CN113267472A - SPR sensor chip and preparation method thereof - Google Patents
SPR sensor chip and preparation method thereof Download PDFInfo
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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
Technical Field
The invention relates to an SPR sensor chip and a preparation method thereof, belonging to the technical field of biomedical detection.
Background
Since the end of 2019, the COVID-19 global epidemic caused by the new coronavirus (SARS-CoV-2) has spread continuously. The early clinical manifestations of new coronary pneumonia are similar to severe acute respiratory syndrome, middle east respiratory syndrome coronavirus diseases, namely fever, headache, myalgia, arthralgia, and lymphadenectasis. SARS-CoV-2 has the characteristics of moderate mortality, high infection rate, long incubation period and the like, and can cause long-term infection. The novel coronavirus (SARS-CoV-2) has been found in a variety of environments, such as aqueous systems, frozen foods, food packaging. Therefore, rapid diagnosis of the COVID-19 virus is critical to effectively control viral transmission and treat patients. Similar to other coronaviruses, the novel coronavirus (SARS-CoV-2) is mainly composed of four structural proteins, namely, a spinous process protein (S), a membrane protein (M), an envelope protein (E), and a nucleocapsid protein (N-gene). Proteins (e.g., S proteins) and viral RNA can be used as targets for qualitative and quantitative analysis of the novel coronavirus (SARS-CoV-2). Alternatively, antibodies, such as IgM and IgG, from a patient sample may be detected to study the patient's history of infection. Various techniques for analyzing the novel coronavirus (SARS-CoV-2) have been developed, such as real-time Polymerase Chain Reaction (PCR), colorimetric analysis, Surface Plasmon Resonance (SPR), and local SPR, electrochemical methods, optical/chemiluminescent immunosensors, fluorescence techniques, and wearable sensors.
Most techniques for analyzing the novel coronavirus (SARS-CoV-2) are based on the specific recognition between antibodies and the different proteins and RNAs in SARS-CoV-2. Compared with an antibody, the DNA aptamer serving as a powerful probe has the advantages of high specificity, strong affinity, rapid and reliable synthesis, easiness in coupling and the like. Thus, DNA aptamers can be used for different in vitro and in vivo diagnostics.
Surface Plasmon Resonance (SPR) is a widely used technique for qualitative and quantitative analysis, and is commonly used to construct immunoassays, multiplex detection of biomolecules, and in situ detection of multiple chemical and biological analyte interactions. Various nano materials, such as functional polymer, nano particle, graphene and MXene nano sheetAnd MoS2Have been used as sensing platforms for the preparation of SPR sensors. MXenes is a typical two-dimensional material that has attracted much attention because of its similar structure and properties to graphene. MXenes has a nano-sheet structure, unique surface chemical properties, high conductivity and excellent biocompatibility. These properties make MXenes effective as a platform for the development of various biosensors. MXene nanoplatelets are used for immobilization of probes and development of SPR biosensors. However, the thickness of the sensitive layer of the SPR biosensor is 200nm, MXene nanosheets are easy to aggregate due to the pi-pi stacking effect, and large-size aggregates are formed. Due to the limitation of the thickness of the SPR biosensor, the MXene nanosheet is difficult to obtain an SPR sensor chip with high sensitivity and high response speed.
Disclosure of Invention
The invention aims to provide an SPR sensor chip which takes sulfydryl functionalized MXene quantum dots as a sensor platform and has high sensitivity and high response speed.
Another object of the present invention is to provide a method for manufacturing an SPR sensor chip.
In order to achieve the above object, the technical solution of the SPR sensor chip of the present invention is:
the SPR sensor chip comprises 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 invention designs and constructs a novel SPR sensor chip by using sulfydryl functionalized MXene quantum dots as a biosensing platform of the SPR sensor chip. The size of zero-dimensional Quantum Dots (QDs) prepared from two-dimensional large-size MXene nano-sheets is very small and is about 5nm, which meets the test requirements of SPR instruments, and due to the combination of quantum confinement, edge effect and surface functionalization, the MXene quantum dots can be used as sensitive nano-materials to construct SPR sensor chips.
The sulfydryl functionalized MXene quantum dots have the characteristics of sulfydryl functionalization, a high conjugated structure and a graphene-shaped MXene phase, can form Au-S bonds through self-assembly to show a strong combination effect with an Au chip when being used as an SPR sensing platform, and simultaneously has a small size and a large specific surface area, and shows strong biological affinity and an amplified SPR effect on an aptamer chain.
According to the invention, a large amount of sulfydryl functionalized MXene quantum dots with smaller sizes are uniformly coated on the surface of the gold chip through the self-assembly effect between the sulfydryl functionalized MXene quantum dots and the SPR gold chip, and then a large amount of aptamers are fixed on the MXene quantum dots through pi-stacking, electrostatic adsorption and hydrogen bonding. Compared with the SPR sensor chip reported previously, the SPR sensor chip based on the sulfydryl functionalized MXene quantum dots with smaller size and larger specific surface area 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.
The sulfydryl functionalized MXene quantum dots can be combined with MXene quantum dots through chemical bonds, such as a coupling agent containing sulfydryl through a chemical coupling reaction (forming chemical bonds), or can be combined with MXene quantum dots through non-chemical bonds, such as a compound containing sulfydryl through pi-stacking to form MXene quantum dots with sulfydryl functional groups on the surfaces. Preferably, the sulfydryl functionalized MXene quantum dot is an MXene quantum dot which is formed by combining a non-chemical bond with the MXene quantum dot and contains sulfydryl functional groups on the surface.
More preferably, the sulfydryl functionalized MXene quantum dot is an MXene quantum dot which is combined with the MXene quantum dot through pi-pi stacking and contains sulfydryl functional groups on the surface.
Preferably, the mercapto-functionalized MXene quantum dot is obtained by a preparation method comprising the following steps: and (3) dispersing the MXene quantum dots and the thiol compound in a dispersion medium to obtain the mercapto-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 dispersion treatment is 10-30 ℃; the dispersion treatment is to perform ultrasonic treatment on the mixture and then perform stirring treatment; the mixture consists of MXene quantum dots, thiol compounds and a dispersion medium; the mixture is obtained by mixing MXene quantum dots, thiol compounds and a dispersion medium; the dispersion medium is water.
More preferably, the temperature of the dispersion treatment is 25 ℃.
Preferably, the power of the ultrasonic wave is 80-200W, and the time of the ultrasonic treatment is 0.2-2 h.
More preferably, the power of the ultrasound is 120W, and the time of the ultrasonic treatment is 0.5 h.
Preferably, the stirring speed is 600-1000 r/min, and the stirring treatment time is 4-8 h.
More preferably, the stirring speed is 800r/min, and the stirring treatment time is 6 h.
Preferably, the concentration of the MXene quantum dots in a 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 compound is a C15-20 alkyl thiol.
Preferably, the thiol compound is n-octadecyl mercaptan.
Preferably, the mercapto-functionalized MXene quantum dot is mercapto-functionalized Nb2C MXene quantum dots.
Preferably, the aptamer is an aptamer for the targeted detection of N-gene of SARS-CoV-2. The aptamer for targeted detection of SARS-CoV-2 can form G-quadruplet with N-gene of novel coronavirus (SARS-CoV-2), and the aptamer chain changes the conformation of the aptamer when binding with the N-gene, so that the contact area or distance between a probe molecule (aptamer) and a chip is changed, and further the SPR signal (RU) is changed, and the N-gene of SARS-CoV-2 can be detected through the change of the SPR signal. In general, one refractive index unit (RU) corresponds to 10-6Refractive index change of about 1pg mm-2The bound protein (binding protein) of (a), therefore, the load of the SPR chip can be calculated by the refractive index unit (RU). And previously reportedCompared with the SPR biosensor, the SPR biosensor chip based on the sulfydryl functionalized MXene quantum dots has the advantages of simple structure and high stability, shows superior sensing performances such as higher sensitivity, quick response and practicability in a complex environment, and can be widely applied to N-gene detection in various environments such as human serum, seawater and marine products.
These advantages are mainly due to several factors: the sulfydryl functionalized MXene quantum dots can be uniformly deposited on an SPR gold chip and can adsorb a large amount of aptamers, so that high sensitivity is obtained; the highly specific recognition of N-gene by aptamers for targeted detection of SARS-CoV-2 allows for a faster response of SPR sensors to N-gene.
Preferably, the aptamer is an N58 aptamer. The high specific recognition of the N58 aptamer with N-gene makes the prepared SPR sensor chip have faster response to N-gene.
The technical scheme of the preparation method of the SPR sensor chip is as follows:
a method for preparing an SPR sensor chip comprises the following steps:
(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.
The invention forms Au-S covalent bond through the self-assembly function between mercapto-functionalized MXene quantum dot and gold atom on the gold chip, a large amount of mercapto-functionalized MXene quantum dot and gold layer form uniform MXene quantum dot layer on the gold chip after being connected through Au-S bond, and SPR sensor chip is formed after an aptamer is fixed on the MXene quantum dot layer.
Preferably, the mercapto-functionalized MXene quantum dot is mercapto-functionalized Nb2C MXene quantum dots.
Preferably, in the step (1), the concentration of the suspension of the sulfydryl functionalized MXene quantum dots is 0.1 mg-mL-1。
And the incubation is to contact the gold chip containing the MXene quantum dot layer with the aptamer solution to enable the MXene quantum dots to adsorb and fix the aptamer and reach an equilibrium state.
Drawings
FIG. 1: nb2A synthesis schematic diagram of the C-SH quantum dots;
FIG. 2: based on Nb2A preparation schematic diagram of the C-SH quantum dot SPR sensor chip;
FIG. 3: nb2C quantum dots and Nb2An infrared spectrogram of the C-SH quantum dots;
FIG. 4: nb2C quantum dot, Nb2C-SH quantum dots and AptN58/Nb2XPS spectrogram of the C-SH quantum dots;
FIG. 5: (a) is Nb2A low-magnification transmission electron microscope image of the C-SH quantum dots, wherein (b) is Nb2High-magnification transmission electron microscope image of C-SH quantum dot, wherein (C) is Nb2High resolution TEM images of C-SH quantum dots;
FIG. 6: (a) for aptamers at Nb at different aptamer concentrations2Delta RU values before and after immobilization on the C-SH quantum dots and delta RU values before and after N-gene detection of the prepared SPR sensor chip, (b) delta RU values before and after N-gene detection of the SPR sensor chip prepared for phosphate buffer solutions with different pH values (an aptamer solution is prepared, an N-gene solution is prepared, and a phosphate buffer solution base solution with the same pH value is used for N-gene detection);
FIG. 7: based on Nb2SPR sensor chip for detecting N-gene (0.05,0.1,0.5,1, 10,50 and 100ng mL) with different concentrations by using C-SH quantum dots-1) The change curve of the delta RU value along with the detection time;
FIG. 8: based on Nb2SPR sensor chip of C-SH quantum dot detects delta RU (delta RU-RU) values before and after N-geneN-gene-RUN58 aptamer) And N-gene concentration, the inset is a linear relationship between the logarithm of the concentration of the N-gene and the Δ RU;
FIG. 9: based on Nb2SPR sensor chip for detecting interferent (the concentration is 100ng mL) of C-SH quantum dot-1) And N-gene (concentration 1ng mL)-1) Δ RU value of (a) versus detection time.
Detailed Description
The following further describes embodiments of the present invention with reference to the drawings.
Materials used in the examples of the present invention: the N58 aptamer is provided by Shanghai biological engineering GmbH, with the sequence 5'-GCT GGA TGT CAC CGG ATT GTC GGA CAT CGG ATT GTC TGA GTC ATA TGA CAC ATC CAG C-3';
the resistivity of the ultrapure water is not less than 18.2M omega cm.
The phosphate buffer solution was prepared by the following method: 8.00g NaCl, 0.20g KCl, 1.44g Na2HPO4、1.8g K2HPO4Dissolving in 800mL of ultrapure water (not less than 18.2M omega cm), adjusting the pH value of the solution to 7.4 by hydrochloric acid, and finally performing constant volume to 1L by using the ultrapure water (not less than 18.2M omega cm) to obtain a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4), wherein the prepared phosphate buffer solution is stored at 4 ℃ before use.
The preparation of the SPR sensor chip in example 2 and the testing of the SPR sensor chips in Experimental examples 3 and 4 were all performed by BiacoreTMThe X100 instrument (GE-Healthcare Bio-Sciences AB, USA) is carried out at 25 ℃.
The first specific embodiment of the SPR sensor chip of the present invention is as follows:
example 1
The SPR sensor chip comprises a glass substrate and a gold layer arranged on the glass substrate, wherein the surface of the gold layer is provided with mercapto-functionalized Nb modified by Au-S covalent bonds2And the mercapto-functionalized MXene quantum dot is anchored with an N-58 aptamer.
Wherein, the mercapto group is functionalized Nb2The preparation method of the C quantum dot comprises the following steps:
(1) preparation of Nb2C MXene powder: mixing Nb with2The AlC powder reacts with hydrofluoric acid with the mass fraction of 3 percent for 48 hours at the temperature of 55 ℃, and then the mixture is washed by deionized water for 12 times,placing in a vacuum oven at 60 ℃ for drying for 18h to obtain Nb2C MXene powder.
(2) Preparation of Nb2C MXene quantum dots: mixing 15mg of Nb2Dispersing C MXene powder in 20mL of ultrapure water (not less than 18.2M omega cm), stirring at a stirring speed of 800r/min for 0.5h, adding 25% by mass of ammonia water, adjusting the pH value of the system to 6, heating at 100 deg.C for 6h, filtering the supernatant with a 220nm filter membrane, and concentrating the filtrate by reduced pressure distillation to obtain Nb2C MXene quantum dots.
(3) Preparation of mercapto-functionalized Nb2C MXene quantum dots: at 25 ℃, adding Nb2Dispersing the C MXene quantum dots in ultrapure water (not less than 18.2M omega cm), stirring for 12h to obtain the concentration of 1 mg/mL-1Nb of2C MXene Quantum dot suspension, then 2mg of n-octadecyl mercaptan was dispersed in 2mL of prepared Nb2Carrying out ultrasonic treatment on the C MXene quantum dot suspension for 30 minutes under the condition that the ultrasonic power is 120W, then stirring for 6 hours under the condition that the stirring speed is 800r/min, finally filtering by using a 220nm filter membrane, and concentrating the filtrate to obtain the mercapto-functionalized Nb2C MXene quantum dot, mark Nb2C-SH quantum dots, Nb2The synthesis scheme of the C-SH quantum dots is shown in figure 1.
Secondly, the specific embodiment of the preparation method of the SPR sensor chip of the invention is as follows:
example 2
The preparation method of the SPR sensor chip of this embodiment is the preparation method of the SPR sensor chip of embodiment 1, and includes the following specific steps:
(1) pretreatment of gold chips
The gold chip is applied with H with the volume ratio of 70:302SO4(98% by mass) and H2O2(30% by mass) for 1 minute, then washing with ultrapure water (18.2 M.OMEGA.. multidot.cm), and then washing with N2Drying in a stream; the gold chip is a glass plate coated with a gold film with a thickness of about 50nm (the size of the glass plate is 10 multiplied by 12 multiplied by 0.3 mm).
(2) Preparation of modified gold chip
Functionalization of thiol groups with Nb2Dispersing the C MXene quantum dots in 10mL of ultrapure water (not less than 18.2M omega cm), stirring for 6h at the stirring speed of 800r/min to obtain the concentration of 0.1 mg/mL-1Mercapto-functionalized Nb of2C MXene quantum dot suspension, 10 μ L of prepared mercapto-functionalized Nb2C MXene quantum dot suspension is dripped on the surface of the gold layer of the gold chip, and then mercapto-functionalized Nb is dripped2Placing the gold chip of the suspension of the C MXene quantum dots in a 4 ℃ environment (a refrigerator with the temperature of 4 ℃), standing for 12h, and carrying out sulfydryl functionalization on the Nb2Au-S bonds are formed between the C MXene quantum dots and the gold layer, and uniform Nb is formed on the gold chip2And C MXene quantum dot layer to obtain the modified gold chip. Mounting the modified gold chip on a surface plasma resonance biochemical analyzer, fixing a matched flow cell, introducing phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) at flow rate of 5 μ L/min-1And washing the modified gold chip (removing the excessive sulfydryl functionalized MXene quantum dots which are not combined through Au-S bonds) for 0.5 hour to complete the balance of the baseline (the change range of the Delta RU is less than 0.3 RU/min).
(3) Preparation of SPR sensor chip
The modified gold chip was placed in N58 aptamer solution (100 nmol/L) for 5. mu.L min-1After flowing for 0.5 hour, incubating (incubation is to contact the gold chip containing the MXene quantum dot layer with the aptamer solution to make the MXene quantum dots adsorb and fix the aptamer and reach an equilibrium state), after obtaining a stable baseline (the change range of the Delta RU is less than 0.3RU/min), washing the chip by flowing phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) to remove the unfixed aptamer to obtain the SPR sensor chip, and marking the quantum dots on the SPR sensor chip as AptN58aptamer/Nb2The preparation schematic diagram of the C-SH quantum dot and SPR sensor chip is shown in figure 2. The preparation method of the N58 aptamer solution with the concentration of 100nmol/L comprises the following steps: to a stock solution of N58 aptamer at a concentration of 100. mu. mol/L, a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) was added to prepare a solution of N58 aptamer at a concentration of 100 nmol/L.
Experimental example 1 structural characterization
1. Infrared spectroscopy
Infrared Spectroscopy of the Nb prepared in example 12C MXene quantum dot and Nb2The C-SH quantum dots are characterized, and the obtained result is shown in figure 3.
As can be seen from FIG. 3, Nb2C MXene quantum dot and Nb2The C-SH quantum dots are modified with a sufficient number of oxygen-containing functional groups. 3496. 3131 and 3034cm-1The spectral bands of (A) are respectively attributed to Nb2O-H stretching, N-H stretching and C-H stretching of the C quantum dots indicate the existence of hydroxyl and amino. 1741 and 1634cm due to C ═ O stretching vibration generated by carbonyl and carboxyl-1There are two typical strong bands. Furthermore, 1400cm-1The spectral band belongs to a C-O-C characteristic band, 450-950 cm-1The broad band of (a) is the typical vibrational peak of Nb-O. For Nb2C-SH quantum dot, 3296cm-1The nearby broad peaks are caused by stretching vibrations of O-H and N-H, and the Nb-O vibration mode changes to higher wave numbers (from 612 cm) after coupling with N-octadecyl mercaptan by pi-stacking-1To 667cm-1) The results prove that Nb is successfully prepared2And C-SH quantum dots.
X-ray photoelectron spectroscopy (XPS)
Nb pair by X-ray photoelectron spectroscopy (XPS)2C MXene Quantum dot, Nb2C-SH quantum dots and AptN58/Nb2The C-SH quantum dots are characterized, and the obtained results are shown in figure 4.
Nb2In the XPS spectrum of the C quantum dot, 4 peaks corresponding to 286.1, 532.1, 407.1 and 232.1eV of Binding Energy (BEs) positions are respectively assigned to C1s, O1s, N1s and Nb3 d. For Nb2The peak at 168.1eV of the C-SH quantum dot corresponds to the Binding Energy (BEs) of S2 p. Whether the N58 aptamer could be immobilized in Nb was investigated by XPS before SPR measurement2On Au chip decorated with C quantum dots, from AptN58 aptamer/Nb2A binding energy peak of P2P can be observed in an XPS spectrum of the C-SH quantum dots. Clearly, the clear XPS signal for P2P is from the phosphate group of the N58 aptamer.
Experimental example 2 morphology characterization
TEM of sample obtained in example 1Prepared Nb2The microstructure and morphology of the C-SH quantum dots are characterized, and the result is shown in FIG. 5. In FIG. 5, (a) is Nb2A low-magnification transmission electron microscope image of the C-SH quantum dots, wherein (b) is Nb2High-magnification transmission electron microscope image of C-SH quantum dot, wherein (C) is Nb2High resolution TEM images of C-SH quantum dots.
At Nb2The quantum dots can be clearly observed in the low-magnification transmission electron microscope image (fig. 5a) of the C-SH quantum dots, and the average transverse dimension is 2.3nm to 5.4nm (fig. 5 b). At Nb2Clear stripes were visible in high resolution TEM images of C-SH quantum dots (FIG. 5C), indicating that Nb is present2Single crystal characteristics of C-SH quantum dots. The distance between adjacent lattice fringes was 0.217nm, [001 ] for carbon]And (5) kneading.
Experimental example 3 preparation of SPR sensor chip and optimization of detection conditions
In order to obtain the SPR sensor chip with the optimal sensing performance, the influence of parameters such as aptamer concentration, pH value of phosphate buffer solution and the like on the sensing performance of the prepared SPR sensor chip is researched. Adding phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) into N58 aptamer stock solution with the concentration of 100 mu mol/L to prepare N58 aptamer solutions with the concentrations of 1, 10,50, 100 and 200nmol/L respectively; at a concentration of 1. mu.g mL-1The N-gene stock solution was added with a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) to prepare a solution having a concentration of 1ng mL-1The N-gene solution of (1). SPR sensor chips based on N58 aptamer solutions with different concentrations were then prepared according to the method of example 2, and the detection concentration was 1ng mL-1The test results of the N-gene solution of (1) are shown in FIG. 6. FIG. 6a shows aptamer concentration versus aptamer concentration at Nb2The effect of immobilization on C-SH quantum dots and the effect on N-gene detection. All SPR measurements were performed in phosphate buffered saline (PBS, 0.01mol/L, pH 7.4). The results showed that the SPR response of the SPR sensor chip after aptamer immobilization (refractive index Unit [ RU ] as the aptamer concentration was increased from 1nmol/L to 100nmol/L]) Value (Δ RU ═ RUApt–RUNb2C QDs) Increasing from 124RU to 517 RU. Detection of N-gene with increasing aptamer concentration from 1nmol/L to 100nmol/LTo Δ RU (Δ RU ═ RUN-gene-RUApt) Increasing from 21.6RU to 129.2 RU. The Δ RU values used to characterize aptamer immobilization and detection of N-gene reached an equilibrium value when the aptamer concentration was greater than 100 nmol/L. This result indicates that the optimal N58 aptamer concentration for constructing SPR sensor chips and detecting N-gene is 100 nmol/L.
Finally, the pH of the phosphate buffer solution was optimized. With 1mol/L NaOH solution and 1mol/L H2SO4The pH of a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) was adjusted to prepare base solutions of phosphate buffer solutions having pH 5.5, 6.5, 7.4, 8.5, and 9.5. Phosphate buffer solution base solutions with pH values of 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, respectively, to prepare N58 aptamer solutions each with a concentration of 100 nmol/L. The base solutions of phosphate buffer solutions at pH 5.5, 6.5, 7.4, 8.5 and 9.5 were added to a concentration of 1 μ g mL, respectively-1In the N-gene stock solution, the preparation concentration is 1ng mL-1The N-gene solution of (1). The prepared base solution of phosphate buffer solution, the N58 aptamer solution and the N-gene solution are stored at 4 ℃ before use. Then, SPR sensor chips prepared based on 100 mu mol/L N58 aptamer solutions prepared based on different pH values of phosphate buffer solution base solutions were prepared according to the method of example 2, and the prepared concentration of 1ng mL based on different pH values of phosphate buffer solution base solutions (used for cleaning the SPR sensor chips) was detected-1The N-gene solution of (1). In the same test process (including preparation of the SPR sensor chip and detection of the N-gene), a phosphate buffer solution base solution with the same pH value is used for preparing the N58 aptamer solution, preparing the N-gene solution and cleaning the SPR sensor chip. As can be seen from FIG. 6b, the highest Δ RU value was detected for N-gene when phosphate buffered solution pH 7.4 was used, indicating that the optimal pH for phosphate buffered solution was 7.4. Thus, preparation based on Nb2The optimal aptamer concentration of the SPR sensor chip of the C-SH quantum dots is 100nmol/L, and the optimal pH value of the phosphate buffer solution is 7.4.
Experimental example 4 sensing Performance of SPR sensor chip
The N-gene solution and the interfering substance solution used in this experimental example were prepared by the following methods:
at a concentration of 1. mu.g mL-1The N-gene stock solution of (2) was added with a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) to prepare concentrations of 0.05,0.1,0.5,1, 10,50 and 100ng mL, respectively-1N-gene solution of (1);
all concentrations are 500ng mL-1The stock solutions of Flu A, Flu B, P1, CPN, IgG, PSA, and BSA in (1) were added with a phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) to prepare 100ng mL of the solutions-1The prepared solution of Flu A, Flu B, P1, CPN, IgG, PSA, and BSA, the N-gene solution, and the interfering substance solution were stored at 4 ℃ before use.
The real samples used in this experimental example were processed as follows:
human serum was diluted 10-fold with phosphate buffered saline (PBS, 0.01mol/L, pH 7.4) to prepare human serum samples containing different concentrations of N-gene for real sample analysis and stored at 4 ℃ prior to use.
Different amounts of N-gene were added to seawater (obtained from Xiamen sea area) to obtain seawater samples for real sample analysis, which were stored at 4 ℃ prior to use.
Seafood (frozen shrimp) was purchased from zhengzhou seafood market, 2g of frozen shrimp was added to 4 ml of Trichloroacetic Acid (TA) with a mass fraction of 3%, and stirred for 10 minutes. Then, the supernatant of the extract was obtained by centrifugation at a centrifugation speed of 12000 rpm for 10 minutes. Adding 1.0mol/L NaOH solution into the supernatant, adjusting the pH value to 7, then diluting by 10 times with deionized water, adding N-gene, diluting to a certain concentration to obtain a seafood sample, analyzing the seafood sample, and storing at 4 ℃ before use.
1. Sensitivity of the reaction
Under the best detection conditions, Nb-based2The sensing performance of the SPR sensor chip of the C-SH quantum dots on the N-gene is shown in FIGS. 7-9. As shown in FIG. 7, with N-gene concentrations (0.05,0.1,0.5,1, 10,50 and 100ng mL) after 1080s injection-1) From 0.05ng mL-1Increase to 100ng mL-1The SPR response also gradually increased from 40RU to 270 RU. As described above, the N58 aptamer chain binds to the N-gene of SARS-CoV-2 to form a G-quadruplex. This property further increases the gold layer thickness of the Au chip of the SPR sensor, thereby changing the refractive index of the capping layer. After 1080s injection, the Nb-based sample is rinsed with phosphate buffer2The SPR response value of the SPR sensor chip of the C-SH quantum dots is reduced to only 15RU, which is quite low. This result indicates a strong binding between the N-gene and the aptamer chain. Based on Nb2SPR sensor chip of C-SH quantum dot detects delta RU (delta RU-RU) values before and after N-geneN-gene-RUApt；RUN-geneTo detect the RU value of the SPR sensor chip immobilized with the N58 aptamer after N-gene, RUAptRU value of SPR sensor chip fixed with N58 aptamer before N-gene) as detected signal, the Δ RU value and N-gene concentration (from 0.05ng mL)-1To 100ng mL-1) Is proportional to the logarithm of (1), as shown in fig. 8, the linear regression equation is 70.26logCN-gene+121.09, where the correlation coefficient (R)2) 0.9923 (see inset of fig. 8). With a signal-to-noise ratio of 3, the limit of detection (LOD) is as low as 4.9pg mL-1。
Based on Nb2The reasons that the SPR sensor chip of the C-SH quantum dot shows low detection Limit (LOD) and fast response when detecting N-gene mainly include the following aspects: (i) nb2The strong self-assembly interaction between the sulfydryl on the C-SH quantum dots and the surface of the Au chip enables the SPR sensor chip to have outstanding stability in aqueous solution. (ii) Aptamers and highly conjugated Nb2The C-SH quantum dots can completely cover the Nb by pi-stacking, hydrogen bonds and van der Waals force action2And the C-SH quantum dots are modified on the Au chip, so that the SPR sensor chip has stronger biological affinity and higher detection efficiency on N-gene. (iii) The high specific recognition between the aptamer and the N-gene can reduce other interferents based on Nb2Nonspecific adsorption on SPR sensor chips of C-SH quantum dots.
2. Selectivity is
By detecting N-gene and differences separatelyIncluding other respiratory viruses (e.g., CPN, influenza Flu A, influenza Flu B and P1) and proteins in human serum (e.g., IgG, PSA and BSA), to test prepared Nb-based antibodies2The selectivity of the SPR sensor chip of the C-SH quantum dots during the detection of the N-gene is 100 ng/mL-1The concentration of N-gene (1 ng. mL)-1) 100 times of the total weight of the powder. The Δ RU values for each of the interferents and N-gene (RU values obtained by detecting each of the interferents or N-gene-RU value of gold chip after aptamer immobilization) are shown in FIG. 9. The results show that the Δ RU values obtained by detecting interferents are negligible and much lower than those based on Nb2And the delta RU value obtained when the SPR sensor chip of the C-SH quantum dots detects the N-gene. This result indicates that Nb is based on2The sensor chip of the C-SH quantum dots has outstanding selectivity in detecting N-gene.
3. Applicability of the invention
By using Nb-based2The SPR sensor chip of the C-SH quantum dots is used for detecting N-gene in seawater, seafood (frozen shrimps) and human serum to verify the applicability of the SPR sensor chip. Adding N-gene with different concentrations into different samples to form N-gene with different concentrations (the concentrations are respectively 0.05,0.1,0.5,1, 10,50, 100 pg.mL)-1) Using a sample solution based on Nb2Detecting the SPR sensor chip of the C-SH quantum dots to obtain a delta RU (delta RU-RU) value before and after detecting the N-gene in different samples by the SPR sensor chipN-gene-RUApt；RUN-geneTo detect the RU value of the SPR sensor chip immobilized with the N58 aptamer after N-gene, RUAptTo detect the RU value of the SPR sensor chip with the N58 aptamer immobilized thereto before N-gene), the concentration of the detected N-gene was derived from the calibration curve, and all the results are summarized in table 1. As can be seen from Table 1, the recovery of N-gene detected in seawater was 96.74% to 113.9%, with a lower RSD value of 0.41% to 2.67%. The detection recovery rate of N-gene in marine products is 97.96 percent to 106.1 percent, and the detection recovery rate of N-gene in human serum is 97.76 percent to 110.2 percent. The marine products have RSD values corresponding to human serum ranging from 0.46% to 3.24% and 0.29% to 3.36%, respectively. These results demonstrate Nb-based2SPR sensor of C-SH quantum dotsThe chip can be used for analyzing N-gene in different environments.
TABLE 1 test results of the applicability of SPR sensor chips
In summary, the embodiments of the present invention use Nb2The C-SH quantum dots are used as a sensitive layer of an aptamer chain anchoring an N-gene target, and a novel SPR sensor chip is designed and constructed and is used for N-gene specific detection of novel coronavirus (SARS-CoV-2). Nb prepared by the invention2The C-SH quantum dots have the characteristics of sulfydryl functionalization, high conjugated structure and graphene-shaped MXene phase, can form Au-S bonds through self-assembly to show strong combination effect with Au chips, and simultaneously show strong biological affinity and amplified SPR effect on aptamer chains. Thus, the concentration range of N-gene is 50pg mL-1To 100ng mL-1Based on mercapto-functionalized Nb2The SPR sensor chip of the C quantum dots has an ultralow detection Limit (LOD) of 4.9pg mL when used for detecting N-gene-1. This limit of detection (LOD) is close to that of most reported biosensors that detect N-genes. Thiol-based functionalized Nb due to specific binding of N58 aptamers to N-gene2The SPR sensor chip of the C quantum dots also shows high selectivity when detecting N-gene. Mercapto-based functionalized Nb2The SPR sensor chip of the C quantum dots shows wide applicability when detecting N-gene in different samples (including seawater, seafood and human serum). The invention lays a foundation for preparing the SPR sensor chip for detecting the N-gene and provides a new strategy for the early sensitive analysis of the N-gene.
Claims (10)
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.
2. The SPR sensor chip of claim 1, wherein said mercapto-functionalized MXene quantum dots are obtained by a preparation method comprising the steps of: and (3) dispersing the MXene quantum dots and the thiol compound in a dispersion medium to obtain the mercapto-functionalized MXene quantum dots.
3. The SPR sensor chip of claim 2, wherein the mass ratio of MXene quantum dots to thiol compounds is 1: 1-3.
4. The SPR sensor chip according to claim 2, wherein the temperature of said 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.
5. The SPR sensor chip of claim 2, wherein said thiol compound is C15-20 alkyl thiol.
6. The SPR sensor chip of claim 5, wherein said thiol compound is n-octadecyl thiol.
7. The SPR sensor chip according to claim 1 or 2, wherein said mercapto-functionalized MXene quantum dots are mercapto-functionalized Nb2C MXene quantum dots.
8. The SPR sensor chip of claim 1, wherein said aptamer is an aptamer for targeted detection of N-gene of SARS-CoV-2.
9. The SPR sensor chip of claim 8, wherein said aptamer is the N58 aptamer.
10. A method of manufacturing the SPR sensor chip according to any one of claims 1 to 9, 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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