CN112649605B

CN112649605B - Based on NaBiF4ECL biosensor for up-conversion of nanoparticles

Info

Publication number: CN112649605B
Application number: CN202011468438.4A
Authority: CN
Inventors: 刘丹青; 吕星星; 赵超越; 黄金美; 郭英
Original assignee: Harbin University of Science and Technology
Current assignee: Ziyao Biological Suzhou Co ltd
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2022-11-01
Anticipated expiration: 2040-12-14
Also published as: CN112649605A

Abstract

An ECL biosensor for detecting pathogenic bacteria, in particular to NaBiF₄：Yb³⁺/Er³⁺Preparation of UCNPs, construction of an ECL immunosensor, and preparation of Escherichia coli O157: ECL detection of H7 belongs to the technical field of biology/sensors. The invention aims to solve the problems that the synthesis conditions of the up-conversion nano material for ECL detection are harsh, the cost is high, the sensitivity is poor and the environmental pollution is easily caused. NaBiF which can be synthesized at room temperature under mild conditions₄：Yb³⁺/Er³⁺The up-conversion nanoparticles are used for ECL detection of pathogenic bacteria (Escherichia coli O157: H7). In NaBiF₄：Yb³⁺/Er³⁺Gold nanoparticles, escherichia coli O157: h7 antibody, and the biosensor was optimized to construct a biosensor for detecting e.coli O157: novel ECL immunosensor for H7. The ECL immunosensor prepared by the invention is 200-100000CFU mL^‑1Shows a linear range for e.coli O157: high sensitivity response of H7, with a minimum detection limit of 138CFU mL^‑1。

Description

Based on NaBiF4ECL biosensor for up-conversion of nanoparticles

Technical Field

The invention belongs to the technical field of biosensors; an ECL biosensor for detecting pathogenic bacteria, in particular to NaBiF₄：Yb³⁺/Er³⁺Preparation of UCNPs, construction of an ECL immunosensor, and preparation of Escherichia coli O157: ECL detection of H7.

Background

Food-borne pathogens contaminate food and drinking water and pose a significant threat to human and animal health. In order to rapidly cope with outbreaks of diseases caused by food-borne or water-borne pathogens, there is an urgent need to develop a new technology capable of rapidly and inexpensively detecting pathogens anywhere. However, many conventional pathogen detection methods require bacterial culture and enrichment steps due to the low concentration of bacteria in the actual sample, with pretreatment times in excess of 18 hours. Modern detection methods such as Polymerase Chain Reaction (PCR), surface Plasmon Resonance (SPR), immunoassay, etc. are much faster, but either require expensive equipment or are not sensitive enough, which severely limits their use in the field.

Electrochemiluminescence (ECL) is an electrochemical luminescence reaction controlled by electrode potential, and has the advantages of high sensitivity, low cost, low signal-to-noise ratio and easiness in control. It is widely used in the fields of medicine and food analysis, clinical diagnosis, environmental pollutant monitoring, immunodetection and the like. ECL emitters play an important role in ECL systems. To date, various fluorescent substances, such as ruthenium (II) complex and luminol, and various nanomaterials, such as noble metal nanoclusters, semiconductor quantum dots, graphene Quantum Dots (GQDs), and carbon nanodots, have been developed in the early days for use in the search and research of ECL systems, and novel and effective ECL materials are in the trend. The rare earth doped up-conversion nanoparticles (UCNPs) have the anti-Stokes optical property, can absorb two or more low-energy photons and emit high-energy photons, and have the advantages of good biocompatibility, good emission stability, less photobleaching, deeper tissue penetration and the like. They are increasingly used in biomedical fields, such as bioanalysis and in vivo imaging. UCNPs, a promising luminescent material, have been recently reported as a new generation of ECL luminescent reagents, and have a series of potential advantages of stable cathode signal, high luminescence intensity, ideal ECL emission signal and long fluorescence lifetime, which will expand the application range of ECL.

Disclosure of Invention

The invention aims to solve the problems that the synthesis conditions of the up-conversion nano material for ECL detection are harsh, the cost is high, the sensitivity is poor and the environmental pollution is easily caused. Bismuth is a low-cost, environmentally friendly metal, and thus a novel NaBiF₄The type up-conversion nano particle is expected to replace NaYF₄UCNPs, and thus are potentially novel ECL emitters.

In order to solve the problems, the ECL biosensor for detecting pathogenic bacteria of the invention relates to NaBiF₄：Yb³ ⁺/ Er³⁺Preparation of UCNPs, construction of an ECL immunosensor, and preparation of Escherichia coli O157: the ECL detection of H7 is specifically carried out according to the following steps:

step one, completely dissolving Bi (NO) in 10 ml of Ethylene Glycol (EG) solution₃)₃·5H₂O(0.78mmol)， Yb(NO₃)₃·5H₂O(0.2mmol)，Er(NO₃)₃·5H₂O (0.02 mmol) and NaNO₃(2 mmol) A clear solution 1 was prepared. Reacting NH₄F (14 mmol) was added to 25mL EG solution to prepare solution 2;

step two, slowly dripping the solution 1 into the solution 2 under violent stirring, and reacting for 10 minutes at room temperature along with violent magnetic stirring to obtain white emulsion;

step three, centrifugally cleaning the white emulsion for several times, and drying the white emulsion in vacuum at 60 ℃ to obtain white powder;

step four, dispersing a certain amount of UCNPs into a mixed solution of Nafion, isopropanol and water (volume ratio of 2^-1；

Step five, continuously polishing the exposed glassy carbon electrode by using alumina slurry with the particle sizes of 1.0, 0.3 and 0.05 mu m to ensure that the surface of the electrode is smooth, and then washing the electrode by using deionized water;

step six, adding 1mmol L of the mixture^-1K₃[Fe(CN)₆](including 0.2mol L^-1KNO₃) Testing cyclic voltammetry until the peak potential difference is less than 80mV;

step seven, 10 mu L of UCNPs solution (0.5 mgmL)^-1) Dripping the solution on the surface of a GCE electrode, and drying at room temperature to obtain a UCNPs modified electrode (UCNPs/GCE);

step eight, dripping 10 mu L of gold nanoparticle solution on the surface of UCNPs/GCE, drying at room temperature, and then dripping 10 mu L of escherichia coli O157: h7 polyclonal antibody (10. Mu.g.mL)^-1) And incubated overnight at 4 ℃;

step nine, after washing with PBS, the antibody-modified electrode was incubated with BSA solution (1%, w/w) for 1 hour to block non-specific sites. The prepared immunosensor was then stored at 4 ℃ for further use;

step ten, mixing 10 μ L of O157: h7 E.coli suspension (concentration range from 0 to 500000CFU ml)^-1) Incubating the immunosensor with the prepared biosensors at 37 ℃ for 40 minutes, respectively, to immobilize the bacteria on the surface of the immunosensor;

step eleven, after carefully washing with PBS solution to remove the uncaptured bacteria, ECL detection is carried out on the biosensor in an ECL detection cell with 0.1mol L of electrolyte^-1PBS contains 0.1mol L^-1K₂S₂O₈(pH 7.4)；

Further defining, in step eleven, the scan potential is in the range of 0 to-2.5V and the scan rate is 100mV s^-1. The voltage of the photomultiplier tube (PMT) was set to 800V.

The NaBiF synthesized by the method of the invention at room temperature by a rapid and environment-friendly method₄：Yb³⁺/Er³⁺Up-converting nanoparticles (UCNPs) can be used for detecting pathogenic bacteria, and have the advantages of higher ECL strength, quick response and excellent stability.

NaBiF in the invention₄：Yb³⁺/Er³⁺UCNP at K₂S₂O₈The presence showed high ECL intensity and stable cathodic signal.

The invention optimizes the method based on NaBiF₄：Yb³⁺/Er³⁺ECL biosensor Conditioning of UCNP results in an optimal UCNPs concentration of 0.5mg mL^-1The ideal incubation time is 45 minutes.

ECL in the invention against Escherichia coli O157: h7 has high sensitivity and the detection range is 200-100000CFU mL^-1Within the range, the lowest detection limit is 138CFU mL^-1。

Drawings

FIG. 1 is NaBiF₄：Yb³⁺/Er³⁺SEM pictures of UCNPs;

FIG. 2 is NaBiF₄：Yb³⁺/Er³⁺XRD pattern and hexagonal system NaBiF of UCNPs₄Standard peak comparison of (c).

FIG. 3 is NaBiF₄：Yb³⁺/Er³⁺UCNPs/GCE in the presence of 0.1M K₂S₂O₈In 0.1M PBS (pH 7.4) at a scanning potential of 100mV s^-1And ECL potential curve at PMT voltage of 800V.

FIG. 4 shows a graph containing 0.1M K₂S₂O₈In 0.1M PBS (pH = 7.4), auNPs/NaBiF₄：Yb³⁺/Er³⁺ECL signal stability for 20 consecutive cycles of the UCNPs/GCE electrode.

FIG. 5 is a plot of the continuous cyclic voltammetry with 0.1M K₂S₂O₈0.1M PBS (pH = 7.4), from AuNPs/NaBiF₄：Yb³⁺/Er³⁺The UCNPs/GCE electrode emits ECL and the cycle is 20 times continuously. The scanning rate is 100mV s^-1The PMT voltage is 800V.

FIG. 6 is the concentration of UCNPs solution versus NaBiF₄：Yb³⁺/Er³⁺Effect of ECL Strength of UCNPs/GCE

FIG. 7 is the incubation time versus E.coli O157: h7 20 antibody/Au/NaBiF₄：Yb³⁺/Er³⁺Effect of ECL Strength of UCNPs/GCE.

Fig. 8 is a detection standard curve of the constructed ECL biosensor. Error bar = RSD (n = 5).

FIG. 9 shows the peak intensity of ECL versus E.coli O157 incubated with E.coli Top 10: H7/Au/NaBiF₄： Yb³⁺/Er³⁺Relationship between the logarithm of the E.coli Top10 concentration of UCNPs/GCE.

Detailed Description

Implementation 1:

step one, completely dissolving Bi (NO) in 10 ml of Ethylene Glycol (EG) solution₃)₃·5H₂O(0.78mmol)， Yb(NO₃)₃·5H₂O(0.2mmol)，Er(NO₃)₃·5H₂O (0.02 mmol) and NaNO₃(2 mmol) toClear solution 1 was prepared. Reacting NH₄F (14 mmol) was added to 25mL EG solution to prepare solution 2;

step two, slowly dripping the solution 1 into the solution 2 under the condition of vigorous stirring, and reacting for 10 minutes at room temperature along with vigorous magnetic stirring to obtain white emulsion;

step three, after centrifugal cleaning is carried out on the white emulsion for several times, vacuum drying is carried out at the temperature of 60 ℃ to obtain white powder;

step seven, 10 mu L of UCNPs solution (0.5 mgmL)^-1) Dripping onto the surface of GCE electrode, and drying at room temperature to obtain UCNPs modified electrode (UCNPs/GCE);

step eleven, after carefully rinsing with PBS solution to remove uncaptured bacteria, the biosensor was subjected to ECL detection in an ECL detection cell0.1mol L of electrolyte^-1PBS contains 0.1mol L^-1K₂S₂O₈(pH 7.4)；

Further defined, the scan potential in step eleven is in the range of 0 to-2.5V and the scan rate is 100mV s^-1. The voltage of the photomultiplier tube (PMT) was set to 800V.

The following tests are adopted to verify the effect of the invention:

1.NaBiF₄：Yb³⁺/Er³⁺characterization of UCNPs

As observed from SEM, the NaBiF produced₄：Yb³⁺/Er³⁺UPNC has a uniform particle size and good dispersibility, and the particle size is about 300nm (FIG. 1). NaBiF₄：Yb³⁺/Er³⁺The elemental composition of UPNCs was determined by elemental scanning analysis, with a uniform distribution of all elements. The presence of Yb and Er elements directly proves that NaBiF₄：Yb³⁺/Er³⁺Successful doping of dopants in UPNC. Due to Er doped in the system³⁺Less, the elements in the element scan image are sparsely distributed.

XRD technique for analysis of NaBiF₄：Yb³⁺/Er³⁺The crystalline phase structure of UCNPs. XRD spectrogram and NaBiF of prepared sample₄：Yb³⁺/Er³⁺(JCPDS 41-0796) was consistent and there were no diffraction peaks of other impurities, confirming that we have obtained NaBiF with hexagonal crystal structure₄(FIG. 2). Due to doped Er³⁺Low content (Er only accounts for 2% in rare earth elements), so Er³⁺Is generally indistinguishable. The combination of the element map results proves that the pure-phase NaBiF is successfully prepared by the simple method₄：Yb³⁺/Er³⁺Upconversion luminescent nanoparticles.

Study of NaBiF₄：Yb³⁺/Er³⁺Coated GCE electrodes were incubated in 0.1M PBS solution (pH 7.4, containing 0.1M K)₂S₂O₈As a co-reactant). (FIG. 3) NaBiF when the potential is cycled negatively between 0 and-2.5V₄： Yb³⁺/Er³⁺UCNPs show strong ECL emission signals at-2.5V, inThere is an initial potential at about-1.3V.

For the ECL process, electrons are first injected into UCNPs at a negative potential, then NaBiF is injected₄: yb, er are reduced to negatively charged radicals. At the same time, the co-reactant S₂O₈ ^2-Electrochemical reduction to strong oxidant-sulfate anion (SO 4)^-·) Further with NaBiF₄：Yb，Er^-·The reaction generates excited NaBiF₄：Yb，Er*。NaBiF₄: yb, er return to the ground state and release photons, thereby generating a strong ECL signal. Based on the redox (Ox-red) pathway, naBiF₄: the ECL mechanism of Yb and Er UCNP is as follows:

NaBiF₄:Yb,Er+e^-→NaBiF₄:Yb,Er^-· (1)

S₂O₈ ^2-+e^-→SO₄ ^2-+SO₄ ^-· (2)

NaBiF₄:Yb,Er^-·+SO₄ ^-·→NaBiF₄:Yb,Er^*+SO₄ ^2- (3)

NaBiF₄:Yb,Er^*→NaBiF₄:Yb,Er+hv (4)

characterization of ECL biosensor construction

The ECL signal of bare GCE is very small when NaBiF₄：Yb³⁺/Er³⁺The ECL intensity increased dramatically to about 16250a after successful modification of the GCE surface by UCNPs. The gold nanoparticles can improve the conductivity of the modified electrode and promote NaBiF in ECL reaction₄：Yb³⁺/Er³⁺Thus a further increase in ECL strength was observed after deposition of the gold nanoparticles. In E.coli O157: after the H7 antibody was immobilized on the electrode surface by strong interaction with gold nanoparticles, the ECL strength decreased to about 14600, indicating that the electron transfer kinetic resistance of the attached antibody was large, thereby decreasing the diffusion rate of the ECL reagent at the electrode interface. Coli O157: h7 toAfter work was immobilized on the biosensor, the ECL strength also decreased significantly. All these results clearly indicate the construction steps of the biosensor.

The construction procedure of the ECL biosensor was also confirmed by the change of ECL signal during stepwise modification of materials and biomolecules. (FIG. 4) the ECL signal of the bare GCE is small (curve a). When NaBiF₄：Yb³⁺/Er³⁺The ECL strength increased dramatically to about 16250a after successful modification of the GCE surface by UCNPs. (curve b). The gold nanoparticles can improve the conductivity of the modified electrode and promote NaBiF in ECL reaction₄：Yb³⁺/Er³⁺Thus a further increase in ECL strength was observed after gold nanoparticle deposition (17618 au, curve c). ECL strength was significantly reduced to about 14600a. (curve d) E.coli O157: the H7 antibody is fixed on the surface of the electrode through strong interaction with the amine group of the gold nanoparticle, and the electron transfer kinetic resistance of the protein membrane is shown to reduce the diffusion rate of the ECL reagent on the electrode interface. 3000CFU mL will be bound by antigen-antibody binding^-1Escherichia coli O157: after H7 was successfully immobilized on the biosensor, the ECL strength also decreased significantly. All these results clearly demonstrate the successful manufacture of the biosensor.

AuNPs/NaBiF₄：Yb³⁺/Er³⁺The UCNPs functionalized glassy carbon electrode was scanned continuously for 20 cycles in the negative potential-2.5 to 0V range, with no significant drop in ECL signal (fig. 5). Thus, the prepared ECL biosensor shows excellent stability and would be a promising candidate for further ECL detection.

3. Escherichia coli O157: detection of H7

To obtain the best ECL performance, the effect of incubation time and UCNPs concentration on ECL signal was investigated. ECL Strength from 0.1 to 0.5mg mL with solution concentration^-1Increased, then greater than 0.5mg mL in UCNPs solution^-1Time decreased (fig. 6). Therefore, 0.5mg mL was selected^-1The UCNPs concentration to prepare a biosensor. For incubation time, prepared sensors were exposed to 10000CFU mL^-1Escherichia coli O157: h7 suspension of differentTime to study the effect of incubation time on ECL signal. It can be observed that as the incubation time increased from 0 min to 45 min, the ECL signal first decreased and then leveled off starting at 45 min and 60 min, indicating that at the 45 min incubation time, e.coli O157 assembled on the electrode surface: h7 reaches saturation (fig. 7). Therefore, 45 minutes is the ideal incubation time.

Under optimized conditions, the constructed biosensor is researched to detect Escherichia coli O157: h7, E.coli O157: relationship between H7 concentration and ECL intensity (fig. 8). The ECL intensity of the biosensor gradually decreases with increasing bacterial concentration and ranges from 200 to 100000CFU mL^-1Interval, ECL intensity has good linear relation with bacterial concentration, R =0.995, regression equation is I_ECL＝22645.8-3621.7log(C_bacteria) In which I_ECLRepresents the ECL Strength, C_bacteriaRepresents Escherichia coli O157: concentration of H7. The lowest detection limit was 138CFU mL^-1. When Escherichia coli O157: the concentration of H7 reaches 100000CFU mL^-1Above, the ECL intensity drop of the sensor tends to flatten out, since all binding sites on the sensor are bound by e.coli O157: h7 occupancy.

To evaluate the reproducibility of the prepared biosensors, concentrations of 3000, 5000 and 100000CFU mL were applied four times in succession, respectively^-1Escherichia coli O157: h7 was detected. RSD were 2.2%,1.5% and 0.8%, respectively, indicating excellent reproducibility of the ECL biosensor. The stability of the immunosensor with respect to long-term storage was also investigated. After storage in a refrigerator at 4 ℃ for two weeks, the ECL strength of the stored biosensor remained 93.5% of the initial strength, indicating good long-term stability.

In the developed E.coli O157: in the selective evaluation of H7 biosensors, 18 e.coli Top10 were used as comparative evaluation since it did not have O157 and H7 antigens. For concentrations between 1000 and 10000CFU mL^-1Coli Top10, the sensor showed little obvious ECL response, no significantly attenuated ECL intensity change. With further increase of concentration, in 10000-500000CFU mL^-1In this range, the ECL strength of E.coli Top10 was slightly decreased by physical adhesion (FIG. 9).

The above results show that based on NaBiF₄：Yb³⁺/Er³⁺The functionalized biosensor of the up-conversion nanoparticles has good selectivity and excellent stability, so that the biosensor has good selectivity and excellent stability when detecting O157: has great potential in the aspect of H7 Escherichia coli.

Claims

1. Based on NaBiF₄：Yb³⁺/Er³⁺Use of UCNPs for escherichia coli O157: an ECL biosensor for H7 detection, which is characterized in that the preparation and detection method is carried out according to the following steps:

step one, continuously polishing a bare glassy carbon GCE electrode by using alumina slurry of 1.0, 0.3 and 0.05 mu m to ensure that the surface of the electrode is smooth, and then washing the electrode by using deionized water;

step two, adding 1mmol L^-1K₃[Fe(CN)₆]Comprising 0.2mol of L^-1 KNO₃Testing cyclic voltammetry until the peak potential difference is less than 80mV;

step three, mixing 10 mu L of 0.1-1.0mg mL^-1NaBiF of (1)₄：Yb³⁺/Er³⁺Dropping UCNPs solution on the surface of GCE electrode, and drying at room temperature to obtain NaBiF₄：Yb³⁺/Er³⁺UCNPs-modified electrodes;

in 10 ml of ethylene glycol EG solution, 0.78mmol of Bi (NO) was completely dissolved₃)₃·5H₂O，0.2mmol Yb(NO₃)₃·5H₂O，0.02mmol Er(NO₃)₃·5H₂O and 2mmol NaNO₃Preparation of clear solution 1, 14mmol of NH₄F is added into 25mLEG solution to prepare solution 2; slowly dropwise adding the solution 1 into the solution 2 under vigorous stirring, and reacting for 10 minutes at room temperature along with vigorous magnetic stirring to obtain white emulsion;

centrifugally cleaning the white emulsion, and drying the white emulsion in vacuum at 60 ℃ to obtain white powder;

adding a certain amount of NaBiF₄：Yb³⁺/Er³⁺UCNPs were dispersed in a mixed solution of Nafion, isopropanol and water at a volume ratio of 2₄：Yb³⁺/Er³⁺The concentration of UCNPs in the biosensor was 0.5mg mL^-1；

Step four, dripping 10 mu L of gold nanoparticle Au NPs solution on the surface of UCNPs/GCE, drying at room temperature, and dripping 10 mu L of 10 mu g mL of gold nanoparticle Au NPs solution on the surface^-1Escherichia coli O157: h7 polyclonal antibody, and incubated overnight at 4 ℃;

AuNPs/NaBiF₄：Yb³⁺/Er³⁺the UCNPs functionalized glassy carbon electrode continuously scans for 20 cycles in the range of negative potential-2.5 to 0V, and ECL signals of the UCNPs functionalized glassy carbon electrode are kept stable;

step five, after washing with PBS, the antibody-modified electrode was incubated with 1% (w/w) BSA solution for 1 hour to block non-specific sites, and then the prepared immunosensor was stored at 4 ℃ for further use;

step six, the concentration of 10 mu L ranges from 0 to 500000CFU ml^-1O157 of (2): the H7 e.coli suspensions were incubated with the prepared biosensors for 45 minutes at 37 ℃ to separate O157: h7 escherichia coli is fixed on the surface of the immunosensor;

step seven, carefully wash with PBS solution to remove uncaptured e.coli O157: h7, carrying out ECL detection on the biosensor in an ECL detection cell with 0.1mol L of electrolyte^-1PBS contains 0.1mol L^-1 K₂S₂O₈，pH 7.4；

Biosensor at 200 to 100000CFU mL^-1Shows a linear range for e.coli O157: high sensitivity response of H7, with a minimum detection limit of 138CFU mL^-1。