CN113267472B - SPR sensor chip and preparation method thereof - Google Patents

Abstract

The invention relates to an SPR sensor chip and a preparation method thereof, belonging to the technical field of biomedical detection. The SPR sensor chip comprises a glass substrate and a gold layer arranged on the glass substrate, wherein sulfydryl functionalized MXene quantum dots modified through Au-S covalent bonds are arranged on the surface of the gold layer, and aptamers are anchored on the sulfydryl functionalized MXene quantum dots. The method uniformly coats the mercapto-functionalized MXene quantum dots on the surface of the gold chip by virtue of the self-assembly effect between the mercapto-functionalized MXene quantum dots and the SPR gold chip, and then fixes the aptamer on the MXene quantum dots by virtue of pi-pi accumulation, electrostatic adsorption and hydrogen bonding. Compared with the SPR sensor chip reported before, the SPR sensor chip based on the sulfydryl functionalized MXene quantum dots has the advantages of simple structure and high stability, and shows superior sensing performances such as higher sensitivity, quick response and practicability in a complex environment.

Description

A kind of SPR sensor chip and preparation method thereof

technical field

The invention relates to an SPR sensor chip and a preparation method thereof, belonging to the technical field of biomedical detection.

Background technique

The early clinical manifestations of COVID-19 are similar to those of severe acute respiratory syndrome and MERS-CoV, namely fever, headache, myalgia, arthralgia, and swollen lymph nodes. SARS-CoV-2 has the characteristics of moderate mortality, high infection rate, and long incubation period, which can lead to long-term infection. The novel coronavirus (SARS-CoV-2) has been found in multiple environments such as water systems, frozen foods, and food packaging. Therefore, rapid diagnosis of the COVID-19 virus is crucial for effective control of virus transmission and treatment of patients. Similar to other coronaviruses, the new coronavirus (SARS-CoV-2) is mainly composed of four structural proteins, namely spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein ( N-gene). Proteins (such as S protein) and viral RNA can be used as targets for qualitative and quantitative analysis of the novel coronavirus (SARS-CoV-2). In addition, antibodies such as IgM and IgG can also be tested from patient samples to study the patient's infection history. Various techniques have been developed to analyze the novel coronavirus (SARS-CoV-2), such as real-time polymerase chain reaction (PCR), colorimetric analysis, surface plasmon resonance (SPR) and localized SPR, electrochemical methods, optical Methods, optical/chemiluminescence immunosensors, fluorescence techniques, and wearable sensors.

Most of the techniques for analyzing the novel coronavirus (SARS-CoV-2) are based on the specific recognition between antibodies and different proteins and RNAs in SARS-CoV-2. Compared with antibodies, DNA aptamers, as a powerful probe, have the advantages of high specificity, strong affinity, fast and reliable synthesis, and easy coupling. Therefore, DNA aptamers can be used for different in vitro and in vivo diagnostics.

As a widely used qualitative and quantitative analytical technique, surface plasmon resonance (SPR) is often used to construct immunoassays, multiplex detection of biomolecules, and in situ detection of multiplex chemical and biological analyte interactions. Various nanomaterials, such as functional polymers, nanoparticles, graphene, MXene nanosheets, and _MoS2 , have been used as sensing platforms for fabricating SPR sensors. MXenes, a typical two-dimensional material, have attracted extensive attention due to their similar structures and properties to graphene. MXenes have a nanosheet structure, unique surface chemistry, high electrical conductivity, and excellent biocompatibility. These properties make MXenes effective as a development platform for various biosensors. MXene nanosheets were used to immobilize probes and develop SPR biosensors. However, the thickness of the sensitive layer of the SPR biosensor is ∼200 nm, and the MXene nanosheets are easy to aggregate due to the π-π* stacking effect, forming large-sized aggregates. Limited by the thickness of SPR biosensors, it is difficult to obtain SPR sensor chips with high sensitivity and fast response using MXene nanosheets.

Contents of the invention

The object of the present invention is to provide a SPR sensor chip with high sensitivity and fast response speed using mercapto-functionalized MXene quantum dots as a sensor platform.

Another object of the present invention is to provide a method for preparing an SPR sensor chip.

For achieving the above object, the technical scheme of the SPR sensor chip of the present invention is:

A SPR sensor chip, comprising a glass substrate and a gold layer disposed on the glass substrate, the surface of the gold layer has mercapto-functionalized MXene quantum dots modified by Au-S covalent bonds, on the mercapto-functionalized MXene quantum dots Anchored with aptamers.

The present invention uses mercapto-functionalized MXene quantum dots as the biosensing platform of the SPR sensor chip, and designs and constructs a novel SPR sensor chip. The size of zero-dimensional quantum dots (QDs) prepared from two-dimensional large-size MXene nanosheets is very small, around 5 nm, which meets the test requirements of SPR instruments, and due to the combination of quantum confinement, edge effects, and surface functionalization, MXene quantum dots Dots can be used as sensitive nanomaterials to build SPR sensor chips.

The thiol-functionalized MXene quantum dots have the characteristics of thiol-functionalization, highly conjugated structure, and graphene-like MXene phase. When used as an SPR sensing platform, they can not only form Au-S bonds through self-assembly, but also show strong interactions with Au chips. The binding effect of the aptamer chain, which has a smaller size and a larger specific surface area, shows a strong bioaffinity and amplified SPR effect on the aptamer chain.

In the present invention, through the self-assembly effect between the thiol-functionalized MXene quantum dots and the SPR gold chip, a large number of smaller-sized thiol-functionalized MXene quantum dots are uniformly coated on the surface of the gold chip, and then stacked by π-π* , electrostatic adsorption and hydrogen bonding to immobilize a large number of aptamers on MXene quantum dots. Compared with previously reported SPR sensor chips, the SPR sensor chip based on thiol-functionalized MXene quantum dots with smaller size and larger specific surface area has the advantages of simple structure and high stability, and exhibits excellent performance in complex environments. Superior sensing performance such as high sensitivity, fast response and practicality.

The thiol-functionalized MXene quantum dots can be combined with MXene quantum dots through chemical bonds, such as combining thiol-containing coupling agents through chemical coupling reactions (forming chemical bonds), or through non-chemical bonds, such as mercapto-containing compounds through π- The π* stacking effect combines with MXene quantum dots to form MXene quantum dots with thiol functional groups on the surface. Preferably, the thiol-functionalized MXene quantum dots are MXene quantum dots with thiol functional groups on the surface formed by combining with MXene quantum dots through non-chemical bonds.

More preferably, the thiol-functionalized MXene quantum dots are MXene quantum dots with thiol functional groups on the surface formed by combining with MXene quantum dots through π-π* stacking interaction.

Preferably, the thiol-functionalized MXene quantum dots are obtained by a preparation method comprising the following steps: dispersing the MXene quantum dots and the thiol compound in a dispersion medium to obtain the thiol-functionalized MXene quantum dots.

Preferably, the mass ratio of the MXene quantum dots to the thiol compound is 1:1-3.

Preferably, the temperature of the dispersing treatment is 10-30° C.; the dispersing treatment is firstly subjecting the mixture to ultrasonic treatment, followed by stirring treatment; the mixture is composed of MXene quantum dots, thiol-based compounds and a dispersion medium; the The mixture is obtained by mixing MXene quantum dots, a thiol compound and a dispersion medium; the dispersion medium is water.

More preferably, the temperature of the dispersion treatment is 25°C.

Preferably, the power of the ultrasound is 80-200W, and the time of the ultrasound treatment is 0.2-2h.

More preferably, the power of the ultrasound is 120W, and the time of the ultrasound treatment is 0.5h.

Preferably, the stirring speed is 600-1000 rpm, and the stirring treatment time is 4-8 hours.

More preferably, the stirring speed is 800r/min, and the stirring treatment time is 6h.

Preferably, the concentration of the MXene quantum dots in the dispersion medium is 0.8-1.2 mg·mL ^-1 .

More preferably, the concentration of the MXene quantum dots in the dispersion medium is 1 mg·mL ^-1 .

Preferably, the thiol-based compound is a C15-20 alkylthiol.

Preferably, the thiol-based compound is n-octadecyl mercaptan.

Preferably, the thiol-functionalized MXene quantum dots are thiol-functionalized Nb ₂ C MXene quantum dots.

Preferably, the aptamer is an aptamer for targeted detection of the N-gene of SARS-CoV-2. The aptamer used for targeted detection of SARS-CoV-2 can form a G-quadruplex with the N-gene of the new coronavirus (SARS-CoV-2), and the aptamer chain changes when it binds to the N-gene The conformation of the aptamer leads to a change in the contact area or distance between the probe molecule (aptamer) and the chip, which in turn causes a change in the SPR signal (RU), through which the N of SARS-CoV-2 can be detected. -gene. In general, one refractive index unit (RU) corresponds to a refractive index change of 10 ^-6 and a conjugate (binding protein) of about 1 pg mm ^-2 , therefore, the loading capacity of the SPR chip can be calculated by the refractive index unit (RU) . Compared with previously reported SPR biosensors, the SPR biosensor chip based on thiol-functionalized MXene quantum dots has the advantages of simple structure and high stability, and shows high sensitivity, fast response and practicability in complex environments. With superior sensing performance, it can be widely used in N-gene detection in various environments such as human serum, seawater, and seafood.

These advantages are mainly attributed to several factors: thiol-functionalized MXene quantum dots can not only be uniformly deposited on the SPR gold chip, but also can adsorb a large number of aptamers, thereby obtaining higher sensitivity; for targeted detection of SARS-CoV The highly specific recognition of the nucleic acid aptamer of -2 and the N-gene makes the SPR sensor have a faster response to the N-gene.

Preferably, the aptamer is N58 aptamer. The highly specific recognition of N58 aptamer and N-gene makes the prepared SPR sensor chip have a faster response to N-gene.

The technical scheme of the preparation method of the SPR sensor chip of the present invention is:

A preparation method for an SPR sensor chip, comprising:

(1) The suspension of mercapto-functionalized MXene quantum dots is contacted with the gold layer of the gold chip to form an Au-S covalent bond to obtain a modified gold chip; the gold chip includes a glass substrate and a gold layer arranged on the glass substrate ;

(2) Incubate the modified gold chip in the aptamer solution.

In the present invention, Au-S covalent bonds are formed through the self-assembly between thiol-functionalized MXene quantum dots and gold atoms on a gold chip, and a large number of thiol-functionalized MXene quantum dots are connected to the gold layer through Au-S bonds to form Au-S covalent bonds. A uniform MXene quantum dot layer is formed on the chip, and an SPR sensor chip is formed after aptamers are fixed on the MXene quantum dot layer. The SPR sensor chip prepared by the present invention has the advantages of simple structure and high stability, and shows excellent performance in complex environments. Superior sensing performance such as high sensitivity, fast response and practicality.

Preferably, the thiol-functionalized MXene quantum dots are thiol-functionalized Nb ₂ C MXene quantum dots.

Preferably, in step (1), the concentration of the suspension of thiol-functionalized MXene quantum dots is 0.1 mg·mL ^-1 .

The incubation is to contact the gold chip containing the MXene quantum dot layer with the aptamer solution, so that the MXene quantum dots adsorb and fix the aptamer and reach an equilibrium state.

Description of drawings

Figure 1: Schematic diagram of the synthesis of Nb ₂ C-SH quantum dots;

Figure 2: Schematic diagram of the preparation of a Nb ₂ C-SH quantum dot SPR sensor chip;

Figure 3: Infrared spectra of Nb ₂ C quantum dots and Nb ₂ C-SH quantum dots;

Figure 4: XPS spectra of Nb ₂ C quantum dots, Nb ₂ C-SH quantum dots and Apt _N58 /Nb ₂ C-SH quantum dots;

Figure 5: (a) is the low-magnification TEM image of Nb ₂ C-SH quantum dots, (b) is the high-magnification TEM image of Nb ₂ C-SH quantum dots, (c) is the Nb ₂ C-SH quantum dots High-resolution TEM images of

Figure 6: (a) is the ΔRU value of the aptamer before and after immobilization on Nb ₂ C-SH quantum dots at different aptamer concentrations and the ΔRU value of the prepared SPR sensor chip before and after detection of N-gene, (b) The ΔRU value of the SPR sensor chip prepared for the phosphate buffer solution of different pH values before and after detection of N-gene (use the phosphate buffer solution base of the same pH value when preparing the aptamer solution and preparing the N-gene solution and detecting N-gene liquid);

Figure 7: The change curve of ΔRU value with detection time for different concentrations of N-gene (0.05, 0.1, 0.5, 1, 10, 50 and 100ng mL ^-1 ) detected by the SPR sensor chip based on Nb ₂ C-SH quantum dots;

Figure 8: The relationship between the ΔRU value (ΔRU=RU _N-gene -RU _{N58 aptamer} ) and the N-gene concentration before and after the detection of N-gene by the SPR sensor chip based on Nb ₂ C-SH quantum dots, the inset figure is ΔRU and N -Linear relationship between the logarithms of the gene concentration;

Figure 9: The variation curve of the ΔRU value of the Nb ₂ C-SH quantum dot-based SPR sensor chip detecting interfering substances (both at a concentration of 100ng mL ^-1 ) and N-gene (at a concentration of 1ng mL ^-1 ) with detection time.

Detailed ways

Embodiments of the present invention will be further described below in conjunction with the accompanying drawings.

Materials used in the examples of the present invention: N58 aptamer provided by Shanghai Sangon Bioengineering Co., Ltd., the sequence is 5'-GCT GGA TGT CAC CGG ATT GTC GGA CAT CGG ATT GTC TGA GTC ATA TGA CACATC CAG C-3 ';

The resistivity of ultrapure water is ≥18.2MΩ·cm.

Phosphate buffer solution was prepared by the following method: Dissolve 8.00g NaCl, 0.20g KCl, _{1.44g Na2HPO4} , _{1.8g K2HPO4} in 800mL ultrapure water (≥18.2MΩ·cm), adjust the solution with hydrochloric acid pH value to 7.4, and finally dilute to 1L with ultrapure water (≥18.2MΩ·cm) to obtain a phosphate buffer solution (PBS, 0.01mol/L, pH=7.4), the prepared phosphate buffer solution is used Stored at 4°C before.

The preparation of the SPR sensor chip in Example 2 and the testing of the SPR sensor chip in Experimental Example 3 and Experimental Example 4 were all carried out at 25°C by Biacore ^TM X100 instrument (GE-Healthcare Bio-Sciences AB, USA).

One, the specific embodiment of SPR sensor chip of the present invention is as follows:

Example 1

The SPR sensor chip of this embodiment includes a glass substrate and a gold layer disposed on the glass substrate, the surface of the gold layer has mercapto-functionalized Nb ₂ C MXene quantum dots modified by Au-S covalent bonds, and the mercapto-functionalized The N-58 aptamer is anchored on the MXene quantum dots.

Wherein, the preparation method of sulfhydryl functionalized Nb ₂ C quantum dots comprises the following steps:

(1) Preparation of Nb ₂ C MXene powder: react Nb ₂ AlC powder with hydrofluoric acid with a mass fraction of 3% at 55°C for 48h, then rinse with deionized water for 12 times, and dry in a vacuum oven at 60°C for 18h , to obtain Nb ₂ C MXene powder.

(2) Preparation of Nb ₂ C MXene quantum dots: Disperse 15 mg of Nb ₂ C MXene powder in 20 mL of ultrapure water (≥18.2 MΩ cm), stir for 0.5 h at a stirring speed of 800 rpm, and then add Ammonia water with a mass fraction of 25%, adjust the pH value of the system to 6, and then heat at 100°C for 6h. Finally, after the supernatant is filtered through a 220nm filter membrane, the filtrate is concentrated by vacuum distillation to obtain Nb ₂ C MXene quantum dots .

(3) Preparation of thiol-functionalized Nb ₂ C MXene quantum dots: Disperse Nb ₂ C MXene quantum dots in ultrapure water (≥18.2MΩ·cm) at 25°C and stir for 12h to obtain a concentration of 1mg·mL ^-1 Nb ₂ C MXene quantum dot suspension, and then disperse 2 mg of n-octadecyl mercaptan in 2 mL of the prepared Nb ₂ C MXene quantum dot suspension, and sonicate for 30 minutes under the condition of ultrasonic power of 120 W, Then stir for 6 hours at a stirring speed of 800 rpm, and finally, filter with a 220nm filter membrane, and concentrate the filtrate to obtain thiol-functionalized Nb ₂ C MXene quantum dots, which are labeled as Nb ₂ C-SH quantum dots, Nb ₂ C The schematic diagram of the synthesis of -SH quantum dots is shown in Figure 1.

Two, the specific embodiment of the preparation method of SPR sensor chip of the present invention is as follows:

Example 2

The preparation method of the SPR sensor chip of the present embodiment is the preparation method of the SPR sensor chip in Example 1, and the specific steps are:

(1) Pretreatment of gold chips

The gold chip was washed with _H2SO4 (98% mass fraction) and _H2O2 (30% mass fraction) at a volume ratio of _70:30 for 1 _minute , and then washed with ultrapure water (≥18.2MΩ·cm) Clean and then dry in _N2 flow; the gold chip is a glass sheet coated with a gold film with a thickness of about 50nm (the size of the glass sheet is 10×12×0.3mm).

(2) Preparation of modified gold chips

Disperse thiol-functionalized Nb ₂ C MXene quantum dots in 10 mL of ultrapure water (≥18.2 MΩ·cm), and stir for 6 h at a stirring speed of 800 rpm to obtain mercapto groups with a concentration of 0.1 mg·mL ^-1 Functionalized Nb ₂ C MXene quantum dot suspension, drop 10 μL of the prepared suspension of thiol-functionalized Nb ₂ C MXene quantum dots on the gold layer surface of the gold chip, and then drop the thiol-functionalized Nb ₂ C MXene quantum dots The gold chip of the suspension was placed in a 4°C environment (a refrigerator with a temperature of 4°C) and stood for 12 hours. Au-S bonds were formed between the thiol-functionalized Nb ₂ C MXene quantum dots and the gold layer, and a uniform layer was formed on the gold chip. Nb ₂ C MXene quantum dot layer to obtain a modified gold chip. The modified gold chip was installed on the surface plasmon resonance biochemical analyzer, and the supporting flow cell was fixed, and phosphate buffer solution (PBS, 0.01mol/L, pH=7.4) was passed through at a flow rate of 5 μL·min ^-1 . The chip was rinsed (to remove excess thiol-functionalized MXene quantum dots not bound by Au-S bonds) for 0.5 hours to complete the equilibrium of the baseline (the variation range of ΔRU was less than 0.3RU/min).

(3) Preparation of SPR sensor chip

The modified gold chip was incubated in N58 aptamer solution (concentration: 100nmol/L) at a flow rate of 5 μL min ^-1 for 0.5 hours (the incubation was to contact the gold chip containing the MXene quantum dot layer with the aptamer solution, Make the MXene quantum dots adsorb and fix the aptamers and reach equilibrium), after obtaining a stable baseline (the variation range of ΔRU is less than 0.3RU/min), use a flowing phosphate buffer solution (PBS, 0.01mol/L, pH= 7.4) Clean the chip to remove unfixed aptamers to obtain an SPR sensor chip, and mark the quantum dots on the SPR sensor chip as Apt _N58aptamer /Nb ₂ C-SH quantum dots. The schematic diagram of the preparation of the SPR sensor chip is shown in Figure 2 . Among them, the preparation method of the N58 aptamer solution with a concentration of 100 nmol/L is as follows: add phosphate buffer solution (PBS, 0.01 mol/L, pH=7.4) to the N58 aptamer stock solution with a concentration of 100 μmol/L, and prepare A solution of N58 aptamer with a concentration of 100 nmol/L was formed.

Experimental Example 1 Structural Characterization

1. Infrared spectroscopy

The Nb ₂ C MXene quantum dots and Nb ₂ C-SH quantum dots prepared in Example 1 were characterized by infrared spectroscopy, and the obtained results are shown in FIG. 3 .

It can be seen from Figure 3 that both the Nb ₂ C MXene quantum dots and the Nb ₂ C-SH quantum dots are decorated with a sufficient number of oxygen-containing functional groups. The bands at 3496, 3131 and 3034cm ^-1 are respectively attributed to the OH stretching, NH stretching and CH stretching of Nb ₂ C quantum dots, indicating the presence of hydroxyl and amino groups. There are two typical strong bands at 1741 and 1634 cm ^-1 due to C=O stretching vibrations generated by carbonyl and carboxyl groups. In addition, the band at 1400cm ^-1 belongs to the characteristic band of COC, and the broad band at 450-950cm ^-1 is the typical vibration peak of Nb-O. For Nb ₂ C-SH quantum dots, the broad peak around 3296 ^cm is caused by the stretching vibrations of OH and NH, and Nb-O vibrations after coupling with n-octadecylmercaptan through π-π* stacking The mode changes to a higher wave number (from 612cm ^-1 to 667cm ^-1 ), and the result proves that Nb ₂ C-SH quantum dots are successfully prepared.

2. X-ray photoelectron spectroscopy (XPS)

The Nb ₂ C MXene quantum dots, Nb ₂ C-SH quantum dots and Apt _N58 /Nb ₂ C-SH quantum dots were characterized by X-ray photoelectron spectroscopy (XPS), and the obtained results are shown in FIG. 4 .

In the XPS spectrum of Nb ₂ C quantum dots, the four peaks corresponding to the binding energy (BEs) positions of 286.1, 532.1, 407.1 and 232.1eV are assigned to C1s, O1s, N1s and Nb3d, respectively. For Nb ₂ C-SH quantum dots, the peak at 168.1 eV corresponds to the binding energy (BEs) of S2p. Before the SPR measurement, XPS was used to investigate whether the N58 aptamer could be immobilized on the Nb ₂ C quantum dot-modified Au chip, and the binding energy of P2p could be observed from the XPS spectrum of Apt _{N58 aptamer} /Nb ₂ C-SH quantum dots peak. Obviously, the clear XPS signal of P2p comes from the phosphate group of N58 aptamer.

Experimental Example 2 Morphological Characterization

The microstructure and morphology of the Nb ₂ C-SH quantum dots prepared in Example 1 were characterized by TEM, and the results are shown in FIG. 5 . In Figure 5, (a) is the low-magnification TEM image of Nb ₂ C-SH quantum dots, (b) is the high-magnification TEM image of Nb ₂ C-SH quantum dots, and (c) is the Nb ₂ C-SH quantum dots High-resolution TEM images of spots.

The quantum dots can be clearly observed in the low-magnification TEM image of Nb ₂ C-SH quantum dots ( FIG. 5 a ), with an average lateral size of 2.3 nm to 5.4 nm ( FIG. 5 b ). Clear fringes can be seen in the high-resolution TEM image of Nb ₂ C-SH quantum dots (Fig. 5c), indicating the single-crystal character of Nb ₂ C-SH quantum dots. The distance between adjacent lattice fringes is 0.217 nm, corresponding to the [001] plane of carbon.

Experimental example 3 SPR sensor chip preparation and optimization of detection conditions

In order to obtain the SPR sensor chip with the best sensing performance, the present invention studies the influence of parameters such as aptamer concentration and pH value of phosphate buffer solution on the sensing performance of the prepared SPR sensor chip. Add phosphate buffer solution (PBS, 0.01mol/L, pH=7.4) to the N58 aptamer stock solution with a concentration of 100 μmol/L to prepare N58 aptamers with concentrations of 1, 10, 50, 100 and 200 nmol/L Body solution; add phosphate buffer solution (PBS, 0.01mol ^/ L, pH=7.4) to the N-gene stock solution with a concentration of 1μg mL-1 to prepare an N-gene solution with a concentration of 1ng mL ^-1 . Then, according to the method of Example 2, SPR sensor chips based on different concentrations of N58 aptamer solutions were prepared, and then the N-gene solution with a concentration of 1 ng mL ^-1 was detected. The test results are shown in Figure 6. Figure 6a shows the effect of aptamer concentration on the immobilization of aptamer on Nb ₂ C-SH quantum dots and the effect on N-gene detection. All SPR measurements were performed in phosphate buffered saline (PBS, 0.01 mol/L, pH=7.4). The results show that as the aptamer concentration increases from 1 nmol/L to 100 nmol/L, the SPR response (refractive index unit [RU]) value of the aptamer-immobilized SPR sensor chip (ΔRU = RU _Apt – RU _{Nb2C QDs} ) increased from 124RU to 517RU. As the concentration of aptamer increased from 1nmol/L to 100nmol/L, the ΔRU (ΔRU=RU _N-gene -RU _Apt ) obtained by detecting N-gene increased from 21.6RU to 129.2RU. When the aptamer concentration was greater than 100nmol/L, the ΔRU value used to characterize aptamer immobilization and detect N-gene reached an equilibrium value. This result indicated that the optimal concentration of N58 aptamer for constructing SPR sensor chip and detecting N-gene was 100nmol/L.

Finally, the pH of the phosphate buffer solution was optimized. Use 1mol/L NaOH solution and 1mol/L H ₂ SO ₄ solution to adjust the pH of phosphate buffer solution (PBS, 0.01mol/L, pH=7.4), and prepare phosphate with pH=5.5, 6.5, 7.4, 8.5 and 9.5 Buffer solution base fluid. Phosphate buffer solution base solutions with pH=5.5, 6.5, 7.4, 8.5 and 9.5 were added to the N58 aptamer stock solution with a concentration of 100 μmol/L to prepare N58 aptamer solutions with a concentration of 100 nmol/L. Phosphate buffer solution base solutions with pH=5.5, 6.5, 7.4, 8.5 and 9.5 were added to the N-gene stock solution with a concentration of 1 μg mL ^-1 to prepare N-gene solutions with a concentration of 1 ng mL ^-1 . The prepared phosphate buffer solution base solution, N58 aptamer solution and N-gene solution were all stored at 4°C before use. Then according to the method of Example 2, the SPR sensor chip of the N58 aptamer solution with a concentration of 100 μmol/L prepared based on the phosphate buffer solution base solution with different pH values was prepared, and then the phosphate buffer solution base solution with different pH values was used (for cleaning the SPR sensor chip) Detect the N-gene solution with a concentration of 1ng mL ^-1 prepared based on phosphate buffer solution base solution with different pH values. Among them, in the same group of testing process (including the preparation of SPR sensor chip and detection of N-gene), the phosphate buffer solution with the same pH value was used when preparing the N58 aptamer solution, preparing the N-gene solution and cleaning the SPR sensor chip. liquid. It can be seen from Figure 6b that when using the phosphate buffer solution with a pH of 7.4, the ΔRU value for detecting N-gene is the highest, indicating that the optimal pH value of the phosphate buffer solution is 7.4. Therefore, the best aptamer concentration for preparing SPR sensor chip based on Nb ₂ C-SH quantum dots is 100nmol/L, and the best pH value of phosphate buffer solution is 7.4.

Sensing performance of experimental example 4SPR sensor chip

The N-gene solution and interfering substance solution used in this experimental example were prepared by the following method:

Add phosphate buffered saline (PBS, 0.01mol/L, pH=7.4) to the N-gene stock solution with a concentration of 1 μg mL ^-1 to prepare concentrations of 0.05, 0.1, 0.5, 1, 10, 50 and 100 ng mL ^{- 1} N-gene solution;

Phosphate buffered saline (PBS, 0.01mol/L, pH=7.4) was added to the stock solution of Flu A, Flu B, P1, CPN, IgG, PSA, and BSA at a concentration of 500ng mL ^-1 , and the prepared concentration was 100ng Flu A, Flu B, P1, CPN, IgG, PSA, BSA solutions in mL ^-1 , the prepared N-gene solution and interfering substance solutions were stored at 4°C before use.

The real samples used in this experiment example were processed by the following methods:

Dilute human serum 10 times with phosphate buffered saline solution (PBS, 0.01mol/L, pH=7.4) to prepare human serum samples containing different concentrations of N-gene for real sample analysis and store at 4°C before use .

Different amounts of N-gene were added to seawater (obtained from the sea area of Xiamen) to obtain seawater samples, which were used for real sample analysis and stored at 4°C before use.

Seafood (frozen shrimp) is purchased from Zhengzhou seafood market, and the frozen shrimp of 2g is added in the mass fraction of 4 milliliters and is 3% trichloroacetic acid (TA), stirs 10 minutes. Then, centrifuge at a centrifugal speed of 12000 rpm for 10 minutes to obtain the supernatant of the extract. Add 1.0mol/L NaOH solution to the supernatant to adjust pH=7, then dilute 10 times with deionized water, then add N-gene, dilute to a certain concentration, and obtain seafood samples for real sample analysis, before use Store at 4°C.

1. Sensitivity

Under the optimal detection conditions, the N-gene sensing performance of the SPR sensor chip based on Nb ₂ C-SH quantum dots was discussed, and the results are shown in Figure 7-9. As shown in Figure 7, after 1080s of injection, as the N-gene concentration (0.05, 0.1, 0.5, 1, 10, 50 and 100ng mL ^-1 ) increased from 0.05ng mL ^-1 to 100ng mL ^-1 , the SPR response It is also gradually enhanced, from 40RU to 270RU. As mentioned earlier, the N58 aptamer chain will bind to the N-gene of SARS-CoV-2 to form a G-quadruplex. This characteristic further increases the gold layer thickness of the Au chip of the SPR sensor, which in turn changes the refractive index of the cover layer. After 1080s of sample injection, the SPR sensor chip based on Nb ₂ C-SH quantum dots was washed with phosphate buffer solution, and the SPR response value dropped only 15RU, which is quite low. This result indicated a strong binding between the N-gene and the aptamer chain. The ΔRU value before and after N-gene detection (ΔRU=RU _N-gene -RU _Apt ; RU _N-gene is the N58 aptamer immobilized after detection of N-gene with the SPR sensor chip based on Nb ₂ C-SH quantum dots The RU value of the SPR sensor chip, RU _Apt is the RU value of the SPR sensor chip immobilized with the N58 aptamer before the detection of the N-gene) as the detected signal, the ΔRU value and the N-gene concentration (from 0.05ng mL ^-1 to 100ngmL ^-1 ), as shown in Figure 8, the linear regression equation is ΔRU=70.26logC _N-gene +121.09, and the correlation coefficient (R ² ) is 0.9923 (inset of Figure 8). With a signal-to-noise ratio of 3, the limit of detection (LOD) was as low as 4.9pg mL ^-1 .

The reasons why the SPR sensor chip based on Nb ₂ C-SH quantum dots show low detection limit (LOD) and fast response when detecting N-gene are mainly as follows: (i) Nb ₂ C-SH quantum dots The strong self-assembly interaction between the sulfhydryl groups and the surface of the Au chip endows the SPR sensor chip with outstanding stability in aqueous solution. (ii) The interaction between the aptamer and the highly conjugated Nb ₂ C-SH quantum dots can completely cover the Nb ₂ C-SH quantum dots through π-π* stacking, hydrogen bonding and van der Waals forces. Au chip, so that the SPR sensor chip has a strong biological affinity and a high detection efficiency for N-gene. (iii) The highly specific recognition between the aptamer and the N-gene can reduce the non-specific adsorption of other interfering substances on the Nb ₂ C-SH quantum dot-based SPR sensor chip.

2. Selective

The prepared N-gene and different interferors, including proteins in other respiratory viruses (such as CPN, influenza Flu A, influenza Flu B, and P1) and human serum (such as IgG, PSA, and BSA), were tested based on the N-gene alone. The selectivity of the Nb ₂ C-SH quantum dot SPR sensor chip when detecting N-gene, the concentration of each interfering substance is 100ng·mL ^-1 , which is 100 times of the N-gene concentration (1ng·mL ^-1 ). The ΔRU value of each interferer and N-gene detected (the RU value obtained by detecting each interferer or N-gene-the RU value of the gold chip after immobilizing the aptamer) is shown in FIG. 9 . The results show that the ΔRU value obtained by detecting interfering substances is negligible, which is much lower than the ΔRU value obtained when N-gene is detected by the SPR sensor chip based on Nb ₂ C-SH quantum dots. This result indicates that the sensor chip based on Nb ₂ C-SH quantum dots has outstanding selectivity in detecting N-gene.

3. Applicability

The applicability of the SPR sensor chip was verified by using the SPR sensor chip based on Nb ₂ C-SH quantum dots to detect N-gene in seawater, seafood (frozen shrimp), and human serum. Add different concentrations of N-gene to different samples to form sample solutions containing different concentrations of N-gene (0.05, 0.1, 0.5, 1, 10, 50, 100 pg·mL ^-1 ), using Nb ₂ The SPR sensor chip of C-SH quantum dot detects, obtains the ΔRU value (ΔRU=RU _N-gene -RU _Apt of SPR sensor chip detection before and after the N-gene in different samples; RU _N-gene is after detecting N-gene The RU value of the SPR sensor chip that is fixed with N58 aptamer, RU _Apt is the RU value of the SPR sensor chip that is fixed with N58 aptamer before detecting N-gene), according to the calibration curve, deduce the N-gene of detection Concentrations, all results are summarized in Table 1. It can be seen from Table 1 that the detection recovery rate of N-gene in seawater is 96.74% to 113.9%, with a relatively low RSD value of 0.41% to 2.67%. The detection recovery rate of N-gene in seafood is 97.96% to 106.1%, and the detection recovery rate of N-gene in human serum is 97.76% to 110.2%. The corresponding RSD values of seafood and human serum ranged from 0.46% to 3.24% and 0.29% to 3.36%, respectively. These results demonstrate that the SPR sensor chip based on Nb ₂ C-SH quantum dots can be used to analyze N-genes in different environments.

Table 1 Applicability test results of SPR sensor chips

In summary, the embodiment of the present invention uses Nb ₂ C-SH quantum dots as the sensitive layer of the aptamer chain anchoring the N-gene target, and designs and constructs a new type of SPR sensor chip for the novel coronavirus N-gene specific detection of virus (SARS-CoV-2). The Nb ₂ C-SH quantum dots prepared by the present invention have the characteristics of sulfhydryl functionalization, high conjugated structure and graphene-like MXene phase, and can not only form Au-S bonds through self-assembly, but also show strong bonding with Au chips function, while showing strong bioaffinity and amplified SPR effect on the aptamer chain. Therefore, the SPR sensor chip based on sulfhydryl-functionalized Nb ₂ C quantum dots has an ultra-low detection limit (LOD) of 4.9 in the N-gene concentration range of 50 pgmL ^-1 to 100 ng mL ^-1 pg mL ^-1 . This limit of detection (LOD) is close to that of most reported biosensors for N-gene detection. Due to the specific binding of N58 aptamer to N-gene, the SPR sensor chip based on sulfhydryl-functionalized Nb ₂ C quantum dots also showed high selectivity when detecting N-gene. The SPR sensor chip based on sulfhydryl-functionalized Nb ₂ C quantum dots showed wide applicability when detecting N-gene in different samples (including seawater, seafood and human serum). The invention lays the foundation for the preparation of the SPR sensor chip for detecting N-gene, and provides a new strategy for the early sensitive analysis of N-gene.

Claims (7)

1. The SPR sensor chip is characterized by comprising a glass substrate and a gold layer arranged on the glass substrate, wherein the surface of the gold layer is provided with mercapto-functionalized MXene quantum dots modified by Au-S covalent bonds, and aptamers are anchored on the mercapto-functionalized MXene quantum dots;

the sulfydryl functionalized MXene quantum dot is prepared by the following steps: carrying out dispersion treatment on the MXene quantum dots and the thiol compound in a dispersion medium to obtain mercapto-functionalized MXene quantum dots; the mass ratio of the MXene quantum dots to the thiol compound is 1 to 3; the thiol compound is C15-20 alkyl thiol.

2. The SPR sensor chip according to claim 1, wherein the temperature of the dispersion treatment is 10 to 30 ℃; the dispersion treatment is to perform ultrasonic treatment on the mixture and then perform stirring treatment; the mixture consists of MXene quantum dots, a thiol compound and a dispersion medium.

3. The SPR sensor chip of claim 1, wherein said thiol compound is n-octadecyl thiol.

4. The SPR sensor chip of claim 1, wherein said mercapto-functionalized MXene quantum dots are mercapto-functionalized Nb ₂ C MXene quantum dots.

5. The SPR sensor chip of claim 1, wherein said aptamer is an aptamer for targeted detection of N-gene of SARS-CoV-2.

6. The SPR sensor chip of claim 5, wherein said aptamer is an N58 aptamer.

7. A method for preparing the SPR sensor chip according to any one of claims 1 to 6, comprising:

(1) Enabling the suspension of the sulfydryl functionalized MXene quantum dots to be in contact reaction with a gold layer of a gold chip to form an Au-S covalent bond, and obtaining a modified gold chip; the gold chip comprises a glass substrate and a gold layer arranged on the glass substrate;

(2) The modified gold chips were incubated in the aptamer solution.

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