Electrochemical luminescence biosensor based on two-dimensional rubrene nanometer luminescent material and preparation method and application thereof

Abstract

The invention relates to the technical field of electrochemiluminescence and biology, in particular to an electrochemiluminescence biosensor based on a two-dimensional rubrene nano-luminescent material and a preparation method and application of the electrochemiluminescence biosensor. The two-dimensional rubrene nano luminescent material (2D-RNPs) is a high-performance luminescent nano material obtained by adopting lamellar graphdiyne (GDY) as a carrier, anchoring a luminescent substance rubrene on the surface of GDY through in-situ assembly and further loading PtNi nano particles. The electrochemical luminescence biosensor for detecting microRNA is constructed by using 2D-RNPs as an electrode modification material and combining a CRISPR / Cas12a system, and the sensor has high sensitivity, high specificity and high stability. The method can be applied to detection of miRNA in serum, cells and exosomes, has the characteristics of low cost, high sensitivity, rapidness and convenience, and provides a new analysis technology for early prevention and precise diagnosis and treatment of clinical diseases.

Description

Technical Field

[0001] The present invention belongs to the technical fields of electrochemiluminescence and biotechnology, and particularly relates to an electrochemiluminescent biosensor based on a two-dimensional rubrene nano-luminescent material, a preparation method thereof, and an application thereof. Background Art

[0002] Breast cancer (BC) is a malignant tumor formed by the uncontrolled proliferation and malignant transformation of epithelial cells in breast tissue. Among female malignant tumors, its incidence and mortality rates both rank first and show a continuous upward trend. Precise diagnosis and timely treatment of breast cancer in the early stage can not only effectively prevent the metastasis of cancer cells to important organs, but also significantly improve the treatment success rate and patient survival rate, and reduce the recurrence rate. Therefore, developing an economical, sensitive, and rapid detection method has important clinical significance for breast cancer diagnosis. Currently, biomedical imaging combined with tissue biopsy is still the mainstream method for breast cancer detection, but it can only identify breast cancer with obvious lesions. In recent years, liquid biopsy technology based on microRNA (miRNA) has shown important value in breast cancer diagnosis, treatment, and prognosis monitoring. The latest research shows that the expression level of miRNA-155 in breast cancer patients is significantly higher than that in healthy people, and it is expected to become a new biomarker for breast cancer diagnosis. However, due to the short sequence, high homology, and low abundance of miRNA, the accurate quantitative detection thereof still faces technical challenges.

[0003] Currently, there are various detection methods for the miRNA field, such as RT-qPCR, fluorescence sensors, colorimetry, etc. As the "gold standard" for miRNA detection, RT-qPCR requires complicated primer design and sample processing procedures, and has strict requirements for the detection environment and the professionalism of operators, resulting in limited promotion. Although the fluorescence sensing platform is relatively convenient to operate, it faces expensive fluorescence labeling costs and low sensitivity, and it is difficult to achieve accurate detection of low-level miRNA. Therefore, there is an urgent need to develop a miRNA detection method with simple operation, high sensitivity, strong specificity, and low cost.

[0004] Electrochemiluminescence (ECL) is an analytical technique that combines the high controllability of electrochemistry and the high signal-to-noise ratio of chemiluminescence. With significant advantages such as simple operation, low cost, low background noise, high sensitivity, wide dynamic range, and fast response speed, it has attracted much attention in the fields of bioanalysis, environmental monitoring, drug analysis, etc. Especially in the detection of low-abundance biomarkers, it has attracted extensive research interest. However, traditional ECL luminophores such as Ru(bpy)3 2+ , luminol, and cadmium-based quantum dots have obvious limitations: Ru(bpy)3 2+Expensive; the ECL signal of luminol is weak in neutral aqueous medium; Cd-containing quantum dots are highly toxic and have poor biocompatibility. Therefore, the development of new nano-materials that are economical, environmentally friendly, and have high ECL performance has become an urgent need in this field. Summary of the Invention

[0005] Based on the above background, the present invention discloses a highly sensitive and highly specific electrochemiluminescence (ECL) biosensor based on two-dimensional rubrene nanocomposite luminescent materials (2D-RNPs) and its construction method. By integrating the CRISPR / Cas12a signal recognition and amplification system with high-performance luminescent nanocomposites, high-precision and high-sensitivity detection of miRNA is achieved. 2D-RNPs (Rub@GDY-PtNi) are synthesized by using lamellar graphdiyne (GDY) as the substrate material, in-situ assembling and anchoring the luminescent substance rubrene (Rub) on the surface of GDY, and further loading PtNi nanoparticles. GDY and PtNi can promote the co-reaction to generate more co-reactant dissolved oxygen (O2) in the ECL system, optimize the reaction path and increase electron transfer to enhance the luminescence efficiency of Rub. Dopamine (DA) can quench the ECL signal of Rub, keeping the ECL signal of Rub@GDY-PtNi at a low level ("on-off"). When the target miRNA is present, it triggers the non-specific trans-cleavage activity of the CRIPSR / Cas12a system. On the one hand, it destroys the quenching effect of DA on the ECL signal, and on the other hand, it initiates a cascade cleavage reaction, causing the ECL signal to be exponentially amplified and restored, and realizing the quantitative analysis of miRNA through the signal "off-on" switch.

[0006] Compared with the existing methods for detecting miRNA, the advantages of the present invention are as follows:

[0007] On the one hand, the Rub@GDY-PtNi nanocomposite material applied in the present invention is a high-performance luminescent material, which significantly improves the luminescence efficiency of Rub by promoting the co-reaction, optimizing the reaction path and enhancing the electron transfer mechanism. These synergistic effects not only improve the reaction efficiency, reduce energy loss, but also enhance the electron transfer efficiency in the material, thus greatly enhancing the luminescence performance of the material. Therefore, compared with other luminescent materials, the Rub@GDY-PtNi nanocomposite material can ensure high detection performance for the target and exhibit excellent sensitivity.

[0008] On the other hand, the present invention realizes a significant leap in specificity by introducing the CRISPR / Cas12a system, successfully overcoming the highly false positive results caused by the single nucleic acid hybridization mechanism in traditional methods. Brief Description of the Drawings

[0009] Figure 1 Schematic diagram of an electrochemiluminescence biosensor based on 2D-RNPs, its preparation method and application in miR-155 detection

[0010] Figure 2 Morphology characterization diagram of 2D-RNPs by low-voltage transmission electron microscopy (TEM)

[0011] Figure 3 Optimization diagram of miR-155 detection by an electrochemiluminescence biosensor based on 2D-RNPs

[0012] Figure 4 Sensitivity performance evaluation diagram of miR-155 detection by an electrochemiluminescence biosensor based on 2D-RNPs

[0013] Figure 5 Specificity performance evaluation diagram of miR-155 detection by an electrochemiluminescence biosensor based on 2D-RNPs

[0014] Figure 6 Stability performance evaluation diagram of miR-155 detection by an electrochemiluminescence biosensor based on 2D-RNPs Detailed implementation manners

[0015] Materials and reagents

[0016] Table 1: Chinese-English comparison table involved in this application

[0017]

[0018]

[0019] Platinum acetylacetonate (Pt(acac)2) and nickel acetylacetonate (Ni(acac)2) were purchased from Titan Co., Ltd. (Shanghai, China), rubrene (Rub) was purchased from J&K Scientific Ltd. (Beijing, China), mono-nitrogen-doped graphdiyne (GDY) was purchased from Xianfeng Nano Co., Ltd. (Jiangsu, China), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), dopamine (DA), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich Chemical Company (St. Louis, Missouri, USA), EnGen LbaCas12a (Cpf1), 10×NEBuffer 3.0 were purchased from New England Biolabs, Inc. (Ipswich, Massachusetts, USA), HPLC-purified crRNA, recombinant RNase inhibitor, HPLC-purified oligonucleotide chains, and enzyme-free water were all purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The nucleic acid sequences are shown in Table 2.

[0020] Table 2: Nucleic acid sequences used in the present invention

[0021]

[0022]

[0023] Instruments and Equipment

[0024] ECL measurements were monitored and recorded on an MPI-E electrochemiluminescence platform (Xi'an Ruimai Analytical Instrument Co., Ltd., China). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed on a CHI660D electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). The electrochemiluminescence detection platform employed a traditional three-electrode system, with a platinum wire auxiliary electrode, an Ag / AgCl reference electrode, and a glassy carbon electrode (GCE, Φ = 4 mm) working electrode. Morphological characterization was performed using a lanthanum hexaboride transmission electron microscope (Lambda, USA).

[0025] Case Study 1: Preparation of 2D-RNPs-based Electrochemiluminescence Biosensor and Feasibility Assessment for MiRNA Detection

[0026] 1.1 Synthesis of PtNi and Rub@GDY-PtNi

[0027] like Figure 1 Octahedral PtNi nanoparticles were synthesized using a solvothermal method. The steps are as follows: 2 mM Pt(acac)2 and 6 mM Ni(acac)2 were weighed and added to 60 mL of N,N-dimethylformamide (DMF). The mixture was stirred thoroughly to form a homogeneous green solution. The green solution was transferred to a glass-lined stainless steel autoclave. The sealed autoclave was heated from room temperature to 120°C at a rate of approximately 10°C / min, followed by a constant temperature reaction for 42 hours. After the reaction was completed and naturally cooled to room temperature, the product was washed multiple times with 50% ethanol diluted in deionized water and then redispersed in deionized water. The synthesis steps of Rub@GDY-PtNi are as follows: 2.5 mg of rubrene powder was dissolved in 2 mL of nitrogen-doped graphyne dispersion (0.1 mg / mL), shaken to mix thoroughly, and 0.5 mL of tetrahydrofuran (THF) was added. The mixture was stirred at room temperature for 1 hour. Sodium dodecyl sulfate (SDS, 5 × 10 - 3M), stirred continuously for 30 min, and then added 2 mL of PtNi nanoparticles dispersed in deionized water. Stir for 1 h. After washing with deionized water several times, disperse in 2 mL of water and store at 4°C for subsequent use.

[0028] 1.2 Preparation of quenching probe S3-DA

[0029] 19.17 mg of EDC and 11.51 mg of NHS were completely dissolved in 1 mL of MES buffer (0.1 M, pH = 6.0) respectively to prepare two solutions with a final concentration of 100 mM. Next, 40 μL of EDC (100 mM), 10 μL of NHS (100 mM), and 10 μL of carboxyl-modified DNA strand (S3, 2.5 μM) were added to 140 μL of PBS (0.1 M, pH = 7.4), and stirred at 4 °C for 2 hours to activate the carboxyl groups on the S3 single strand. Then, the above mixed solution was continuously shaken and stirred overnight in a 4 °C refrigerator with 200 μL of DA solution (10 mM) to successfully synthesize S3-DA.

[0030] Among them, the nucleotide sequence S2 is shown in SEQ ID NO.4 respectively, and S3 is shown in SEQ ID NO.5.

[0031] 1.3 Construction of an electrochemiluminescence biosensor based on 2D-RNPs

[0032] The GE was polished with 0.05 μM alumina for 5 min, sonicated with deionized water, and repeated three times to obtain clean GE, which was dried with nitrogen and reserved. As Figure 1 Shown in B, 10 μL of the Rub@GDY-PtNi solution diluted 10 times with the original solution was dropped on the electrode and dried into a film at 37 °C. 10 μL of S2-NH2 (1 μM) was dropped on the film surface, capped and incubated at 4 °C for 8 h. After gently rinsing with PBS, 10 μL of BSA (1%) was used to react at room temperature for 1 h to block the non-specific sites on the GE surface. Then 10 μL of S3-DA (2.5 μM) was dropped and reacted at 37 °C for 1 h. The quenching of the luminescent substrate by DA kept the ECL signal at a low level ("on-off" switching). The prepared electrochemiluminescence sensor was gently rinsed with PBS and stored at 4 °C for later use.

[0033] 1.4 Feasibility verification of detecting miRNA with an electrochemiluminescence biosensor based on 2D-RNPs

[0034] The CRISPR / Cas12a system was constructed by a one-pot method, which is simple to operate and has low contamination. As Figure 1As shown in C. When a certain concentration of miR-155 is introduced, it binds to the toehold region (lower part of the neck) of the hairpin probe (Hp) through base complementary pairing, triggering the unwinding of the Hp stem-loop structure and releasing the single-stranded DNA in the upper part of the neck. This single-stranded DNA and the DNA activation strand (S1) jointly bind to crRNA to activate the CRISPR / Cas12a system. The activated Cas12a, through non-specific trans-cleavage activity, on the one hand cleaves the single-stranded part of dsDNA-DA to disrupt the quenching effect of DA on the luminescent substrate, and on the other hand cleaves the loop of Hp, resulting in the self-disassembly of the hairpin. The released target can participate in the recognition process again, triggering a cascade cleavage reaction to exponentially amplify and restore the ECL signal ("off-on" switching). Finally, the high-sensitivity detection of miR-155 is achieved through the change in ECL signal intensity.

[0035] Furthermore, in the CRISPR / Cas12a system described in Experimental Example 1.4, the sequences of crRNA, Hp, S1, and miR-155 are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.6.

[0036] Furthermore, in Example 1.4, the usage concentration of Hp is 1 nM, the usage concentration of S1 is 3 nM, and the usage concentrations of crRNA and Cas12a are both 50 nM. The reaction conditions are 37 °C for 30 min.

[0037] Furthermore, the ECL signal detection is carried out in a working solution of PBS (0.1 M, pH = 7.4). Parameter settings: Cyclic voltammetry, scanning at a potential of -1.2 - 1.2 V, and the photomultiplier tube is 600 V.

[0038] Example 2: Morphological characterization of the composite substrate by low-voltage transmission electron microscopy (TEM)

[0039] To verify whether the luminescent substrate described in Example 1 was successfully synthesized, low-voltage transmission electron microscopy (TEM) was used to take the morphology of the composite substrate. As Figure 2 shown, GDY (left) presents an ideal lamellar structure with rough edges. After in-situ growth of Rub, the edges and surface transform into a smooth hierarchical lamellar structure with uniform interlayer spacing and clear interfaces. It shows that Rub has successfully grown on the surface of GDY, forming a Rub@GDY composite material (middle). After further mixing and stirring with pre-synthesized PtNi nanoparticles for several hours, particle adsorption can be clearly seen on the lamellar surface of Rub@GDY-PtNi (right), confirming that PtNi has been successfully loaded on the surface of the composite substrate through physical adsorption. The results of this series of morphological observations provide intuitive microscopic structural evidence for the layer-by-layer synthesis mechanism of the luminescent substrate.

[0040] Case 3: Optimization of miR-155 Detection Using a 2D-RNP-Based Electrochemiluminescence Biosensor

[0041] In this experiment, gradient experiments were carried out on four key parameters: the concentration of the quenching probe, the reaction time between S2 and S3, the cleavage time of the CRISPR / Cas12a system, and the concentration of Cas12a. Using 1 nM miR-155 as the standard detection concentration, the effects of various factors on the performance of the sensor were systematically explored. Using the detection of 1 nM miR-155 as the standard concentration, optimization was carried out. As Figure 3 shown in a, when the concentration of DA was 10 mM, the ECL signal of the substrate material had decreased to the plateau phase, indicating that the optimal concentration of the quenching probe used in this sensor was 10 mM. As Figure 3 shown in b, taking 60 min as the node, the ECL signal had decreased to the ideal background signal value, indicating that the hybridization time of the quenching probe S3-DA with S2 could effectively reduce the background signal at 60 min. As Figure 3 shown in c, taking the addition of CRISPR / Cas12a containing 1 nM miR-155 to the electrode as the node, the cleavage time was optimized. The results showed that the signal recovered to the plateau phase after 30 min. As Figure 3 shown in d, controlling the above conditions, the concentration of Cas12a was optimized. The results showed that Cas12a with a concentration of 60 nM restored the ECL signal to the plateau phase.

[0042] Case 4: Sensitivity Performance Evaluation of Exogenous miR-155 Detection Using a 2D-RNP-Based Electrochemiluminescence Biosensor

[0043] To explore the detection sensitivity performance of this electrochemiluminescence biosensor, seven groups of miR-155 with different concentrations (1×10 -4 、1×10 -3 、1×10 -2 、1×10 -1 、1、1×10 1 、1×10 2 、1×10 3 pM) were set as the experimental groups, and the group without adding miR-155 was set as the control group. After the CRISPR / Cas12a system containing miR-155 was incubated at 37 °C for 30 min, as Figure 4 shown, the average ECL signals of the three experiments in the experimental groups showed different degrees of increase. There was a linear relationship between the miR-155 concentration and the ECL signal intensity. The formula obtained by linear fitting was: y = 1139.388lgC + 6057.627, R 2= 0.991. After calculation, the detection limit reached 36.6 aM. The results showed that this electrochemiluminescence biosensor had good sensitivity performance for the detection of miRNA.

[0044] Experimental Case 5: Specificity Performance Evaluation of an Electrochemiluminescence Biosensor Based on 2D-RNPs for the Detection of miR-155

[0045] To avoid the influence of other homologous miRNAs on the sensor detection in a complex environment, different miRNAs were used to evaluate the specificity performance of this electrochemiluminescence biosensor. The ECL signals of 1 nM miR-155 and 10 nM let-7a, miR-122, miR-378, miR-429, and a mixture of the above miRNAs were compared under the same conditions. As Figure 5 shown, only when miR-155 was present could the sensor have a significant ECL signal. This indicated that the sensor had good specificity for miRNA detection.

[0046] The nucleic acid sequences of the let-7a, miR-122, miR-378, and miR-429 are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0047] Experimental Case 6: Stability Performance Evaluation of an Electrochemiluminescence Biosensor Based on 2D-RNPs for the Detection of miR-155

[0048] To comprehensively explore the detection stability of this sensor, different tests were carried out using 10 pM and 1 pM miR-155, respectively. The within-batch and between-batch differences were determined with 10 pM miR-155 to evaluate the reproducibility of the sensor; the same electrode was continuously measured with 1 pM miR-155 to investigate its stability. As Figure 6 shown in a, the relative standard deviation (RSD) within the batch was 1.93%, and the between-batch RSD was 1.67%. The lower RSD values indicated that the sensor had a small degree of dispersion in the detection results within different batches and within the same batch, showing good reproducibility. As Figure 6 shown in b, when the same electrode was continuously measured with 1 pM miR-155, the RSD of the ECL signal value was 1.27%, indicating that the sensor had small signal fluctuations during continuous detection and good stability.

Claims

1. An electrochemiluminescence biosensor based on two-dimensional rubrene nano-luminescent material, its preparation method and application, characterized in that, Including the following steps: (1) Prepare the luminescent substrate Rub@GDY-PtNi a. Synthesize uniform octahedral PtNi nanoalloy particles using the solvothermal method; b. Using the lamellar GDY as a carrier, anchor Rub on GDY through in-situ assembly, then add the synthesized PtNi nanoalloy particles to successfully adsorb PtNi on the surface of Rub@GDY. After washing, resuspend in deionized water to obtain the luminescent substrate Rub@GDY-PtNi; (2) Prepare the quenching probe S3-DA Use EDC and NHS to stir at 4°C for 2 h to activate the carboxyl group on S3, add DA and stir overnight at 4°C to successfully form an amide bond between the carboxyl group and the amino group on DA, and successfully prepare the quenching probe S3-DA; (3) Construct an electrochemiluminescence sensor Drop the Rub@GDY-PtNi solution described in S1 onto the purified glassy carbon electrode GE for incubation and drying, and bind it with the DNA strand (S2) modified with amino groups and the quenching probe S3-DA; note that the non-specific binding sites on the luminescent substrate need to be blocked with BSA buffer before this step; successfully construct a low-background electrochemiluminescence sensor; Among them, the nucleotide sequences of S2 are shown as SEQ ID NO.4 respectively, and S3 is shown as SEQ ID NO.

5.

2. The construction method of an electrochemiluminescence sensor according to claim 1, characterized in that: The concentration of S3 in the S3-DA is 2.5 μM, and the concentration of DA is 10 mM.

3. The construction method of an electrochemiluminescence sensor according to claim 1, characterized in that: The concentration of the DNA strand (S2) is 1 μM, the reaction time is 10 h, and the reaction time of S2 and S3-DA is 1 h.

4. An electrochemiluminescence sensor, characterized in that, The electrochemiluminescence sensor is constructed by the method described in claim 1 or 2.

5. A material for improving the detection sensitivity of an electrochemical sensor, characterized in that, The material includes the luminescent substrate Rub@GDY-PtNi described in claim 1 or / and the quenching probe S3-DA described in claim 1.

6. The electrochemiluminescence sensor according to claim 4 integrates the CRISPR / Cas12a signal recognition and amplification system for application to miRNA.

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