Preparation method of aptamer biosensor for rapid detection of dual tumor markers

Abstract

The present invention belongs to the field of clinical diagnostic technology and relates to a method for preparing an aptamer biosensor for rapid detection of dual tumor markers. The present invention prepares a composite nanofiber membrane of polyacrylonitrile and transition metal salt precursors based on a coaxial electrospinning process. The carbon nanofiber structure uniformly loaded with cobalt metal nanoparticles obtained by a thermal reduction method has a large specific surface area and a unique three-dimensional network electron transmission channel, which significantly improves the electron transfer capacity and the loading amount of aptamer molecules. The biosensor based on the core-shell nanofiber network structure exhibits excellent electrocatalytic ability and signal stability. The preparation process of the biosensor is simple and controllable, and it is easy to achieve large-scale and productized production. It can realize the rapid identification and detection of trace miRNA-155 and miRNA-21 nucleic acid molecules in real serum within 2 minutes, and has important application value in disease diagnosis and clinical biomedicine.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical diagnosis and relates to a method for preparing an aptamer biosensor for rapid detection of dual tumor markers. Background Art

[0002] Pancreatic cancer is a malignant tumor that originates in the pancreatic ductal epithelium and acinar cells, accounting for 95% of all pancreatic malignancies and earning it the nickname "King of Cancer." The pancreas is located deep within the abdominal cavity, and even small masses cannot be detected by standard ultrasound scans, requiring only CT scans. Its early symptoms are subtle and lack specificity, making them easily overlooked, leading to a yearly increase in pancreatic cancer mortality. Therefore, early screening and diagnosis of pancreatic cancer are crucial to ensure that patients receive earlier, faster, and more systematic treatment, thereby improving their survival and cure rates. Tumor marker testing is widely used as a routine and accessible technique for early screening and postoperative assessment. miRNA-155 and miRNA-21 are clinically recognized as important markers for the early diagnosis of pancreatic cancer. Simultaneous testing of both can effectively increase the probability of early detection of pancreatic cancer.

[0003] Currently, the more established miRNA detection technologies include Northern blot analysis, microarray analysis, and quantitative real-time PCR (qRT-PCR). However, these technologies are limited by the large instrumentation, high cost, and time required, making them inadequate for in situ, immediate detection. Electrochemical biosensors, due to their high sensitivity, fast response time, low cost, and portability, are gaining increasing attention in cancer biomarker detection. High-performance biosensors based on nanomaterials have been widely used for early diagnosis and progression monitoring of cancer. However, it is worth noting that these reported electrochemical biosensors for miRNA detection generally suffer from the following limitations: 1) Previously reported electrochemical methods rely primarily on enzymatic signal amplification to improve sensitivity, which limits rapid detection and is costly. 2) These electrochemical biosensors are primarily focused on detecting a single tumor biomarker. Since tumor biomarker expression levels can be affected by other factors (such as inflammation and infection), false-positive diagnoses are likely to occur. Therefore, developing sensing nanomaterials with both high catalytic activity and excellent conductivity and designing efficient strategies for detecting multiple cancer markers are crucial steps in constructing aptamer biosensors for the simultaneous detection of two tumor markers to improve early cancer diagnosis. Currently, no effective biosensor for the simultaneous detection of miRNA-155 and miRNA-21 has been reported. Summary of the Invention

[0004] Aiming at the problems existing in traditional pancreatic cancer early detection equipment, the present invention proposes a method for preparing a novel aptamer biosensor for rapid detection of dual tumor markers.

[0005] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0006] A method for preparing an aptamer biosensor for rapid detection of dual tumor markers, the specific steps of which are as follows:

[0007] (1) Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane: The transition metal salt was dissolved in DMF solvent to obtain spinning solution A for preparing the organic-inorganic composite nanofiber membrane, and polyacrylonitrile was dissolved in DMF to obtain spinning solution B. Spinning solutions A and B were degassed by vacuum filtration. The two prepared spinning solutions A and B were respectively loaded into syringes and coaxially electrospun under certain parameters such as spinning voltage, distance from the spinning nozzle to the receiver, spinning solution flow rate, ambient temperature, and ambient humidity. The resulting composite nanofiber membrane was vacuum dried to remove the residual volatile solvent in the membrane.

[0008] (2) Preparation of Co nanoparticles@carbon nanofibers (Co NPs@CNFs): The transition metal salt precursor / polyacrylonitrile composite nanofiber membrane prepared above was placed in a muffle furnace. PAN was first partially oxidized in an oxygen atmosphere to maintain the three-dimensional nanofiber network structure. The material was then transferred to a tube furnace and pyrolyzed at high temperature under an inert gas atmosphere. The resulting powder was washed with deionized water, filtered, and vacuum dried to obtain the Co NPs@CNFs composite material.

[0009] (3) Preparation of dual tumor marker aptamer biosensor: The Co NPs@CNFs composite material was dispersed in deionized water to obtain a dispersion. The dispersion was drop-coated on the surface of a gold electrode and dried in air. The CoNPs@CNFs modified electrode was incubated in a specific single-stranded DNA probe solution, and then the aptamer CApt solution corresponding to miRNA-155 and miRNA-21 was added. The low-temperature reaction allowed the aptamer CApt to specifically bind to the single-stranded DNA probe, and then the solution was washed with deionized water. The prepared aptamer sensor was stored at low temperature for further use.

[0010] Preferably, the transition metal salt in step (1) is any one of cobalt acetylacetonate (Co (acac) 2), cobalt chloride (CoCl 2), cobalt nitrate (Co (NO 3) 2), and cobalt acetate (Co (Ac) 2); the concentration of the transition metal salt in the spinning solution A is 3-5 wt% by mass; the concentration of polyacrylonitrile (PAN) in the spinning solution B is 10-16 wt% by mass; the spinning voltage is 16-22 KV, the distance from the spinning nozzle to the receiver is 20-22 cm, the flow rate of the spinning solution A is 1-2 mL / h, the flow rate of the spinning solution B is 0.8-1.5 mL / h, the ambient temperature is 20-25°C, the ambient humidity is 40-50%, the vacuum degree of vacuum drying is 0.1 Mp, the vacuum drying temperature is 60°C, and the vacuum drying time is 12 h.

[0011] Preferably, in step (2), the temperature of partial oxidation of the composite nanofiber membrane material in the oxygen atmosphere of the muffle furnace is 210-280°C, the time is 3-8 h, the heating rate is 2°C / min, and the cooling rate is 3°C / min.

[0012] Preferably, in step (2), the partially oxidized composite nanofiber membrane material is subjected to high-temperature cracking in an inert gas atmosphere in a tubular furnace, the inert gas atmosphere is nitrogen or argon, the high-temperature cracking temperature is 600-1000°C, the time is 3-5h, the heating rate is 3-5°C / min, the inert gas flow rate is 10-15mL / min, the vacuum drying time is more than 12h, and the vacuum drying temperature is 60°C.

[0013] Preferably, in step (3), the dispersion concentration of the Co NPs@CNFs composite material in deionized water is 2-6 mg / mL; the volume of the Co NPs@CNFs composite material drop-coated on the surface of the gold electrode is 2-10 μL, and the drying time is 12-24 h; the concentration of the specific single-stranded DNA probe solution is 1-10 μM, and the incubation time is 12-24 h; the concentration of the aptamer CApt solution corresponding to miRNA-155 and miRNA-21 is 1-20 μM, the addition amount is 20-80 μL, the specific binding time is 12-24 h, and the temperature is 4°C; the low-temperature storage temperature is 4°C.

[0014] This invention uses a coaxial electrospinning process to prepare a composite nanofiber membrane of polyacrylonitrile and a transition metal salt precursor. To enhance the catalytic activity and conductivity of the sensor-modified material, carbon nanofibers uniformly loaded with cobalt metal nanoparticles (Co NPs@CNFs) are obtained via thermal reduction. This core-shell nanofiber structure possesses a large specific surface area and a unique three-dimensional network of electron transport channels, significantly enhancing electron transfer capacity and aptamer loading. A biosensor based on this core-shell nanofiber network exhibits excellent electrocatalytic performance and signal stability. The fabrication process is simple and controllable, making it easy to scale up and commercialize. The biosensor can rapidly identify and detect trace amounts of miRNA-155 and miRNA-21 in real serum within 2 minutes. It can also analyze other miRNAs, providing a new and efficient analytical method for the rapid and sensitive detection of multiple cancer biomarkers and for early cancer screening and postoperative follow-up in clinical practice. It has important applications in disease diagnosis and clinical biomedicine.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are:

[0016] 1. Based on electrochemical biosensing technology, this invention prepares a highly stable biosensor electrode based on high-performance Co NPs@CNFs electrospun core-shell nanofiber membrane materials. The electrode exhibits excellent electrocatalytic properties and electron transfer capabilities, enabling highly sensitive detection of two pancreatic cancer markers, miRNAs, in real blood samples with a low detection limit.

[0017] 2. The aptamer electrochemical biosensor modified with high-performance Co NPs@CNFs electrospun core-shell nanofiber membrane material can achieve rapid recognition and simultaneous detection of two miRNAs cancer markers within 2 minutes, greatly improving the accuracy and efficiency of early cancer diagnosis.

[0018] 3. The aptamer electrochemical biosensor can still show high sensitivity, long-term stability, strong specificity and anti-interference ability for two miRNAs cancer markers in real serum samples with complex components.

[0019] 4. The preparation process of this aptamer electrochemical biosensor is simple and controllable, and it is easy to achieve large-scale and commercial production, which greatly simplifies the detection steps of cancer markers and shortens the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the scanning electron microscope image of Co (acac) 2 / PAN composite nanofiber membrane.

[0021] Figure 2 Scanning electron microscope image of electrospun core-shell nanofiber membrane Co NPs@CNFs. DETAILED DESCRIPTION

[0022] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0023] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0024] In the following examples, conventional equipment or conventional operations in chemical experiments were used unless otherwise specified.

[0025] Example 1

[0026] like Figure 1 and Figure 2 As shown, this embodiment provides a method for preparing a novel aptamer biosensor for rapid detection of dual tumor markers.

[0027] The steps are as follows.

[0028] (1) Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane: Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane synthesis solutions A and B: Dissolve cobalt acetylacetonate (Co(acac)2) in DMF solvent and stir at room temperature for 6 hours to obtain a spinning solution A with a mass concentration of 3 wt%. Dissolve polyacrylonitrile (PAN) in DMF and stir at room temperature for 12 hours to obtain a spinning solution B with a mass concentration of 10 wt%. Spinning solutions A and B were degassed by vacuum filtration. 4 mL of each solution was loaded into a 5 mL syringe. Coaxial electrospinning was performed under the following electrospinning parameters: spinning voltage of +22 kV and -3 kV, a nozzle-to-receiver distance of 20 cm, a flow rate of 1 mL / h for solution A and 0.8 mL / h for solution B, an ambient temperature of 25°C, and an ambient humidity of 45%. (The spinning process was time-controlled and stopped when the specified time was reached or when one of the spinning solutions was depleted.) The resulting composite nanofiber membrane was vacuum-dried at 60°C for 12 h to remove any residual volatile solvent, with a vacuum of 0.1 MPa, to obtain a Co(acac)2 / PAN composite nanofiber membrane.

[0029] (2) Preparation of Co nanoparticles @ carbon nanofibers (Co NPs @ CNFs): The Co (acac) 2 / PAN composite nanofiber membrane material prepared above was placed in a muffle furnace, and PAN was partially oxidized at 210 °C in an oxygen atmosphere to maintain the three-dimensional network structure of the nanofiber. The heating rate of the muffle furnace was 2 °C / min, and the cooling rate was 3 °C / min. After 8 h of oxidation reaction, the Co (acac) 2 / PAN composite nanofiber membrane product was collected. After the oxidation reaction was completed and the temperature dropped to room temperature, the material was transferred to a tube furnace and placed in an inert gas N2 atmosphere at 800 °C for high-temperature cracking reaction for 5 h. The nitrogen flow rate was 10 mL / min, and the tube furnace heating rate was 5 °C / min. After the cracking reaction was completed, the temperature was naturally cooled to room temperature. After the cracking reaction, the product was collected, washed with a large amount of deionized water, filtered, and placed in a vacuum drying oven at 80 °C for dehydration and drying for 12 h with a vacuum degree of 0.1 MPa to obtain a Co NPs @ CNFs composite material.

[0030] (3) Preparation of dual tumor marker aptamer biosensor: The Co NPs@CNFs composite material in step (2) was dispersed in deionized water to prepare a 2 mg / mL dispersion. 2 μL was drop-coated on the surface of the gold electrode. After drop-coating, the electrode was naturally dried in air for 12 h. The CoNPs@CNFs modified electrode was reacted in a 1 μM specific single-stranded DNA probe (ssDNA) fixed solution for 12 h at a reaction temperature of 4 °C. The fixed solution was: 10 mM Tris-HCl, 1 mM EDTA, 10 mM TCEP and 0.1 M NaCl (pH 7.4). At this time, the -SH modified ssDNA probe molecules were firmly bound to the sensing material through the Co-S bond. After the reaction was completed, the ssDNA probe physically adsorbed on the surface was washed away with a washing buffer, wherein the washing buffer was: 10 mMTris-HCl (pH 7.4). The ssDNA-loaded modified electrode was then immersed in a 1 mM MCH solution for 2 hours to block unbound active sites. The modified electrode, immobilized with the capture probe ssDNA, was then immersed in a hybridization solution containing 5 μM MB-modified miRNA-155 complementary aptamer (cApt A) and 5 μM Fc-modified miRNA-21 complementary aptamer (cApt B) for 2 hours at 4°C in 10 mM PBS (pH 7.4) containing 0.25 M NaCl. After the capture probe ssDNA had fully hybridized with cApt A and cApt B, the Co NPs@CNFs-modified electrode surface was rinsed again with wash buffer. Finally, the modified electrode was removed and rinsed with deionized water to obtain the miRNAs aptamer biosensor. Upon contact with the target miRNA-155 and miRNA-21, the cApt-immobilized signal tag was released from the hybridized DNA complex and bound to the corresponding target. This phenomenon resulted in a significant decrease in the peak current density of the signal marker DPV. A linear relationship was established between the signal change and the concentrations of miRNA-155 and miRNA-21, enabling quantitative analysis of miRNA-155 and miRNA-21.

[0031] miRNA-155, miRNA-21, aptamers, and complementary single-stranded DNA were provided by Shanghai Sangon Biotechnology Co., Ltd. The designed sequences of miRNA-155, miRNA-21 aptamers, and complementary single-stranded DNA (ssDNA) are as follows.

[0032] 5′-UUA AUG CUA AUC GUG AUAGGG GU-3′.

[0033] :5′-UAG CUU AUC AGA CUG AUG UUG A-3′.

[0034] Adaptor: 5′-AC CCC UAU CAC GAU UAG CAU UAA-3′.

[0035] Adaptor: 5′-U CAA CAU CAG UCU GAU AAG CUA-3′.

[0036] 5′-SH -TAG CTT ATC AGA CTG ATG TTG A AAAAA TTA ATG CTA ATC GTG ATAGGG GT-3′.

[0037] The sensor's miRNA detection performance was tested using the differential pulse technology of the Shanghai Chenhua CHI660E electrochemical workstation. The voltage variation range was -0.6-0.6V, the increment was 5mV, the amplitude was 25mV, and the frequency was 5Hz. The working electrode, reference electrode, and platinum electrode modified with the Co NPs@CNFs composite material were connected to the corresponding electrode wires on the electrochemical workstation, and the electrode area was immersed in the detection system. The detection limit of the obtained aptamer biosensor for miRNA-155 was as low as 0.3fM, and the linear range could reach 0.8-10 6 fM, the detection limit of miRNA-21 was as low as 0.03 fM, and the linear range was 0.01-10 4 fM, and the detection results of both can be obtained within 2 minutes. After completing the standard curve test, the Co NPs@CNFs-based aptamer biosensor electrode was stored in PBS buffer solution at 4°C for 30 days, and its response signal was 96% of the initial signal, indicating that the aptamer biosensor has excellent stability.

[0038] Before the prepared aptamer biosensor detected the levels of miRNA-155 and miRNA-21 in real blood samples, the whole blood was first separated through a hollow fiber membrane to obtain serum, which was then added to the detection system. The test results are shown in Table 1. The aptamer biosensor can accurately report the levels of miRNA-155 and miRNA-21 in whole blood. Its detection results are consistent with those of commercial miRNA analyzers, with a small detection error, demonstrating excellent anti-interference ability and broad practical application prospects.

[0039] Example 2

[0040] Unless otherwise specified in this embodiment and subsequent embodiments, the conditions are consistent with those in Example 1. This embodiment provides a method for preparing a novel aptamer biosensor for rapid detection of dual tumor markers, and the steps are as follows.

[0041] (1) Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane: Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane synthesis solutions A and B: Dissolve cobalt chloride (CoCl2) in DMF solvent and stir at room temperature for 6 hours to obtain a spinning solution A with a mass concentration of 5wt%. Dissolve polyacrylonitrile PAN in DMF and stir at room temperature for 12 hours to obtain a spinning solution B with a mass concentration of 12wt%. Spinning solutions A and B were degassed by vacuum filtration. 4mL of each of the prepared spinning solutions A and B were loaded into 5mL syringes respectively. Coaxial electrospinning was carried out under the following electrospinning parameters: +16KV, -3KV spinning voltage, 22cm distance from the spinning nozzle to the receiver, 2mL / h flow rate of spinning solution A, 1.5mL / h flow rate of spinning solution B, 20℃ ambient temperature, and 50% ambient humidity. The composite nanofiber membrane obtained by the reaction was vacuum dried at 60° C. for 12 h to remove the residual volatile solvent in the membrane, thereby obtaining a CoCl 2 / PAN composite nanofiber membrane material.

[0042] (2) Preparation of Co nanoparticles @ carbon nanofibers (Co NPs @ CNFs): The CoCl2 / PAN composite nanofiber membrane material prepared above was placed in a muffle furnace, and PAN was partially oxidized at 280°C in an oxygen atmosphere to maintain the three-dimensional network structure of the nanofiber. The heating rate of the muffle furnace was 2°C / min, and the cooling rate was 3°C / min. After 3 hours of oxidation reaction, the CoCl2 / PAN composite nanofiber membrane product was collected. After the oxidation reaction was completed and the temperature dropped to room temperature, the material was transferred to a tube furnace and placed in an inert gas N2 atmosphere at 1000°C for high-temperature pyrolysis for 3 hours. The nitrogen flow rate was 15 mL / min, and the tube furnace heating rate was 3°C / min. After the pyrolysis reaction was completed, the temperature was naturally cooled to room temperature. After the pyrolysis reaction, the product was collected, washed with a large amount of deionized water, filtered, and placed in a vacuum drying oven at 80°C for 12 hours with a vacuum degree of 0.1 MPa to obtain a Co NPs @ CNFs composite material.

[0043] (3) Preparation of dual tumor marker aptamer biosensor: The Co NPs@CNFs composite material in step (2) was dispersed in deionized water to prepare a 4 mg / mL dispersion. 4 μL was drop-coated on the surface of the gold electrode and dried in air for 20 h. The Co NPs@CNFs modified electrode was reacted in a 6 μM specific single-stranded DNA probe (ssDNA) fixed solution for 24 h at a reaction temperature of 4 °C. The fixed solution was 10 mM Tris-HCl, 1 mM EDTA, 10 mM TCEP and 0.1 M NaCl (pH 7.4). At this time, the -SH modified ssDNA probe molecules were firmly bound to the sensing material through Co-S bonds. After the reaction was completed, the ssDNA probe physically adsorbed on the surface was washed off with a washing buffer, wherein the washing buffer was 10 mM Tris-HCl (pH 7.4). The modified electrode loaded with ssDNA was then immersed in a 1 mM MCH solution for 2 h to block the unbound active sites. The modified electrode, immobilized with the capture probe ssDNA, was then immersed in a hybridization solution containing 5 μM MB-modified miRNA-155 complementary aptamer (cApt A) and 5 μM Fc-modified miRNA-21 complementary aptamer (cApt B) at 4°C for 2 h in 10 mM PBS (pH 7.4) containing 0.25 M NaCl. After the capture probe ssDNA had fully hybridized with cApt A and cApt B, the Co NPs@CNFs-modified electrode surface was rinsed again with wash buffer. Finally, the modified electrode was removed and rinsed with deionized water to obtain the miRNAs aptamer biosensor. Upon contact with the target miRNA-155 and miRNA-21, the cApt-immobilized signal tag was released from the hybridized DNA complex and bound to the corresponding target. This phenomenon resulted in a significant decrease in the DPV peak current density of the signal tag. A linear relationship was formed between the signal change and the concentration of miRNA-155 and miRNA-21, thus achieving quantitative analysis of miRNA-155 and miRNA-21.

[0044] miRNA-155, miRNA-21, aptamer and complementary single-stranded DNA were all provided by Shanghai Sangon Biotechnology Co., Ltd. The design sequences of miRNA-155, miRNA-21, aptamer and complementary single-stranded DNA (ssDNA) were consistent with those in Example 1.

[0045] The sensor's miRNA detection performance was tested using differential pulse technology on a Shanghai Chenhua CHI660E electrochemical workstation. The voltage range was -0.6-0.6V, the increment was 5mV, the amplitude was 25mV, and the frequency was 5Hz. The working electrode, reference electrode, and platinum electrode modified with Co NPs@CNFs composite material were connected to the corresponding electrode wires on the electrochemical workstation, and the electrode area was immersed in the detection system. The detection limit of the obtained aptamer biosensor for miRNA-155 was as low as 0.35fM, and the linear range could reach 1-10 7 fM, the detection limit of miRNA-21 is as low as 0.7 fM, and the linear range can reach 10-10 6 fM, and both detection results can be obtained within 3 minutes. After completing the standard curve test, the Co NPs@CNFs-based aptamer biosensor electrode was stored in PBS buffer solution at 4°C for 30 days, and its response signal was 93% of the initial signal, indicating that the aptamer biosensor has excellent stability.

[0046] Before the prepared aptamer biosensor detected the levels of miRNA-155 and miRNA-21 in real blood samples, the whole blood was first separated through a hollow fiber membrane to obtain serum, which was then added to the detection system. The test results are shown in Table 1. The aptamer biosensor can accurately report the levels of miRNA-155 and miRNA-21 in whole blood. Its detection results are consistent with those of commercial miRNA analyzers, with a small detection error, demonstrating excellent anti-interference ability and broad practical application prospects.

[0047] Example 3

[0048] This embodiment provides a method for preparing a novel aptamer biosensor for rapid detection of dual tumor markers, and the steps are as follows.

[0049] (1) Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane: Preparation of transition metal salt precursor / polyacrylonitrile composite nanofiber membrane synthesis solutions A and B: Dissolve cobalt nitrate (Co (NO3)2) in DMF solvent and stir at room temperature for 6 hours to obtain a spinning solution A with a mass concentration of 3 wt%. Dissolve polyacrylonitrile PAN in DMF and stir at room temperature for 12 hours to obtain a spinning solution B with a mass concentration of 16 wt%. Spinning solutions A and B were degassed by vacuum filtration. 4 mL of each of the two prepared spinning solutions A and B were loaded into 5 mL syringes respectively. Coaxial electrospinning was carried out under the following electrospinning parameters: +20 KV, -3 KV spinning voltage, 22 cm distance from the spinning nozzle to the receiver, 1.5 mL / h flow rate of spinning solution A, 1 mL / h flow rate of spinning solution B, 25 °C ambient temperature, and 45% ambient humidity. The composite nanofiber membrane obtained by the reaction was vacuum dried at 60° C. for 12 h to remove the residual volatile solvent in the membrane, thereby obtaining a Co (NO 3 ) 2 / PAN composite nanofiber membrane material.

[0050] (2) Preparation of Co nanoparticles @ carbon nanofibers (Co NPs @ CNFs): The Co (NO3)2 / PAN composite nanofiber membrane material prepared above was placed in a muffle furnace, and PAN was partially oxidized at 240℃ under an oxygen atmosphere to maintain the three-dimensional network structure of the nanofiber. The heating rate of the muffle furnace was 2℃ / min, and the cooling rate was 3℃ / min. After 6 hours of oxidation reaction, the Co (NO3)2 / PAN composite nanofiber membrane product was collected. After the oxidation reaction was completed and the temperature dropped to room temperature, the material was transferred to a tube furnace and placed in an inert gas N2 atmosphere at 600℃ for high-temperature pyrolysis for 5 hours. The nitrogen flow rate was 12 mL / min, and the tube furnace heating rate was 5℃ / min. After the pyrolysis reaction was completed, the temperature was naturally cooled to room temperature. After the pyrolysis reaction, the product was collected, washed with a large amount of deionized water, filtered, and placed in a vacuum drying oven at 80℃ for dehydration and drying for 12 hours with a vacuum degree of 0.1 MPa to obtain a Co NPs@ CNFs composite material.

[0051] (3) Preparation of dual tumor marker aptamer biosensor: The Co NPs@CNFs composite material in step (2) was dispersed in deionized water to prepare a 6 mg / mL dispersion. 10 μL was dropwise applied to the surface of the gold electrode and dried in air for 24 h. The Co NPs@CNFs modified electrode was reacted in a 10 μM specific single-stranded DNA probe (ssDNA) fixed solution for 18 h at 4°C. The fixed solution consisted of 10 mM Tris-HCl, 1 mM EDTA, 10 mM TCEP, and 0.1 M NaCl (pH 7.4). At this time, the -SH modified ssDNA probe molecules were firmly bound to the sensing material through Co-S bonds. After the reaction was completed, the ssDNA probe physically adsorbed on the surface was washed off with a washing buffer containing 10 mMTris-HCl (pH 7.4). The ssDNA-loaded modified electrode was then immersed in a 1 mM MCH solution for 2 hours to block unbound active sites. The modified electrode, immobilized with the capture probe ssDNA, was then immersed in a hybridization solution containing 5 μM MB-modified miRNA-155 complementary aptamer (cApt A) and 5 μM Fc-modified miRNA-21 complementary aptamer (cApt B) at 4°C for 2 hours in 10 mM PBS (pH 7.4) containing 0.25 M NaCl. After the capture probe ssDNA fully hybridized with cApt A and cApt B, the Co NPs@CNFs-modified electrode surface was rinsed again with wash buffer. Finally, the modified electrode was removed and rinsed with deionized water to obtain the miRNAs aptamer biosensor. Upon contact with the target miRNA-155 and miRNA-21, the cApt-immobilized signal tag is released from the hybridized DNA complex and binds to the corresponding target. This phenomenon results in a significant decrease in the DPV peak current density of the signal tag. This signal change forms a linear relationship with the concentration of miRNA-155 and miRNA-21, enabling quantitative analysis of miRNA-155 and miRNA-21.

[0052] miRNA-155, miRNA-21, aptamer and complementary single-stranded DNA were all provided by Shanghai Sangon Biotechnology Co., Ltd. The design sequences of miRNA-155, miRNA-21, aptamer and complementary single-stranded DNA (ssDNA) were consistent with those in Example 1.

[0053] The sensor's miRNA detection performance was tested using differential pulse technology on a Shanghai Chenhua CHI660E electrochemical workstation. The voltage range was -0.6-0.6V, with a 5mV increment, an amplitude of 25mV, and a frequency of 5Hz. The working electrode, reference electrode, and platinum electrode modified with the Co NPs@CNFs composite material were connected to the corresponding electrode wires on the electrochemical workstation, with the electrode area immersed in the detection system. The resulting aptamer biosensor had a detection limit of as low as 0.6fM for miRNA-155, and a linear range of 1.5-2x10 6 fM, the detection limit of miRNA-21 was as low as 0.34 fM, and the linear range could reach 1-2x10 5 fM, and both detection results can be obtained within 5 minutes. After completing the standard curve test, the Co NPs@CNFs-based aptamer biosensor electrode was stored in PBS buffer solution at 4°C for 30 days, and its response signal was 92% of the initial signal, indicating that the aptamer biosensor has excellent stability.

[0054] Before the prepared aptamer biosensor detected the levels of miRNA-155 and miRNA-21 in real blood samples, the whole blood was first separated through a hollow fiber membrane to obtain serum, which was then added to the detection system. The test results are shown in Table 1. The aptamer biosensor can accurately report the levels of miRNA-155 and miRNA-21 in whole blood. Its detection results are consistent with those of commercial miRNA analyzers, with a small detection error, demonstrating excellent anti-interference ability and broad practical application prospects.

[0055] Example 4

[0056] This example differs from Example 1 in that Co(acac)2 in solution A was replaced with Cu(acac)2, while all other conditions remained unchanged. The resulting aptamer biosensor demonstrated similar detection performance for miRNA-155 and miRNA-21 to that of the biosensor in Example 1.

[0057] Example 5

[0058] This example differs from Example 1 in that the Co(acac)2 / PAN composite nanofiber membrane material was not subjected to lysis treatment and was used directly as the electrode modification material, while all other conditions remained unchanged. Experimental verification showed that the aptamer biosensor prepared based on the Co(acac)2 / PAN composite nanofiber membrane material showed little response to miRNAs and could not meet practical detection requirements.

[0059] Example 6

[0060] This example differs from Example 1 in that PAN was omitted from the preparation of a cobalt nanoparticle-based biosensor, while all other conditions remained unchanged. Experimental verification demonstrated that the cobalt nanoparticle-based biosensor had a detection limit of only 0.4 mM for miRNA-155, with a linear range of only 1-89 mM. Its detection limit for miRNA-21 was only 0.2 mM, with a linear range of only 1-71 mM. These performance reductions significantly degraded the sensor performance compared to Example 1, demonstrating the crucial role of the Co NPs@CNFs composite structure in the detection of miRNAs.

[0061] Example 7

[0062] This example differs from Example 1 in that only carbon nanofibers prepared by carbonizing PAN were used as the sensing material, without the addition of cobalt nanoparticles, to prepare a carbon nanofiber-based aptamer biosensor. All other conditions remained unchanged. Experimental verification revealed that the carbon nanofiber-based aptamer biosensor exhibited a weak response signal to miRNAs, failing to meet practical detection requirements.

[0063] From the comparison results of the above examples, it can be seen that the structure of the prepared composite sensing material plays a crucial role in the detection performance of miRNAs. Due to the low specific surface area, poor electron transfer ability and easy aggregation of cobalt nanoparticles themselves, directly using them to construct miRNAs detection electrodes cannot achieve ideal detection performance and cannot meet the actual detection effect. After introducing PAN carbon nanofiber material, the cobalt nanoparticles are evenly distributed on the surface of the carbon nanofiber, such as Figure 2 As shown, the dispersibility of cobalt nanoparticles can be improved, providing more active sites for loading ssDNA, increasing the ssDNA loading capacity, and significantly improving the sensitivity and linear range of miRNA nucleic acid molecule detection. At the same time, after high-temperature pyrolysis treatment, carbon nanofibers with a three-dimensional network structure are obtained, which greatly enhances the catalytic ability and electron transfer capacity. In the present invention, the carbon nanofibers mainly serve as a carrier for the cobalt nanoparticles and provide excellent electron-carrying channels.

[0064] Examples 1-4 show that high-temperature pyrolysis treatment of different types of transition metal salt precursors / polyacrylonitrile composite nanofiber membranes can obtain core-shell structured Co NPs@CNFs, proving that this method has good universality.

[0065] The Co(acac)2 / PAN composite nanofiber membrane prepared in Example 5 was used directly as an electrode modification material without undergoing high-temperature pyrolysis. Because the surface of the material lacks the high-temperature reduced Co element, the single-stranded capture probe ssDNA cannot bind to the electrode modification material. When the targets miRNA-155 and miRNA-21 are added to the detection system, the cApt signal remains unchanged. Therefore, the aptamer biosensor based on the Co(acac)2 / PAN composite nanofiber membrane material exhibits little response to miRNAs and cannot meet practical detection requirements.

[0066] In Example 6, the aptamer biosensor prepared solely from Co nanoparticles with low specific surface area and agglomerated materials was unable to achieve excellent detection performance for miRNA-155 and miRNA-21. In Example 7, only carbon nanofibers prepared by carbonization of PAN were used as the sensing material without compounding cobalt nanoparticles to prepare a carbon nanofiber-based aptamer biosensor, with the other conditions remaining unchanged. Since there was no Co elemental substance that could be reduced at high temperature on the surface of the material, the single-stranded capture probe ssDNA was physically adsorbed only through the extremely large specific surface area of ​​the carbon nanofiber electrode modified material, and then the aptamer modified with the hybridization signal labeling molecule was used for the detection of miRNA-155 and miRNA-21. Since the physical adsorption of the single-stranded capture probe ssDNA resulted in a low ssDNA loading amount and the ssDNA was easily detached, the prepared aptamer biosensor had a weak response signal to miRNAs, and the stability of the sensor could not meet the actual detection requirements. These two examples fully demonstrate that the construction of three-dimensional network core-shell structured Co NPs@CNFs can greatly improve detection performance. At the same time, the regular and appropriately sized sensing materials can also promote the transmission of sensing signals and biorecognition reactions. The aptamer biosensor obtained under the optimized preparation conditions has excellent detection limits and detection ranges for miRNA-155 and miRNA-21: the detection limit of the aptamer biosensor for miRNA-155 is as low as 0.3fM, and the linear range can reach 0.8-10 6 fM, the detection limit of miRNA-21 was as low as 0.03 fM, and the linear range was 0.01-10 4 fM, and the detection results of both can be obtained within 2 minutes.

[0067] In summary, the Co NPs@CNFs composite-based aptamer biosensor prepared in this invention exhibits excellent electrocatalytic activity and electron transfer efficiency for the detection of miRNAs. It is capable of identifying and detecting low-concentration miRNAs and is also suitable for analyzing other types of miRNA nucleic acid molecules. Furthermore, the prepared aptamer biosensor can be stored in PBS buffer at 4°C for 30 days, with its response signal still reaching over 90% of the initial signal, demonstrating excellent stability. This invention provides a new and efficient analytical detection method for the rapid and sensitive detection of multiple cancer biomarkers and for early cancer screening and postoperative follow-up in clinical medicine, with important application value in disease diagnosis and clinical biomedicine.

[0068] Table 1 shows the detection results of serum miRNA-155 and miRNA-21 by the aptamer sensors prepared in Examples 1-3, and the commercial miRNA analyzer was used as a reference data.

[0069] Table 1 Test results of miRNA-155 and miRNA-21 in real blood samples

[0070]

[0071] From the results in Table 1, it can be seen that the aptamer sensors prepared in Examples 1-3 can accurately detect miRNA-155 and miRNA-21 in real serum, have extremely low detection limits and wide linear ranges, and exhibit excellent anti-interference ability and detection accuracy.

[0072] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing an aptamer biosensor for rapid detection of dual tumor markers, characterized in that: Here are the steps: (1) Preparation of composite nanofiber membrane A transition metal salt is dissolved in DMF to obtain a spinning solution A; polyacrylonitrile is dissolved in DMF to obtain a spinning solution B; the spinning solution A and the spinning solution B are vacuum degassed and then added to a syringe for coaxial electrospinning; the solvent is removed by vacuum drying to obtain a composite nanofiber membrane; (2) Preparation of cobalt metal nanoparticles@carbon nanofibers The composite nanofiber membrane prepared in step (1) is placed in a muffle furnace, partially oxidized under an oxygen atmosphere, and then placed in a tubular furnace under an inert gas atmosphere for high-temperature cracking to obtain a powder, which is then washed with deionized water, filtered, and vacuum-dried to obtain cobalt metal nanoparticles@carbon nanofibers; (3) Preparation of dual tumor marker aptamer biosensor Cobalt metal nanoparticles@carbon nanofibers were evenly dispersed in deionized water and drop-coated on the surface of a gold electrode. After drying in air, the mixture was incubated with a specific single-stranded DNA probe solution. Then, the aptamer CApt solution corresponding to miRNA-155 and miRNA-21 was added. After the reaction at low temperature, the mixture was washed with deionized water and stored at low temperature. The transition metal salt in step (1) is any one of cobalt acetylacetonate, cobalt chloride, cobalt nitrate, and cobalt acetate; In step (2), the oxidation reaction temperature is 210-280°C and the time is 3-8 h; the high-temperature cracking temperature is 600-1000°C and the time is 3-5 h.

2. The method for preparing the aptamer biosensor for rapid detection of dual tumor markers according to claim 1, characterized in that: In step (1), the mass concentration of spinning solution A is 3-5wt%; the mass concentration of spinning solution B is 10-16wt%, and the spinning parameters are as follows: spinning voltage is 16-22KV, spinning distance is 20-22 cm, flow rate of spinning solution A is 1-2 mL / h, flow rate of spinning solution B is 0.8-1.5mL / h, ambient temperature is 20-25°C, and ambient humidity is 40-50%; vacuum degree of vacuum drying is 0.1Mp, vacuum drying temperature is 60°C, and vacuum drying time is 12h.

3. The method for preparing the aptamer biosensor for rapid detection of dual tumor markers according to claim 1, characterized in that: In step (2), the heating rate of the muffle furnace is 2 °C / min, and the cooling rate is 3 °C / min.

4. The method for preparing the aptamer biosensor for rapid detection of dual tumor markers according to claim 1, characterized in that: In step (2), the inert gas is nitrogen or argon, the heating rate of the tubular furnace is 3-5°C / min, the inert gas flow rate is 10-15 mL / min; the vacuum drying time is more than 12 h, and the vacuum drying temperature is 60°C.

5. The method for preparing the aptamer biosensor for rapid detection of dual tumor markers according to claim 1, characterized in that: Step (3) The concentration of cobalt metal nanoparticles@carbon nanofibers in deionized water is 2-6 mg / mL, the drop coating volume is 2-10 μL, the drying time is 12-24 h, the concentration of the specific single-stranded DNA probe solution is 1-10 μM, the incubation time is 12-24 h, the concentration of the aptamer CApt solution corresponding to miRNA-155 and miRNA-21 is 1-20 μM, the addition amount is 20-80 μL, the specific binding time is 12-24 h, and the temperature is 4°C; the low-temperature storage temperature is 4°C.

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