CN109100400B - Sensor for detecting concanavalin A, its preparation method and application - Google Patents

Abstract

The invention relates to the technical field of electrochemiluminescence sensors, in particular to a sensor for detecting concanavalin A, its preparation method and application. The sensor for detecting concanavalin A includes a cathode luminescent body, an anode luminescence body and a co-reaction reagent, the cathode luminescence body includes cadmium telluride quantum dots modified by graphene oxide, and the anode luminescence body includes nano gold and nanoplatinum-modified N‑(aminobutyl)‑N‑(ethylisoluminol). The preparation method comprises: the cadmium telluride quantum dots modified by graphene oxide are combined with the recognition unit through π-π stacking, and modified on the surface of the pretreated electrode; the anode emitter combined with the recognition unit is modified, and incubated to obtain the sensor. The invention builds a ratio electrochemiluminescence system based on competitive consumption of oxygen, improves the signal-to-noise ratio, and makes the detection result highly reliable.

Description

Sensor for detecting concanavalin A, its preparation method and application

technical field

The invention relates to the technical field of electrochemiluminescence sensors, in particular to a sensor for detecting concanavalin A, its preparation method and application.

Background technique

Electrogenerated Chemiluminescence (ECL) is a new type of detection technology combining electrochemical and spectroscopic methods. It has the advantages of strong versatility, simple optical device, and good time and space controllability. , Molecular recognition, DNA detection and immune analysis have been widely used.

At present, most ECL detection is based on single-signal detection. These single-signal detection methods are susceptible to distorted results due to changes in the electrode surface and dissociation of reagents induced by non-target substances, especially in complex biological environments such as cells and body fluids. Therefore, overcoming the interference of environmental factors and improving the sensitivity and reliability of analysis are very important for clinical micro and trace analysis.

Concanavalin A (Con A) is a carbohydrate-binding protein and a phytohemagglutinin, which has a strong mitogenic effect, a good effect on promoting lymphocyte transformation, and can precipitate glycogen Originally, it agglutinates red blood cells of sheep, horses, dogs, rabbits, pigs, rats, mice, guinea pigs and other animals and humans, and can also selectively activate suppressive T cells (Ts), which plays an important role in regulating the body's immune response. As a lectin model mediating pathological and physiological responses, ConA is often selected for further clinical testing.

In view of this, the present invention is proposed.

Contents of the invention

The first object of the present invention is to provide a sensor for detecting concanavalin A, said sensor is a ratio-type electrochemiluminescence system constructed based on competitive consumption of oxygen, which improves the signal-to-noise ratio, compared with single signal detection The means can effectively avoid the interference to the detection caused by the instrument, environmental factors, and some human operation factors, so that the reliability of the detection results is high.

The second object of the present invention is to provide a preparation method of the sensor for detecting concanavalin A, the preparation method has simple process, mild reaction conditions and strong operability.

The third object of the present invention is to provide an application of the sensor for detecting concanavalin A in detecting concanavalin A. The sensor has high detection sensitivity to concanavalin A, low detection limit, and can detect concanavalin A. The concanavalin A in the sample is detected in ultra-trace amount, with high reliability.

In order to realize the above-mentioned purpose of the present invention, special adopt following technical scheme:

A sensor for detecting concanavalin A, comprising a cathode luminescent body, an anode luminescence body and co-reaction reagents, the cathode luminescence body includes cadmium telluride quantum dots modified by graphene oxide, and the anode luminescence body includes nano gold And nano-platinum modified N-(aminobutyl)-N-(ethylisoluminol).

The present invention respectively adopts graphene oxide modified cadmium telluride quantum dots as cathode luminous body, N-(aminobutyl)-N-(ethyl isoluminol) modified by nano-gold platinum as anode luminous body, and cathodic luminous body The upper modification can recognize the recognition unit of the target concanavalin A, which is used to bind the target. The graphene-modified cadmium telluride quantum dots are used as the matrix, and the recognition unit is combined through π-π stacking, so that carbohydrates and The biospecific interaction between proteins immobilizes the target analyte Con A; the recognition unit that can recognize the target concanavalin A is also modified on the anode luminescent body, and the target analyte immobilized on the cathodoluminescent complex is further used The specific recognition of Con A and the recognition unit in the anode emitter realizes the immobilization of the anode emitter. With the increase of the concentration of the target analyte Con A, the immobilization amount of the anode emitter increases, and the ECL signal of the cathode emitter decreases due to the competition between the cathode emitter and the anode emitter for co-reaction reagents. The ECL signal is enhanced, so that the opposite changes of the two ECL signals are obtained, and the concentration of the target analyte Con A is detected through the signal ratio.

The N-(aminobutyl)-N-(ethyl isoluminol) modified by the nano-gold and nano-platinum can reduce the ECL signal of the graphene quantum dot while enhancing its own ECL signal, according to different The ratio of the two ECL signals that show opposite changes in the potential realizes the ratiometric detection.

Preferably, the coreactant comprises oxygen. More preferably, the oxygen is dissolved oxygen.

In the sensor of the present invention, the competition process between the anode luminous body and the negative luminous body only consumes the dissolved oxygen in the detection solution, without adding co-reaction reagents such as H ₂ O ₂ , and the detection is convenient.

Preferably, the sensor further includes a recognition unit modified on the cathode luminescent body and the anode luminescent body respectively, and the recognition unit is used to recognize concanavalin A.

Preferably, the recognition unit includes phenoxylated dextran.

Preferably, the cathode emitter and the anode emitter are modified on the surface of the glassy carbon electrode.

The present invention also provides a method for preparing the sensor for detecting concanavalin A, comprising the following steps:

The cadmium telluride quantum dot modified by graphene oxide is combined with the recognition unit through π-π stacking, and is modified on the surface of the pretreated electrode; the anode luminescent body combined with the recognition unit is modified and incubated to obtain the sensor.

The preparation method of the sensor for detecting concanavalin A of the present invention has simple process, mild reaction conditions and strong operability.

Preferably, the graphene oxide-modified cadmium telluride quantum dots are dispersed in water, and the dispersion concentration is 0.5-2.0 mg/mL, preferably 1.0 mg/mL.

Preferably, the recognition unit includes phenoxylated dextran.

Preferably, the concentration of the phenoxylated dextran is 5-30 mg/mL, preferably 10-20 mg/mL, more preferably 15 mg/mL. More preferably, the phenoxylated dextran is made into an aqueous solution of phenoxylated dextran.

Preferably, the volume ratio of the graphene oxide-modified cadmium telluride quantum dot dispersion to the phenoxylated dextran solution is 1:(0.8-1.2), preferably 1:1.

Preferably, the preparation method of the anode emitter comprises: mixing and reacting HAuCl ₄ , N-(aminobutyl)-N-(ethylisoluminol) and H ₂ PtCl ₆ , and stirring to obtain the anode emitter .

Preferably, the preparation method of the anode emitter comprises:

Add the solution of HAuCl ₄ into the solution of N-(aminobutyl)-N-(ethylisoluminol), stir at room temperature for 1-3h; add the solution of H ₂ PtCl ₆ , stir at room temperature for 1-3h to obtain The solutions were mixed and the solid was collected by centrifugation.

Preferably, the anode emitter is mixed with a solution of phenoxylated dextran such that the anode emitter is bound to the phenoxylated dextran. More preferably, the anode emitter is dispersed in water, a solution of phenoxylated dextran is added, and stirred for 1-3 hours.

Preferably, the mass concentration of the HAuCl ₄ solution is 0.5-2%, preferably 1%. More preferably, the solution of HAuCl ₄ is an aqueous solution of HAuCl ₄ .

Preferably, the mass concentration of the H ₂ PtCl ₆ solution is 0.5-2%, preferably 1%. More preferably, the H ₂ PtCl ₆ solution is an aqueous H ₂ PtCl ₆ solution.

Preferably, the concentration of the N-(aminobutyl)-N-(ethylisoluminol) solution is 5-15mmol/L, preferably 10mmol/L. More preferably, the N-(aminobutyl)-N-(ethylisoluminol) solution is an aqueous solution of N-(aminobutyl)-N-(ethylisoluminol).

Preferably, the volume ratio of the HAuCl ₄ solution, the H ₂ PtCl ₆ solution, and the N-(aminobutyl)-N-(ethyl isoluminol) solution is 1:(0.8- 1.2): (1-3), preferably 1:1:2.

Preferably, the centrifugation conditions include: centrifugation at a rotational speed of 10000±2000 rpm/min for 10-20 min.

Preferably, the preparation method of the sensor comprises the steps of:

Graphene oxide-modified cadmium telluride quantum dots were dropped onto the electrode surface, dried, and recognition units were added dropwise to form π-π stacking.

Preferably, after π-π stacking is formed, a solution of bovine serum albumin is drop-coated on the surface of the electrode. More preferably, the mass fraction of the bovine serum albumin solution is 0.5-1.5%, preferably 1%.

Preferably, the volume ratio of the bovine serum albumin solution to the graphene oxide-modified cadmium telluride quantum dot dispersion is 1:(1-1.5).

Preferably, after the bovine serum albumin solution is drop-coated, the solution to be tested containing concanavalin A is added dropwise for incubation.

Preferably, the test solution containing concanavalin A is added dropwise for incubation and then the anode luminescent body bound to the recognition unit is modified. More preferably, the method for modifying the anode emitter combined with the recognition unit comprises: drop-coating the anode emitter combined with the recognition unit on the surface of the incubated electrode, and incubating at 0-10°C for 1-3h, preferably Incubate for 2 h at 4°C.

Preferably, the concentration of concanavalin A in the test solution is ≥3.0×10 ^-5 ng/mL, preferably 3.0×10 ^-5 -10 ng/mL.

Preferably, the preparation method of the graphene oxide-modified cadmium telluride quantum dots comprises: mixing a solution of CdCl ₂ and graphene oxide, adding Na ₂ TeO ₃ , C ₆ H ₅ Na ₃ O ₇ , mercaptopropionic acid and NaBH _4. Refluxing at 120-140° C. for 8-12 hours, washing with ethanol and water and centrifuging to collect solids, and dispersing them in water to obtain the graphene oxide-modified cadmium telluride quantum dots. More preferably, the dispersion concentration of the graphene oxide-modified cadmium telluride quantum dots is 1 mg/mL.

Preferably, the pretreatment method of the electrode surface includes: polishing the glassy carbon electrode with alumina powder of 0.25-0.35 μm and 0.45-0.55 μm respectively, washing in water and ethanol, and drying.

The present invention also provides an application of the sensor for detecting concanavalin A in detecting concanavalin A.

Preferably, the method for detecting concanavalin A comprises the steps of:

(1) After incubating the sensor with a solution containing different concentrations of standard concanavalin A, collect the luminescent signals of the cathode luminescent body and the anode luminescent body, and compare the intensity ratio of the two luminescent signals with that of the standard concanavalin A. The logarithm of solution concentration is carried out linear fitting, obtains working curve;

(2) Incubating the sensor with the test solution, collecting the luminescent signals of the cathodoluminescent body and the anode luminescent body, and calculating the concentration of concanavalin A in the test solution through the working curve.

Compared with prior art, the beneficial effect of the present invention is:

(1) The present invention adopts specific cathodoluminescent bodies and anode luminous bodies respectively, and binds the target analyte Con A immobilized on the cathodoluminous bodies with the specific recognition of the phenoxylated dextran in the anode luminous bodies; With the increase of the concentration of the target analyte Con A, the immobilization amount of the anode luminophore increases accordingly, and the ECL signal of the cathode luminescence decreases due to the competition between the cathode luminescence and the anode luminescence for co-reaction reagents, and the ECL of the anode luminescence The signal is enhanced, so that the opposite changes of the two ECL signals are obtained, and the concentration of the target analyte Con A is detected through the signal ratio, so as to realize the detection of the concentration of concanavalin A by a ratio-type electrochemiluminescence system constructed by competing to consume oxygen , compared with a single signal detection method, it can effectively avoid interference to the detection caused by instruments, environmental factors, and some human operation factors, so that the reliability of the detection results is high;

(2) The present invention also defines the dosage ratio relationship of each substance, which further improves the detection sensitivity of the prepared sensor and reduces the detection limit;

(3) The preparation method of the sensor for detecting concanavalin A according to the present invention has simple process, mild reaction conditions and strong operability;

(4) The detection limit of the sensor of the present invention for detecting concanavalin A can reach 3.0×10 ^-5 ng/mL.

Part of the name description and abbreviation involved in the present invention refer to as follows:

Graphene oxide: GO

Graphene oxide-modified cadmium telluride quantum dots: G-CdTe QDs

Phenoxylated dextran: Dexp

N-(aminobutyl)-N-(ethylisoluminol): ABEI

Nano-gold and nano-platinum modified N-(aminobutyl)-N-(ethylisoluminol): ABEI-Au-Pt

Glassy carbon electrode: GCE

Bovine serum albumin: BSA.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Figure 1 is a schematic diagram of the preparation process of the sensor provided by the embodiment of the present invention and the corresponding detection principle;

Fig. 2 is the TEM image of the G-CdTe QDs prepared by the embodiment of the present invention;

Figure 3 is an enlarged image of the TEM image shown in Figure 2;

Fig. 4 is the SEM image of the ABEI-Au-Pt prepared by the embodiment of the present invention;

Fig. 5 is the XPS figure of the ABEI-Au-Pt that the embodiment of the present invention prepares,

Fig. 6 is the high-resolution curve of each element corresponding to the XPS diagram shown in Fig. 5, wherein, Fig. 6 (a)-(d) is the high-resolution curve of N, C, Au and Pt respectively;

Fig. 7 is the ECL spectrum of ABEI and the ultraviolet absorption spectrum of G-CdTe QDs prepared by the embodiment of the present invention;

Figure 8 is the ECL response curves of different modified electrodes prepared in the embodiment of the present invention,

Among them, a is the glassy carbon electrode modified with G-CdTe QDs; b is the glassy carbon electrode modified with Dexp and G-CdTe QDs; c is the glassy carbon electrode modified with BSA, Dexp and G-CdTe QDs; d is the modified Glassy carbon electrodes with Con A, BSA, Dexp and G-CdTe QDs; e are glassy carbon electrodes modified with Dexp-ABE I-Au-Pt, Con A, BSA, Dexp and G-CdTe QDs;

Figure 9 is the cyclic voltammetry curves of different modified electrodes prepared in the embodiment of the present invention,

Among them, a is the bare glassy carbon electrode; b is the glassy carbon electrode modified with G-CdTe QDs; c is the glassy carbon electrode modified with Dexp and G-CdTe QDs; d is the glassy carbon electrode modified with BSA, Dexp and G-CdTe QDs Glassy carbon electrode; e is glassy carbon electrode modified with Con A, BSA, Dexp and G-CdTe QDs; f is glassy carbon modified with Dexp-ABEI-Au-Pt, Con A, BSA, Dexp and G-CdTe QDs electrode;

Fig. 10 is the change figure of the ECL signal intensity of G-CdTe QDs of the present invention, ABEI with pH;

Fig. 11 is the ECL response curve of the sensor described in the embodiment of the present invention at different concentrations of Con A;

Fig. 12 is the working curve of sensor detection Con A described in the embodiment of the present invention;

Fig. 13 is a variation curve of the ECL signal of the sensor according to the embodiment of the present invention with the number of cycles;

Fig. 14 is a selectivity diagram of the sensor to Con A according to the embodiment of the present invention.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but those skilled in the art will understand that the embodiments described below are some of the embodiments of the present invention, rather than all of them. It is only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The present invention provides a sensor for detecting concanavalin A, comprising a cathodoluminescent body, anodic light emitting body and co-reaction reagents, the cathodoluminescent body includes G-CdTe QDs, and the anodic light emitting body includes ABEI-Au- Pt.

In a preferred embodiment of the present invention, said coreactant comprises oxygen. More preferably, the oxygen is dissolved oxygen.

The cathodoluminescent body that the present invention adopts and the anode luminous body, the possible luminescent mechanism under the condition that co-reaction reagent dissolved oxygen exists is as follows:

G-CdTe QDs+e ^- →G-CdTe QDs ^·- (1)

O ₂ +2G-CdTe QDs ^·- +2H ⁺ →H ₂ O ₂ +2G-CdTe QDs* (2)

G-CdTe QDs*→G-CdTe QDs+hν (3)

ABEI-e ^- → ABEI ⁺ (4)

O ₂ +e ^- → O ₂ ^·- (5)

ABEI ^·+ +O ₂ ^·- →ABEI* (6)

ABEI*→ABEI+hν (7)

In a preferred embodiment of the present invention, the sensor further includes a recognition unit modified on the cathode luminescent body and the anode luminescent body respectively, and the recognition unit is used to recognize concanavalin A. Preferably, the identification unit includes Dexp.

In a preferred embodiment of the present invention, the cathode emitter and the anode emitter are modified on the surface of the glassy carbon electrode.

The present invention also provides a method for preparing the sensor for detecting concanavalin A, comprising the following steps:

The G-CdTe QDs are combined with the recognition unit through π-π stacking, and are modified on the surface of the pretreated electrode; the anode luminescent body combined with the recognition unit is then modified and incubated to obtain the sensor.

In a preferred embodiment of the present invention, the G-CdTe QDs are dispersed in water, and the dispersion concentration is 0.5-2.0 mg/mL.

In a preferred embodiment of the present invention, the identification unit includes Dexp. Preferably, the concentration of Dexp is 5-30 mg/mL. More preferably, the Dexp is made into an aqueous solution of Dexp.

In a preferred embodiment of the present invention, the volume ratio of the G-CdTe QDs dispersion to the Dexp solution is 1:(0.8-1.2), preferably 1:1.

In a preferred embodiment of the present invention, the preparation method of the anode emitter comprises: mixing and reacting HAuCl ₄ , ABEI and H ₂ PtCl ₆ , and stirring to obtain the anode emitter.

In a preferred embodiment of the present invention, the preparation method of the anode emitter comprises:

The solution of HAuCl ₄ was added to the solution of ABEI, stirred at room temperature for 1-3 h; the solution of H ₂ PtCl ₆ was added, stirred at room temperature for 1-3 h to obtain a mixed solution, and the solid was collected by centrifugation.

In a preferred embodiment of the present invention, the anode emitter is mixed with a solution of phenoxylated dextran such that the anode emitter is bound to the phenoxylated dextran. Preferably, the anode emitter is dispersed in water, the solution of Dexp is added, and stirred for 1-3h. Preferably, the dispersion concentration of the anode emitter is 1-2 mg/mL, preferably 1.5 mg/mL. More preferably, the volume ratio of the volume of the anode emitter dispersion to the Dexp solution is 1:(0.8-1.2), preferably 1:1.

In a preferred embodiment of the present invention, the mass concentration of the HAuCl ₄ solution is 0.5-2%, preferably 1%. More preferably, the solution of HAuCl ₄ is an aqueous solution of HAuCl ₄ .

In a preferred embodiment of the present invention, the mass concentration of the H ₂ PtCl ₆ solution is 0.5-2%, preferably 1%. More preferably, the H ₂ PtCl ₆ solution is an aqueous H ₂ PtCl ₆ solution.

In a preferred embodiment of the present invention, the concentration of the ABEI solution is 5-15mmol/L, preferably 10mmol/L. More preferably, the solution of ABEI is an aqueous solution of ABEI.

In a preferred embodiment of the present invention, the volume ratio of the solution of HAuCl ₄ , the solution of H ₂ PtCl ₆ , and the solution of N-(aminobutyl)-N-(ethylisoluminol) 1:(0.8-1.2):(1-3), preferably 1:1:2.

In a preferred embodiment of the present invention, the centrifugation conditions include: centrifugation at a rotational speed of 10000±2000 rpm/min for 10-20 min.

In a preferred embodiment of the present invention, the preparation method of the sensor includes the following steps:

G-CdTe QDs were drop-coated on the electrode surface, dried, and the recognition unit Dexp was dropped to form π-π stacking.

In a preferred embodiment of the present invention, after π-π stacking is formed, a solution of BSA is drop-coated on the surface of the electrode. More preferably, the mass fraction of the BSA solution is 0.5-1.5%, preferably 1%.

In a preferred embodiment of the present invention, the volume ratio of the BSA solution to the G-CdTe QDs dispersion is 1:(1-1.5), preferably 1:1.

In a preferred embodiment of the present invention, after the solution of BSA is drop-coated, the solution to be tested containing Con A is added dropwise for incubation.

In a preferred embodiment of the present invention, the test solution containing Con A is added dropwise for incubation, and then the anode luminescent body bound to the recognition unit is modified. More preferably, the method for modifying the anode emitter combined with the recognition unit is: drop-coat the anode emitter on the surface of the incubated electrode, and incubate at 0-10°C for 1-3h, preferably at 4°C Incubate for 2 hours.

In a preferred embodiment of the present invention, the concentration of concanavalin A in the test solution is ≥3.0×10 ^-5 ng/mL, preferably 3.0×10 ^-5 -10 ng/mL.

In a preferred embodiment of the present invention, the preparation method of G-CdTe QDs includes: mixing a solution of CdCl ₂ and GO, adding Na ₂ TeO ₃ , C ₆ H ₅ Na ₃ O ₇ , mercaptopropionic acid and NaBH ₄ , Reflux reaction at 120-140° C. for 8-12 h, wash with ethanol and water and centrifuge to collect the solid, and disperse in water to obtain the G-CdTe QDs. More preferably, the concentration of the G-CdTe QDs is 0.5-2.0 mg/mL, preferably 1 mg/mL.

In a preferred embodiment of the present invention, the pretreatment method of the electrode surface includes: polishing the glassy carbon electrode with 0.25-0.35 μm and 0.45-0.55 μm alumina powder respectively, washing in water and ethanol, and drying.

The present invention also provides an application of the sensor for detecting concanavalin A in detecting concanavalin A.

In a preferred embodiment of the present invention, the method for detecting concanavalin A comprises the following steps:

(1) After incubating the sensor with a solution containing different concentrations of standard concanavalin A, collect the luminescent signals of the cathode luminescent body and the anode luminescent body, and compare the intensity ratio of the two luminescent signals with that of the standard concanavalin A. The logarithm of solution concentration is carried out linear fitting, obtains working curve;

(2) Incubating the sensor with the test solution, collecting the luminescent signals of the cathodoluminescent body and the anode luminescent body, and calculating the concentration of concanavalin A in the test solution through the working curve.

The reagents and instrument information used in each embodiment of the present invention are as follows:

1. Reagents

Sodium tellurite (Na ₂ TeO ₃ ) and cadmium chloride hemipentahydrate (CdCl ₂ 2.5H ₂ O) were purchased from Alfa Aisha (China) Co., Ltd.;

GO is provided by Nanjing Pioneer Nanotechnology Company;

ABEI was purchased from TCL Development Co., Ltd. (Shanghai, China);

Con A, from CanaValia ensiformis jack beans;

3-Mercaptopropionic acid (MPA) was provided by Sigma Chemical Co., Ltd. (St.Louis, MO, USA);

Dexp comes from Aladdin Shanghai Co., Ltd.;

Chloroauric acid (HAuCl ₄ 4H ₂ O) and chloroplatinic acid (H ₂ PtCl ₆ ) were purchased from Shanghai Chemical Reagent Co., Ltd.;

Bovine serum albumin was purchased from Sigma-Aldrich Shanghai Co., Ltd.;

Phosphate buffered saline solutions (PBS, 0.10mol/L) with various pH values were prepared using Na ₂ HPO ₄ (0.10mol/L) and KH ₂ PO ₄ (0.10mol/L), KCl (0.10mol/L) as support electrolyte use;

All chemical reagents are analytically pure;

All experiments used double distilled water.

2. Instrument

ECL signal detection: MPI-A electrochemiluminescence analyzer, Xi'an Ruimai Electronic Technology Co., Ltd.; the whole detection process is at room temperature, the voltage of the photomultiplier tube is set to 800V, and the scanning range is -1.7V to +0.6V , the scan rate is 0.5V/s; using a three-electrode system, the glassy carbon electrode modified by the present invention is used as a working electrode, the Ag/AgCl electrode is used as a reference electrode, and Pt is used as an auxiliary electrode;

Cyclic voltammetry detection: CHI600D electrochemical workstation, Shanghai CH Instrument Company; using a three-electrode system, the modified glassy carbon electrode of the present invention is used as the working electrode, the Ag/AgCl electrode is used as the reference electrode, and Pt is used as the auxiliary electrode;

Ultraviolet absorption spectrum test: UV-2450 ultraviolet spectrophotometer, Shimadzu Co., Ltd.;

Scanning electron microscopy images: Scanning Electron Microscopy, Hitachi;

Transmission electron microscopy images: Transmission Electron Microscope H-800, Hitachi;

XPS test: Thermo ESCALAB 250 spectrometer, SID-Molecular.

Example 1

This embodiment provides a method for preparing a sensor for detecting concanavalin A, the steps are as follows:

1. Preparation of G-CdTe QDs

Weigh 36.89 mg of CdCl ₂ ·2.5H ₂ O and dissolve in 50 mL of deionized water, add 220 μL of 1 mg/mL GO to the above solution under stirring conditions and continue stirring for 1 h. Then gradually add 1 mL of 0.01M Na ₂ TeO ₃ , 50 mg of C ₆ H ₅ Na ₃ O ₇ 2H ₂ O, 33 μL of MPA and 100 mg of NaBH ₄ with continuous stirring, and heat to reflux at 130°C for 10 h , washed and centrifuged three times with ethanol and distilled water at a volume ratio of 1:1 to collect G-CdTe QDs, dispersed in deionized water and stored at 4 °C for later use, with a dispersion concentration of 1 mg/mL.

2. Preparation of anode emitter Dexp-ABEI-Au-Pt

1 mL of HAuCl ₄ solution with a mass fraction of 1% was added dropwise to 2 mL of 10 mmol/L ABEI solution with stirring, and the reaction was stirred at room temperature for 2 h. Then, 1 mL of H ₂ PtCl ₆ solution with a mass fraction of 1% was added dropwise to the above mixed solution under stirring condition and continued to stir for 2 h. Then the above mixed solution was centrifuged at 10000rpm for 15min, and washed three times with deionized water, and the obtained anode emitter ABEI-Au-Pt was dispersed into double distilled water, the dispersion concentration was 1.5mg/mL, and 1mL was added, and the concentration It was a 15 mg/mL Dexp solution, stirred for 2 hours to obtain the anode emitter Dexp-ABEI-Au-Pt combined with the recognition unit, and stored at 4°C for future use.

3. Preparation of sensors

Please refer to FIG. 1 , which is a schematic diagram of the preparation process of the sensor provided by the embodiment of the present invention and the corresponding detection principle. The preparation process steps are as follows:

Polish the bare glassy carbon electrode (φ=4.0mm) with 0.3 μm and 0.5 μm alumina powder respectively, ultrasonically clean it with ultrapure water and ethanol in turn, dry it in the air, and drop 10 μL of prepared G-CdTe QDs were dried in air; then 10 μL of Dexp solution with a concentration of 15 mg/mL was added dropwise to the electrode surface modified with G-CdTe QDs, through the π-π stacking combination between G-CdTe QDs and Dexp effect, modify Dexp to the electrode surface; then drop 10 μL of BSA solution with a mass fraction of 1% onto the electrode surface modified with Dexp to block the non-specific binding sites of the modified electrode; For biospecific binding, 10 μL of Con A containing the analyte was incubated on the electrode surface for 1 h; then Dexp-ABEI-Au-Pt was added dropwise to the electrode surface and incubated at 4 °C for 2 h to obtain the the above sensor.

Example 2

This embodiment provides a method for preparing a sensor for detecting concanavalin A, the steps are as follows:

1. Preparation of G-CdTe QDs

Same as Example 1

2. Preparation of anode emitter Dexp-ABEI-Au-Pt

1 mL of HAuCl ₄ solution with a mass fraction of 1% was added dropwise to 2 mL of 10 mmol/L ABEI solution with stirring, and the reaction was stirred at room temperature for 2 h. Then, 1 mL of H ₂ PtCl ₆ solution with a mass fraction of 1% was added dropwise to the above mixed solution under stirring condition and continued to stir for 2 h. Then the above mixed solution was centrifuged at 10000rpm for 15min, and washed three times with deionized water, and the obtained anode emitter ABEI-Au-Pt was dispersed into double distilled water, the dispersion concentration was 1.5mg/mL, and 1mL was added, and the concentration It was a 15 mg/mL Dexp solution, stirred for 2 hours to obtain the anode emitter Dexp-ABEI-Au-Pt combined with the recognition unit, and stored at 4°C for future use.

3. Preparation of sensors

Polish the bare glassy carbon electrode (φ=4.0mm) with 0.3 μm and 0.5 μm alumina powder respectively, ultrasonically clean it with ultrapure water and ethanol in turn, dry it in the air, and drop 10 μL of prepared G-CdTe QDs were dried in the air; then 12 μL of Dexp solution with a concentration of 15 mg/mL was added dropwise to the electrode surface modified with G-CdTe QDs, through the π-π stacking combination between G-CdTe QDs and Dexp Dexp was modified to the surface of the electrode; then 8 μL of BSA solution with a mass fraction of 1% was dropped onto the surface of the electrode modified with Dexp to block the non-specific binding sites of the modified electrode; For biospecific binding, 10 μL Con A containing the analyte was incubated on the electrode surface for 1 h; then Dexp-ABEI-Au-Pt was added dropwise to the electrode surface and incubated at 4 °C for 2 h to obtain the the above sensor.

Example 3

This example refers to the preparation method of Example 1, the difference is that when preparing the sensor in step 3, through the biospecific binding between Con A and Dexp, 10 μL of a series of Con A standard solutions containing different concentrations were incubated on the electrode respectively surface, the incubation time was 1 h.

The serial concentrations of Con A standard solution are: 1.0×10 ^-4 ng/mL, 1.0×10 ^-3 ng/mL, 1.0×10 ^{- 2} ng/mL, 0.1ng/mL, 1ng/mL and 10ng/mL, The standard solution is obtained by dissolving Con A in 0.1 mol/L PBS buffer solution with pH=7.4.

Experimental example 1

In order to further confirm and illustrate the preparation process of the present invention, taking Example 1 as an example, a characterization test was carried out on each substance in the preparation process of the sensor, the details are as follows.

Please refer to Figures 2 and 3, which are TEM images of G-CdTe QDs prepared in Example 1 of the present invention, as can be seen from the figure that many granular cadmium telluride quantum dots are dispersed on the surface of graphene oxide, and from the figure In 3, the size of quantum dots can be observed to be about 2.2nm.

Please refer to Figure 4, which is the SEM image of ABEI-Au-Pt prepared in Example 1 of the present invention. From the figure, a flower-like morphology can be observed, and each flower is stacked layer by layer. It is further illustrated that ABEI obtains ABEI-Au-Pt composite nanomaterials by in situ reduction of HAuCl ₄ and H ₂ PtCl ₆ .

Please refer to Fig. 5 and Fig. 6 at the same time, which are respectively the XPS diagram of ABEI-Au-Pt prepared in Example 1 of the present invention and the high-resolution curves of the corresponding elements. It can be seen from the XPS figure that the characteristic peaks at 398.3eV and 293.9eV come from N 1s (corresponding to Figure 6(a)) and C 1s (corresponding to Figure 6(b)) of ABEI, Au 4f (corresponding to Figure 6(c) )) The characteristic peaks appear at 84.25eV and 87.75eV, and the characteristic peaks at 72.1eV belong to Pt 4f (corresponding to Figure 6(d)). These characteristic peaks illustrate the successful preparation of ABEI-Au-Pt nanocomposites.

Please refer to Figure 7, which is the ECL spectrum of ABEI and the ultraviolet absorption spectrum of G-CdTe QDs prepared in the embodiment of the present invention. It can be seen from the figure that the ECL spectrum of ABEI does not overlap with the ultraviolet absorption spectrum of G-CdTe QDs , indicating that there is no ECL energy transfer between ABEI and G-CdTe QDs.

Experimental example 2

In order to further illustrate the ECL and CV performance of the sensor prepared by the present invention, each step of modifying G-CdTe QDs on the bare electrode, and then sequentially modifying Dexp, BSA, analyte Con A and Dexp-ABEI-Au-Pt ECL response signal and cyclic voltammetry curve test, the test results are shown in Figure 8 and Figure 9.

The specific test method for the ECL response signal is: 3mL of 0.10mol/L PBS (pH 7.4) solution is used as the test base solution, and the ECL response signals of different modified electrodes in each step are tested respectively;

It can be seen from Figure 8 that the modified electrodes obtained in each step correspond to a-e in Figure 8; when the bare electrode is modified with G-CdTe QDs (curve 8a), a strong cathode ECL signal appears at the potential of -1.7V, which means that There is no ECL emission at the anode. When the electrode is sequentially modified with Dexp (curve 8b) and BSA (curve 8c), compared with curve 8a, the ECL emission signal of the cathode gradually decreases. When the analyte Con A (curve 8d) was dropped onto the electrode, the cathode signal decreased significantly. The reason for these signal reductions is attributed to Dexp, BSA, and Con A, which are non-electroactive species that hinder electron transfer. When Dexp-ABEI-Au-Pt (curve 8e) was modified onto the electrode surface, the cathode emission decreased, a new anodic ECL emission signal appeared at +0.6 V, and the anodic signal was attributed to ABEI, indicating the successful construction of the sensor.

The test method of the specific cyclic voltammetry curve is: in the solution of 5.0mmol/L [Fe(CN) ₆ ^4- ]/[Fe(CN) ₆ ^3- ], use cyclic voltammetry to characterize, wherein the scanning potential is set to -1.7V to +0.6V.

It can be seen from Fig. 9 that a pair of obvious redox peaks can be observed on the bare electrode (curve 9a). When the electrode is modified with G-CdTe QDs (curve 9b), the redox peak current value is significantly reduced. When Dexp (curve 9c) and BSA (curve 9d) were modified on the electrode, the redox peak current values decreased sequentially. When the analyte Con A (curve 9e) was incubated on the electrode, the peak current value further decreased. These continuously decreasing redox peak current values are due to the hindrance of electron transfer by Dexp, BSA and Con A. When Dexp-ABEI-Au-Pt (curve 9f) is modified on the electrode surface, the peak current value increases significantly, because ABEI, Au and Pt all promote electron transfer.

Experimental example 3

In order to further optimize the working conditions of the sensor, the pH of the PBS solution used in the test was optimized.

G-CdTe QDs and ABEI were placed in PBS solutions with pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, respectively, and the respective ECL response signals were tested. The test results are shown in Figure 10, which are G-CdTe QDs, ECL signal intensity of ABEI as a function of pH.

It can be seen from the figure that the ECL signal intensity of G-CdTe QDs and ABEI all increases with the increase of pH in the range of pH 6.5-8.5, and the biochemical activity of the analyte Con A of the present invention is at pH It is best when it is 7.4, so the optimal pH is 7.4.

Experimental example 4

In this experimental example, the ECL response curve of the sensor according to the present invention is tested at different concentrations of Con A, and the quantitative detection of Con A is finally realized.

Specifically, taking Example 3 as an example, after incubating the sensor with solutions containing different concentrations of standard Con A, the luminescent signals of the cathode luminescent body and the anode luminous body are collected, and the test results are shown in Figure 11, which is an example of the invention. The ECL response curves of the sensor at different Con A concentrations, af in the figure correspond to concentrations of 1.0×10 ^-4 ng/mL, 1.0×10 ^-3 ng/mL, 1.0×10 ^-2 ng/mL, ECL signal of the sensor after incubation with 0.1 ng/mL, 1 ng/mL and 10 ng/mL of Con A. It can be seen from the figure that with the increase of Con A concentration, the ECL emission of G-CdTe QDs at the cathode at −1.7 V gradually decreases, while the ECL emission of ABEI at the anode at +1.6 V gradually increases.

The intensity ratio of the two kinds of ECL signals of cathode and anode and the logarithm of the solution concentration of described standard Con A are carried out linear fitting, obtain working curve, as shown in Figure 12, present good linear relationship; The detection range is 1.0×10 ^-4 ng/mL to 10ng/mL, and its detection limit is as low as 3.0×10 ^-5 ng/mL. The linear regression equation is I _{G-CdTe QDs} /I _ABEI ＝-0.5189+0.6459logc( Among them, I _{G-CdTe QDs} represents the ECL signal of cathode G-CdTe QDs, I _ABEI represents the ECL signal of ABEI-Au-Pt, c represents the concentration of Con A), and the correlation coefficient R is 0.995.

For comparison with other methods for detecting Con A, the detection performance of other methods for detecting Con A is listed in Table 1 below.

Table 1 Detection performance of different Con A detection methods

Remarks: References:

[1] Zou, L., Pang, H.L., Chan, P.H., Huang, Z.S., Gu, L.Q., Wong, K.Y., 2008. Carbohydr. Res. 343, 2932-2938.

[2] Huang, C.F., Yao, G.H., Liang, R.P., Qiu, J.D., 2013. Biosens. Bioelectron.50, 305-310.

[3] Guo, C.X., Boullanger, P., Jiang, L., Liu, T., 2007. Biosens. Bioelectron. 22, 1830-1834.

[4] Zhang, H., Lu, Q.Y., Zuo, F.M., Yuan, R., Chen, S.H., 2017.Sens.Actuator B:Chem.241,887-894.

Experimental example 5

Stability is very important for sensors, and good stability is usually used as one of the criteria for judging ECL performance. The sensor incubated with 1.0×10 ^-3 ng/mL Con A was scanned continuously for 8 times in an air-saturated 0.1mol/L PBS solution with a pH of 7.4, as shown in Figure 13. It can be seen from the figure that There is no obvious difference in the ECL signal, and its relative standard deviation (RSD) is 2.1%, which is within an acceptable range, indicating that the sensor of the present invention has good stability.

In order to test the selectivity of the sensor of the present invention, the mass fraction is 1% bovine serum albumin (BSA), 50ng/mL alpha-fetoprotein (PHA), 50ng/mL prostate-specific antigen (CA) and 0.1U/mL cancer antigen (AFP) was added to 0.1ng/mL Con A as an interfering substance, and the ECL response signal of the sensor of the present invention was compared with and without the presence of interfering substances, as shown in Figure 14 It can be seen from the figure that the above-mentioned interfering substances will not interfere with the detection of Con A, indicating that the sensor of the present invention has good selectivity for Con A.

In order to further test the reproducibility of the sensor of the present invention, prepare 5 identical sensors under the same experimental conditions with reference to the embodiment of the present invention, and test the ECL response signal, the relative standard deviation of the ECL response of 5 sensors ( RSD) were 3.4% (cathode) and 4.7% (anode), indicating that the sensor of the present invention has good reproducibility.

Experimental example 6

The practicability of the sensor of the present invention was tested using the standard recovery method. First, human serum samples were diluted 20-fold with 0.10 mol/L, pH 7.4 PBS solution before testing. Different concentrations of Con A were added to the diluted human serum samples to determine the recovery rate. The test results are shown in Table 2. The recovery rate of Con A ranged from 95.3% to 106%, indicating that this method can be used in actual biological Sample analysis and detection have good practical application potential.

The recovery test result of Con A in table 2 human serum

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (23)

1. a kind of for detecting the sensor of concanavalin A, which is characterized in that including cathodoluminescence body, anode light body and be total to Reaction reagent, the cathodoluminescence body include the cadmium telluride quantum dot of graphene oxide modification, and the anode light body includes receiving N- (aminobutyl)-N- (ethyl different luminol) of meter Jin and Platinum Nanoparticles modification；

The coreaction reagent is dissolved oxygen；

The sensor further includes modifying respectively in the recognition unit of cathodoluminescence body and anode light body, and the recognition unit is used In identification concanavalin A；

The recognition unit is phenoxylation glucan.

2. according to claim 1 for detecting the sensor of concanavalin A, which is characterized in that

By cathodoluminescence body and the modification of anode light body in glassy carbon electrode surface.

3. of any of claims 1 or 2 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that including such as Lower step:

Cadmium telluride quantum dot and the recognition unit of graphene oxide modification by pi-pi accumulation in conjunction with, and modification is in pretreated electricity Pole surface；The anode light body for being combined with recognition unit is modified again, and incubation obtains the sensor.

4. according to claim 3 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that by institute The cadmium telluride quantum dot for stating graphene oxide modification is dispersed in water, dispersion concentration 0.5-2.0mg/mL.

5. according to claim 4 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described The concentration of phenoxylation glucan is 5-30mg/mL.

6. according to claim 5 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described The volume ratio of the solution of the dispersion liquid and phenoxylation glucan of the cadmium telluride quantum dot of graphene oxide modification is 1 ﹕ (0.8-1.2)。

7. according to claim 3 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described The preparation method of anode light body includes: by HAuCl₄, N- (aminobutyl)-N- (ethyl different luminol) and H₂PtCl₆Mixing is anti- It answers, stirs to get the anode light body.

8. according to claim 7 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described The preparation method of anode light body includes:

By HAuCl₄Solution be added N- (aminobutyl)-N- (ethyl different luminol) solution in, be stirred at room temperature reaction 1-3h； H is added₂PtCl₆Solution, 1-3h is stirred at room temperature, obtains mixed solution, solid is collected by centrifugation.

9. according to claim 7 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that anode Illuminator is mixed with the solution of phenoxylation glucan, and anode light body is made to be combined with phenoxylation glucan.

10. according to claim 7 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described HAuCl₄Solution mass concentration be 0.5-2%；

The H₂PtCl₆Solution mass concentration be 0.5-2%；

The concentration of the solution of N- (the aminobutyl)-N- (ethyl different luminol) is 5-15mmol/L；

The HAuCl₄Solution, the H₂PtCl₆Solution, the N- (aminobutyl)-N- (ethyl different luminol) solution Volume ratio be 1 ﹕ (0.8-1.2) ﹕ (1-3).

11. according to claim 3 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described The preparation method of sensor, includes the following steps:

It is dry in the cadmium telluride quantum dot of electrode surface drop coating graphene oxide modification, recognition unit is added dropwise and forms pi-pi accumulation.

12. according to claim 11 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that shape After pi-pi accumulation, in the solution of electrode surface drop coating bovine serum albumin(BSA).

13. according to claim 12 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that institute The mass fraction for stating the solution of bovine serum albumin(BSA) is 0.5-1.5%.

14. according to claim 12 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that institute The volume ratio for stating the dispersion liquid of the solution of bovine serum albumin(BSA) and the cadmium telluride quantum dot of graphene oxide modification is 1 ﹕ (1- 1.5)。

15. 1-14 is described in any item for detecting the preparation method of the sensor of concanavalin A according to claim 1, special Sign is, after the solution of drop coating bovine serum albumin(BSA), the prepare liquid containing concanavalin A is added dropwise and is incubated for；

The anode light body that modification after the prepare liquid containing concanavalin A is incubated for is combined with recognition unit is added dropwise.

16. according to claim 15 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that institute The method for stating the anode light body that modification is combined with recognition unit includes: the electrode table by anode light body drop coating after incubation Face is incubated for 1-3h under the conditions of 0-10 DEG C.

17. according to claim 15 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that institute It states in prepare liquid, concentration >=3.0 × 10 of concanavalin A^-5ng/mL。

18. according to claim 17 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that institute It states in prepare liquid, the concentration of concanavalin A is 3.0 × 10^-5-10ng/mL。

19. according to claim 3 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that described The preparation method of the cadmium telluride quantum dot of graphene oxide modification includes: mixing CdCl₂Solution and graphene oxide, be added Na₂TeO₃、C₆H₅Na₃O₇, mercaptopropionic acid and NaBH₄, in 120-140 DEG C of back flow reaction 8-12h, it is centrifuged using ethyl alcohol and water washing Solid is collected, is dispersed in water, the cadmium telluride quantum dot of the graphene oxide modification is obtained.

20. according to claim 3 for detecting the preparation method of the sensor of concanavalin A, which is characterized in that electrode The preprocess method on surface includes: to polish glass-carbon electrode through 0.25-0.35 μm and 0.45-0.55 μm of alumina powder respectively, It is cleaned in water and ethyl alcohol, it is dry.

21. the application of sensor in detection concanavalin A of any of claims 1 or 2 for detecting concanavalin A, It is characterized in that, the method for detecting concanavalin A includes the following steps:

(1) after being incubated for the sensor using the solution of the standard concanavalin A containing various concentration, acquisition cathodoluminescence body and The luminous signal of anode light body, by the solution concentration of the intensity rate of two kinds of luminous signals and the standard concanavalin A Logarithm carries out linear fit, obtains working curve；

(2) sensor is incubated for using prepare liquid, acquires the luminous signal of cathodoluminescence body and anode light body, passes through work The concentration of concanavalin A in the prepare liquid is calculated in curve.

22. the application of sensor in detection concanavalin A according to claim 21 for detecting concanavalin A, It is characterized in that, in the environment of acquisition luminous signal, pH 6.5-8.5.

23. the application of sensor in detection concanavalin A according to claim 22 for detecting concanavalin A, It is characterized in that, in the environment of acquisition luminous signal, pH 7.4.