mmol respectively, and the ratio of the amount of the trialdehyde phloroglucinol to the amount of the 5,5 '-diamino-2, 2' -bipyridyl is 3: 2.

4. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

the phosphate buffer solution has a concentration of 20mM and a pH of 7.4.

5. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

the concentration of Tp-BpyCOF dripped on the surface of the glassy carbon electrode is 0.5mgmL^-1The mass percentage concentration of the chitosan is 0.002%.

6. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

the Ir (ppy)₃The concentration of (2) was 5 uM.

7. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

said (AC)₂₉And (GT)₂₉At a concentration of 100nM, (AC)₂₉The reaction condition for modifying the single-stranded DNA on the surface of the electrode is room temperature for 12h, (GT)₂₉And (AC)₂₉And reacting at 37 ℃ for 30min to hybridize to form double-stranded DNA.

8. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

Ru(bpy)₃ ²⁺in a concentration of 50mM, electrode and Ru (bpy)₃ ²⁺The reaction time of the solution was 30 min.

9. The method of claim 1 for preparing an electrochemiluminescence sensor using a covalent organic framework as a co-reactant, wherein:

Ir(ppy)₃the electrochemiluminescence of (a) is positioned at 505nm, Ru (bpy)₃ ²⁺The electrochemiluminescence of (a) is at 620 nm.

10. The use of the electrochemiluminescence sensor prepared by the method of claim 1 in arsenite detection is characterized in that:

general (GT)₂₉/(AC)₂₉Soaking the Tp-BpyCOF modified glassy carbon electrode in phosphate buffer solution containing arsenite radicals with different concentrations for 12h, cleaning the surface of the electrode with ultrapure water, and placing the electrode in 50uL50mMRu (bpy)₃ ²⁺The surface of the electrode was washed with ultrapure water for 30min, the electrode was used as a working electrode, and the working electrode, a reference electrode and a counter electrode were placed together in 5uMIr (ppy)₃In the acetonitrile solution, an MPI-B type electrochemical luminescence test system is adopted to test an electrochemical luminescence signal of an electrode in a potential range of-2.5 to +0.2V and under the condition of 505nm and 620nm filters, according to Ir (ppy) at 505nm₃Electrochemical (c) ofChemiluminescence signal and Ru (bpy) at 620nm₃ ²⁺Ratio of electrochemiluminescence signals of (ECL)₅₀₅/ECL₆₂₀) And the relation between the concentration of the arsenite and the concentration of the arsenite realizes the detection of the arsenite, the linear range of the arsenite detection is 0.1ppt-20ppb, and the detection limit is as low as 0.05 ppt.