A new anti-fouling material OPA-CsPbBr3 and its preparation method and sensing application
By doping the highly hydrophilic polymer OPA into the perovskite CsPbBr3 to form OPA-CsPbBr3, and combining it with nanozyme cascade catalysis and CRISPR/Cas13a system, the problem of nonspecific protein adsorption of biosensors in complex environments was solved, and highly sensitive and stable miRNA-let-7a detection was achieved.
Patent Information
- Application Number
- CN202411114939.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-14
- Publication Date
- 2025-10-03
- Estimated Expiration
- 2044-08-14
AI Technical Summary
Existing biosensors suffer from decreased detection accuracy and sensitivity due to nonspecific protein adsorption in complex biological samples, lack anti-fouling properties and have poor conductivity, which limits their application in complex environments.
Polyacrylic acid (OPA) modified with highly hydrophilic polymer octylamine was doped into perovskite CsPbBr3 to form a new anti-fouling material OPA-CsPbBr3, which was then combined with nanozyme cascade catalysis and CRISPR/Cas13a system to construct an electrochemical biosensor.
Highly sensitive and stable miRNA-let-7a detection was achieved in complex biological samples, nonspecific protein adsorption was reduced, and the anti-fouling performance and conductivity of the sensor were improved, with a detection limit as low as 0.34 fmol/L.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of electrochemical detection technology, and specifically relates to a novel anti-fouling material OPA-CsPbBr3, a preparation method thereof, and sensing applications. Background Art
[0002] MicroRNA (miRNA) has been implicated in the development and progression of numerous diseases, including cancer. Therefore, quantitative detection of miRNA biomarkers is crucial for the prevention and treatment of early-stage cancer patients. Over the past few years, various approaches have been developed for cancer detection, with biosensors garnering attention due to their high selectivity, excellent stability, and rapid response. However, biofouling caused by nonspecific protein adsorption is a significant issue, impacting the accuracy and sensitivity of biosensors in complex biological samples (such as blood and serum), limiting their practical application potential in complex environments. Therefore, developing sensing interfaces that resist biofouling and reduce nonspecific protein adsorption is crucial for constructing sensitive and stable electrochemical biosensors.
[0003] The adsorption of nonspecific proteins on the sensing interface is mainly caused by two forces: hydrophobic interaction and electrostatic attraction. Therefore, constructing an anti-fouling interface with good electrical neutrality and hydrophilicity can easily reduce the electrostatic attraction between the sensing interface and the pollutants, forming a hydration layer coating the interface, which can effectively prevent the adhesion of nonspecific proteins. Anti-fouling materials such as polyethylene glycol, zwitterionic polymers, and polypeptides have good hydrophilicity and electrical neutrality, making them ideal interfacial anti-fouling materials. However, their easy oxidation, difficulty in synthesis, instability, and poor conductivity limit their application. Moreover, poor conductivity will inevitably hinder the transfer of electrons on the electrode surface, affecting the sensitivity of the biosensor. Therefore, the development of materials with anti-fouling properties and excellent conductivity is of great significance for the construction of high-performance electrochemical sensing interfaces. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a new anti-fouling material OPA-CsPbBr3 and its preparation method and sensing application to improve the above problems.
[0005] In order to achieve the above object, the present invention provides the following technical solutions:
[0006] A new antifouling material OPA-CsPbBr3 is composed of polyacrylic acid OPA modified with highly hydrophilic polymer octylamine and perovskite CsPbBr3.
[0007] The present invention also provides a method for preparing the novel antifouling material OPA-CsPbBr3 as described above, the method comprising the following steps:
[0008] S101, adding CsBr and PbBr2 into DMF and stirring until the solids are completely dissolved to obtain a perovskite precursor;
[0009] S102, adding OAm and the synthesized amphiphilic OPA polymer to the perovskite precursor, stirring at room temperature for 5 minutes to obtain a mixture, pouring the mixture into toluene and stirring for 10 minutes to obtain a reaction product;
[0010] S103, the reaction product is subjected to high-speed centrifugation to obtain a yellow solid, the yellow solid is washed with toluene, and then dried in a vacuum for 2 hours to obtain a new antifouling material OPA-CsPbBr3.
[0011] Preferably, in step S101, the concentrations of CsBr and PbBr2 are both 0.4 mM, and the volume of DMF is 10 mL.
[0012] Preferably, in step S102 , the volume of the perovskite precursor solution is 1 mL; the volume of OAm is 50 μL; the mass of the amphiphilic OPA polymer is 2 mg; and the volume of toluene is 20 mL.
[0013] The novel antifouling material OPA-CsPbBr3 disclosed in the present invention is mainly formed by doping OPA polymer into CsPbBr3 to obtain an OPA-CsPbBr3 material with excellent conductivity, high hydrophilicity and near-neutral charge. It can also further improve the water stability of CsPbBr3, thus becoming an effective antifouling coating.
[0014] The embodiment of the present invention further provides an application of the novel anti-fouling material OPA-CsPbBr3 as described above in an electrochemical biosensor.
[0015] Preferably, the preparation method of the electrochemical biosensor comprises the following steps:
[0016] S201, dsDNA generation by strand displacement amplification reaction;
[0017] S202, polishing the glassy carbon electrode (GCE) with polishing powder, and then ultrasonically cleaning the surface of the electrode several times with water and ethanol;
[0018] S203, dropping the OPA-CsPbBr3 solution on the cleaned glassy carbon electrode GCE to obtain a uniform nanofilm, and then immobilizing the PNA chain on the nanofilm on the surface of the glassy carbon electrode GCE through an amidation reaction between the -NH2 of PNA and the -COOH of OPA-CsPbBr3;
[0019] S204, Cas13a / crRNA was added to the surface of the glassy carbon electrode immobilized with PNA chains. After reacting for 40 minutes, dsDNA was dropped onto the reaction surface of the glassy carbon electrode and incubated overnight at 4°C to trigger the trans-cleavage function of CRISPR / Cas13a. Then, 10 μL of Ru@PSS NPs / H2 / Thi was applied to the above electrode and reacted for 1 hour to construct an electrochemical biosensor.
[0020] Preferably, in step S201, a strand displacement amplification reaction is prepared and carried out in 10 μL of a system solution containing 1× Klenow fragment (3'→5' outer chain) polymerase KFP reaction buffer, 5 μL of miRNA-let-7a at different concentrations, 1 μL of 1 mM deoxynucleotide triphosphate dNTP, 1 μL of KFP (4 U) and 3 μL of hDNA (5 nM), and the reaction conditions are incubation at 37°C for 1 h.
[0021] The embodiments of the present invention also provide the use of the electrochemical biosensor described above in the detection of miRNA-let-7a.
[0022] The electrochemical biosensor disclosed in the present invention is based on the anti-fouling material OPA-CsPbBr3, integrates nanozyme cascade catalysis and CRISPR / Cas13a system, and constructs an electrochemical biosensor for highly sensitive detection of miRNA-let-7a. By doping OPA into CsPbBr3, a new anti-fouling material OPA-CsPbBr3 with excellent conductivity, high hydrophilicity and near-neutral charge is synthesized for the immobilization of the sensing probe PNA. In the presence of miRNA-let-7a, it triggers the SDA reaction with the help of dNTP and KFP to generate a large amount of dsDNA. The obtained dsDNA can activate the trans-cleavage function of CRISPR / Cas13a, resulting in the cleavage of the "rU / / rU" site of H2 in Ru@PSS NPs / H2 / Thi. PNA can recognize the sheared Ru@PSS NPs / H2 / Thi through base complementary pairing and obtain an electrochemical signal related to miRNA-let-7a. More importantly, the synthesized OPA-CsPbBr3 exhibits glucose oxidase-like activity and is coupled with Ru@PSS NPs (peroxidase-like activity) to achieve nanozyme cascade signal amplification. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:
[0024] Figure 1 : Schematic diagram of the electrochemical biosensor based on nanozyme cascade catalysis; among them, (A) is the preparation process of the anti-fouling material OPA-CsPbBr3; (B) is the synthesis of Ru@PSS NPs / H2 / Thi and the target-induced SDA reaction; (C) is the construction process of the anti-fouling electrochemical biosensor.
[0025] Figure 2 : TEM images (AB), EDS images (C), XRD spectrum (D) and FT-IR images (E) of the anti-fouling material OPA-CsPbBr3 prepared in Example 1.
[0026] Figure 3 : CV characterization of the step-by-step construction process of the electrochemical biosensor; among them, (a) GCE, (b) OPA-CsPbBr3 / GCE, (c) SDA / Cas13a / crRNA / OPA-CsPbBr3 / GCE, and (d) Ru@PSS / Thi / H2 / SDA / Cas13a / crRNA / OPA-CsPbBr3 / GCE.
[0027] Figure 4 :(A) Schematic diagram of nanozyme cascade reaction based on colorimetry;(B) UV-visible absorption spectra of different reaction systems: TMB + Glu, TMB + Glu + Ru@PSS NPs, TMB + Ru@PSS NPs + OPA-CsPbBr3, TMB+ Glu + Ru@PSS NPs + OPA-CsPbBr3.
[0028] Figure 5 : (A) and (B) are the contact angle characterizations of GCE and OPA-CsPbBr3 / GCE, respectively; (C) is the Zeta potential characterization of OPA-CsPbBr3.
[0029] Figure 6 : DPV signal responses of (A) GCE and (B) GCE / OPA-CsPbBr3 incubated with different concentrations of human serum.
[0030] Figure 7:(A) DPV signals of different concentrations of miRNA-let-7a: blank, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM); (B) Calibration curve for measuring the difference in DPV response to the logarithm of miRNA-let-7a concentration in 0.1 M PBS solution (blue) and 100% human serum (red); (C) and (D) are specificity and reproducibility analyses of the anti-fouling electrochemical biosensor. DETAILED DESCRIPTION
[0031] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0032] The raw materials used in the following examples were obtained from the following sources: cesium bromide (CsBr), lead bromide (PbBr2), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), polyacrylic acid (PAA), hydrogen peroxide (H2O2), 3,3′,5,5′-tetramethylbenzidine (TMB), and glucose were purchased from Aladdin (Shanghai, China). Bovine serum albumin (BSA) was obtained from Beijing Nuochuang Chemical Technology Co., Ltd. Ethyl acetate (EA) and N,N-dimethylformamide (DMF) were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. Cis-1-amino-9-octadecene (OAm), tetramethylammonium hydroxide (TMAH), and horseradish peroxidase (HRP) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Ruthenium chloride hydrate (RuCl3·nH2O) was purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Polystyrene sulfonate (PSS) was purchased from Beijing Chuangke Technology Co., Ltd. Thionine (Thi) was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Engen Lba Cas 13a and NEBuffer r2.1 were purchased from New England Biolabs. Klenow fragment (3'→5' extension) polymerase (KFP) was purchased from Shanghai Biotech Biotechnology Co., Ltd. Deoxynucleotide triphosphates (dNTPs), all DNA sequences, and miRNAs were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) as shown in the following table.
[0033]
[0034] All electrochemical measurements in the following examples, including cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were performed on a CHI 760E electrochemical workstation (Shanghai Chenhua) using a conventional three-electrode system. UV-visible absorption spectra were measured using a Lambda 1050+2D UV-visible spectrophotometer (Shanghai, China). Transmission electron microscopy (TEM, Tecnai G2 F30, USA) and elemental mapping (Tecnai G2 F30, USA) were used to characterize the morphology, size, and elemental distribution of the nanomaterials. X-ray diffraction (XRD) analysis of the various samples was performed using a Japanese DMAX-2400 X-ray diffractometer. Fourier transform infrared spectroscopy (FTIR) was performed using a Nicolet iS10, USA.
[0035] Example 1
[0036] Reference Figure 1 The preparation steps of the antifouling material OPA-CsPbBr3 are as follows:
[0037] (1) CsBr (0.4 mM) and PbBr2 (0.4 mM) were added to DMF (10 mL) and stirred until the solids were completely dissolved to obtain the perovskite precursor.
[0038] (2) Add 50 μL of OAm and 2 mg of amphiphilic OPA polymer to the above precursor solution (1 mL), stir at room temperature for 5 min to obtain a mixture, and then add the mixture to toluene (20 mL) and stir for 10 min to obtain the reaction product.
[0039] (3) The reaction product was subjected to high-speed centrifugation to obtain a yellow solid, which was washed with toluene and dried in a vacuum for 2 h to obtain the anti-fouling material OPA-CsPbBr3.
[0040] The properties of the anti-fouling material OPA-CsPbBr3 are analyzed below.
[0041] First, the morphology of the prepared antifouling material OPA-CsPbBr3 was characterized by transmission electron microscopy (TEM), and it was found that it was in the shape of regular nanocubes ( Figure 2 AB).
[0042] Then, an X-ray diffractometer was used to perform X-ray diffraction analysis on the anti-fouling material OPA-CsPbBr3. The X-ray energy spectrum (EDS) element map showed that the Cs, Pb and Br elements in OPA-CsPbBr3 were evenly distributed ( Figure 2C). X-ray diffraction (XRD) analysis showed that the diffraction peaks at 15.2°, 21.49° and 30.37° correspond to the (100), (110) and (200) planes of cubic phase CsPbBr3 (JCPDS 54-0752), respectively. Figure 2 D).
[0043] Then, the antifouling material OPA-CsPbBr3 was analyzed using Fourier transform infrared spectrometer, and Fourier transform infrared spectroscopy (FT-IR) further characterized the surface functional groups of OPA-CsPbBr3. Figure 2 As shown in E, compared with CsPbBr3, 1654 cm -1 The strong peak at 1546 cm corresponds to the carbonyl stretching vibration peak (C=O) of the ligand OPA in OPA-CsPbBr3. -1 The strong peak at octylamine is the amide bond formed by the interaction between the amine end of polyacrylic acid and the carboxylic acid end of polyacrylic acid, which proves that the OPA ligand successfully binds to the CsPbBr3 surface.
[0044] Example 2
[0045] Reference Figure 1 The antifouling material OPA-CsPbBr3 prepared in Example 1 was used to construct an electrochemical biosensor. The specific construction method is as follows:
[0046] (1) Preparation of dsDNA by SDA reaction: The SDA reaction was performed in 10 μL of a system solution containing 1× Klenow fragment (3'→5' outer chain) polymerase (KFP) reaction buffer, 5 μL of miRNA-let-7a at different concentrations, 1 μL of deoxynucleotide triphosphate (1 mM, dNTP), 1 μL of KFP (4 U), and 3 μL of hDNA (5 nM). The dsDNA was obtained by incubation at 37°C for 1 h.
[0047] (2) Polish the glassy carbon electrode (GCE) with polishing powder, and then clean its surface by ultrasonic treatment with water and ethanol several times.
[0048] (3) 5 μL of OPA-CsPbBr3 solution (10 mg / mL) was dropped onto the cleaned GCE to obtain a uniform nanofilm. PNA chains (5 μL, 1 μM) were immobilized on the nanofilm on the surface of the glassy carbon electrode through the amidation reaction between the -NH2 of PNA and the -COOH of OPA-CsPbBr3.
[0049] (4) 10 μL of Cas13a / crRNA was added to the surface of the modified glassy carbon electrode and reacted for 40 min. 10 μL of the product of the SDA reaction (dsDNA) was dropped onto the reaction surface of the glassy carbon electrode and incubated overnight at 4 °C to trigger the trans-cleavage function of CRISPR / Cas13a. Then, 10 μL of Ru@PSS NPs / H2 / Thi was applied to the above glassy carbon electrode and reacted for 1 h to construct an electrochemical biosensor.
[0050] The following will study some characteristics of the prepared antifouling material OPA-CsPbBr3 and electrochemical biosensor:
[0051] Cyclic voltammetry characterization:
[0052] The measurement of CV signals can be used to characterize the construction process of antifouling electrochemical biosensors. Figure 3 As shown, the peak current of the GCE modified with OPA-CsPbBr3 (curve b) is significantly higher than that of the bare GCE (curve a), demonstrating the excellent electrical conductivity of OPA-CsPbBr3. When the SDA product (dsDNA) and Cas13a / crRNA are sequentially modified with the glassy carbon electrode, the peak current decreases significantly due to the large steric hindrance that hinders electron transfer (curve c). When Ru@PSS NPs / H2 / Thi are added to the glassy carbon electrode, the current increases significantly due to the excellent conductivity of Ru@PSS and the electrochemical activity of Thi (curve d). These results demonstrate the effectiveness and feasibility of the electrochemical biosensor proposed in this example during construction.
[0053] Catalytic activity studies based on colorimetry:
[0054] The feasibility of constructing a nanoenzyme cascade system by using OPA-CsPbBr3 (glucose oxidase-like activity) and Ru@PSS NPs (peroxidase-like activity) was investigated using a colorimetric method. OPA-CsPbBr3 catalyzes glucose to produce gluconic acid and H2O2. The generated H2O2 serves as the substrate for the second enzymatic reaction and is catalyzed by Ru@PSS NPs for the oxidation of the substrate TMB, ultimately generating the blue product oxTMB ( Figure 4 A). Figure 4 As shown in Figure 2, compared with other control groups, only the simultaneous presence of TMB, glucose, OPA-CsPbBr3 and Ru@PSS NPs can oxidize TMB to oxTMB, and the product shows a maximum absorption peak at 652 nm, confirming that the enzyme cascade system was successfully constructed.
[0055] Hydrophilicity and Zeta potential characterization of OPA-CsPbBr3:
[0056] Biofouling and nonspecific adsorption pose challenges to the accurate detection of biomolecules in complex biological samples. However, in this embodiment, the electrode surface is modified with an antifouling material having excellent hydrophilicity and neutral charge, which can effectively prevent the nonspecific adsorption of biomolecules.
[0057] Specifically, this embodiment characterizes the hydrophilicity of different electrode surfaces by measuring the static water contact angle, such as Figure 5 As shown in Figure A, the static water contact angle of the OPA-CsPbBr3 modified interface (OPA-CsPbBr3 / GCE) decreases significantly from 53.133° (bare GCE) to 12.137°. This is due to the presence of numerous hydrophilic groups such as -NH2 and -COOH in OPA. The electroneutrality of OPA-CsPbBr3 is subsequently confirmed by the zeta potential, which is close to 0.0 mV. These results demonstrate the excellent hydrophilicity and electroneutrality of OPA-CsPbBr3.
[0058] Evaluation of antifouling performance of modified electrodes:
[0059] The following will study the antifouling performance of the constructed electrode in different concentrations of human serum. Figure 6 As shown, the DPV signal of the bare GCE decreased significantly after exposure to different concentrations of human serum. This is due to the fact that protein adsorption at the electrode interface hinders the electrochemical reaction of the signal molecules. In contrast, the DPV signal of the electrode modified with OPA-CsPbBr3 decreased only slightly after incubation with human serum samples, indicating that the electrochemical biosensor has good anti-fouling performance in complex biological media.
[0060] Investigation of sensor sensitivity, specificity and reproducibility:
[0061] This example will evaluate the performance of the electrochemical biosensor under the optimal experimental conditions. Figure 7 As shown in A, as the concentration of a series of target miRNA-let-7a increased from 100 fmol / L to 10 nmol / L, the corresponding DPV signal increased. In addition, the logarithm of the miRNA-let-7a concentration (log c miRNA-let-7a ) and current (ΔI) have a significant linear relationship ( Figure 7 B), where the linear equation is expressed as ΔI (μA) = 1.03 lg c miRNA-let-7a +5.81 (R 2 = 0.99), with a detection limit as low as 0.34 fmol / L. Notably, even in 100% human serum, the biosensor still exhibited a good linear response to miRNA-let-7a, close to that in pure PBS.
[0062] In addition to sensitivity, high specificity is also particularly important for evaluating biosensors for miRNA-let-7a detection. Therefore, this example selected a series of miRNAs homologous sequences (miRNA-155, miRNA-21, miRNA-122, and miRNA-141) as interfering substances to study the specificity of the biosensor. Figure 7 As shown in Figure C, the DPV response of interfering miRNAs (100 nmol / L) showed no significant difference compared to the blank sample, while the DPV current of miRNA-let-7a (10 nmol / L) and the mixture (containing miRNA-let-7a and its homologous sequence miRNAs) increased significantly. These results demonstrate that the electrochemical biosensor of this example has remarkable specificity.
[0063] In addition, this example further evaluated the reproducibility of the biosensor by measuring the intra-batch and inter-batch precision. Figure 7 As shown in Figure 4, the intra-assay and inter-assay relative standard deviations (RSDs) were 4.9% and 4.8%, respectively, indicating that the biosensor had good reproducibility.
[0064] In summary, the present invention discloses a novel antifouling material (OPA-CsPbBr3) by incorporating the highly hydrophilic polymer OPA into perovskite. This material not only overcomes the poor stability of perovskite, but also exhibits excellent antifouling properties due to OPA's excellent hydrophilicity and charge neutrality, reducing nonspecific adsorption of proteins in complex biological samples. Furthermore, the present invention discloses an electrochemical biosensor based on the antifouling material OPA-CsPbBr3 combined with nanozyme cascade catalysis and the CRISPR / Cas13a system to achieve sensitive detection of miRNA-let-7a in complex biological samples. This electrochemical biosensor exhibits a linear range of 100 fmol / L to 10 nmol / L, with a detection limit as low as 0.34 fmol / L, and has potential applications in bioanalysis, early diagnosis, and clinical treatment of human diseases.
Claims
1. Application of a novel antifouling material, OPA-CsPbBr3, in an electrochemical biosensor. The novel antifouling material, OPA-CsPbBr3, is composed of polyacrylic acid (OPA) modified with a highly hydrophilic polymer, octylamine, and perovskite CsPbBr3. The preparation method of the electrochemical biosensor comprises the following steps: S201, generating dsDNA by preparing a strand displacement amplification reaction (SDA); S202, polishing the glassy carbon electrode (GCE) with polishing powder, and then ultrasonically cleaning the surface of the electrode several times with water and ethanol; S203, dropping the OPA-CsPbBr3 solution on the cleaned glassy carbon electrode GCE to obtain a uniform nanofilm, and then immobilizing the PNA chain on the nanofilm on the surface of the glassy carbon electrode GCE through an amidation reaction between the -NH2 of PNA and the -COOH of OPA-CsPbBr3; S204, Cas13a / crRNA was added to the surface of the glassy carbon electrode with immobilized PNA chain and reacted for 40 minutes. Then, dsDNA was dropped onto the reaction surface of the glassy carbon electrode and incubated overnight at 4°C to trigger the trans-cleavage function of CRISPR / Cas13a. Then, 10 μL of Ru@PSS NPs / H2 / Thi was applied to the above glassy carbon electrode and reacted for 1 hour to construct an electrochemical biosensor.
2. The use according to claim 1, characterized in that In step S201, a strand displacement amplification reaction was prepared in 10 μL of a system solution containing a polymerase KFP reaction buffer containing a 1× Klenow fragment with a 3'→5' outer chain, 5 μL of miRNA-let-7a at different concentrations, 1 μL of 1 mM deoxynucleotide triphosphate dNTP, 4 U of 1 μL KFP, and 3 μL of 5 nM hDNA. The reaction was incubated at 37°C for 1 h.
3. The use according to claim 1, characterized in that The electrochemical biosensor is applied to the detection of miRNA-let-7a.