CN107315044B

CN107315044B - Based on octahedron Cu2O-Au electrochemical aptamer sensor and preparation method thereof

Info

Publication number: CN107315044B
Application number: CN201710456454.3A
Authority: CN
Inventors: 李朝睿; 邱景富; 陈帅
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2017-06-16
Filing date: 2017-06-16
Publication date: 2020-04-28
Anticipated expiration: 2037-06-16
Also published as: CN107315044A

Abstract

The invention provides octahedron-based Cu₂The preparation method of the O-Au electrochemical aptamer sensor is characterized by comprising the following steps: (1) preparation of Cu₂An O-Au nanocomposite; (2) marking of Cu₂An O-Au nanocomposite; (3) preparing an electrochemical aptamer sensor. The electrochemical aptamer sensor provided by the invention has good sensitivity (the detection limit is 23fM), high specificity and acceptable repeatability, and can be used for detection in human serum samples. Furthermore, Cu₂The O-Au nano composite material also has excellent photocatalytic activity, Cu₂The O-Au nano composite material has wide application prospect in the field of electroluminescent sensors.

Description

Based on octahedron Cu2O-Au electrochemical aptamer sensor and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to octahedron-based Cu₂An O-Au electrochemical aptamer sensor and a preparation method thereof.

Background

Thrombin is Na⁺Activated mutant serine proteases, as central proteases in the coagulation cascade. In vascular injury, thrombin is rapidly produced from the precursor active enzyme prothrombin by a series of enzymatic cleavages, and its concentration can vary from pM to mM during coagulation. Thrombin plays a key role in physiological and pathological coagulation and is involved in various diseases such as central nervous system injury, thromboembolic disease, alzheimer's disease and cancer. Therefore, a highly sensitive and specific thrombin detection method is very significant for research and clinical diagnosis.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide an octahedral Cu based alloy₂An O-Au electrochemical aptamer sensor and a preparation method thereof are used for solving the problems of low detection limit on thrombin, long response time, high cost, poor sensitivity and the like in the prior art.

To achieve the above and other related objects, the present invention provides an octahedral Cu-based alloy₂The preparation method of the O-Au electrochemical aptamer sensor comprises the following steps：

(1) Preparation of Cu₂O-Au nanocomposite: mixing Cu₂O and HAuCl₄Mixing the aqueous solutions, and reacting to obtain Cu₂O-Au nano composite material for standby;

(2) marking of Cu₂O-Au nanocomposite: mixing Cu₂O-Au nanocomposite, NH₂Mixing with TBA, adding BSA aqueous solution after the reaction is finished, and reacting to obtain marked Cu₂An O-Au nanocomposite;

(3) preparing an electrochemical aptamer sensor: cu to be marked₂Dripping O-Au nano composite material on the modified electrode, and incubating to obtain the octahedral Cu-based nano composite material₂O-Au electrochemical aptamer sensor.

Preferably, in step (1), Cu₂The preparation method of O comprises the following steps: will contain Cu²⁺Is mixed with an alkaline solution and reacted to obtain Cu (OH)₂Adding a reducing agent to the mixture to react to obtain Cu₂O。

Preferably, in step (1), the reducing agent is selected from hydrazine hydrate.

Preferably, in step (1), Cu is first added₂O is prepared into an aqueous solution, and then Cu is added₂Adding PVP and HAuCl into O aqueous solution₄Aqueous solution, reaction to obtain Cu₂O-Au nanocomposites.

Preferably, in step (1), HAuCl is present in the mixture₄·4H₂The concentration of O is more than or equal to 1.25 mM.

Preferably, in step (1), HAuCl is present in the mixture₄·4H₂The concentration of O is 1.25-7.50 mM.

Preferably, in step (1), HAuCl is present in the mixture₄·4H₂The concentration of O is 3.75-7.50 mM.

Preferably, in step (1), HAuCl is present in the mixture₄·4H₂The concentration of O is 5.00-7.50 mM.

Preferably, in the step (2), Cu is contained in the mixed solution₂The concentration of O-Au is 0.5-3.5 mg/mL.

Preferably, in the step (2), Cu is contained in the mixed solution₂The concentration of O-Au is 1.5-3.5 mg/mL.

Preferably, in the step (2), Cu is contained in the mixed solution₂The concentration of O-Au was 2.0 mg/mL.

Preferably, in step (2), the concentration of TBA in the mixture is not less than 0.5. mu.M.

Preferably, in step (2), the concentration of TBA in the mixture is 0.5 to 3.0. mu.M.

Preferably, in step (2), the concentration of TBA in the mixture is 1.5 to 3.0. mu.M.

Preferably, in step (2), the concentration of TBA in the mixture is 2.0 to 3.0. mu.M.

Preferably, in step (2), Cu is added₂O-Au Complex, NH₂And (3) after the mixed reaction of the TBA and the chlorhematin, adding BSA aqueous solution for reaction after the reaction is finished.

Preferably, in step (2), Cu is added₂O-Au Complex, NH₂And (3) after mixing and reacting the TBA and the toluidine blue, adding hemin for reaction, and after the reaction is finished, adding a BSA (bovine serum albumin) aqueous solution for reaction.

Preferably, in step (2), the reaction temperature is 4 ℃.

Preferably, in step (2), Cu₂O-Au Complex, NH₂After mixing of-TBA and toluidine blue, the reaction time was 12 h.

Preferably, in step (2), the reaction time is 2h after adding hemin.

Preferably, in step (3), the electrode is a modified glassy carbon electrode.

Preferably, in step (3), the electrode is immersed in HAuCl₄In aqueous solution, electrodepositing until gold nano-particles are fixed on the surface of an electrode, and then adding NH₂And (3) dropwise adding a TBA solution onto the AuNPs modified electrode surface, incubating, dropwise adding a BSA solution onto the electrode surface, incubating, dropwise adding a TB standard solution onto the electrode surface, and incubating to obtain the modified electrode.

Preferably, in the step (3), the electrodeposition time is more than or equal to 10 s.

Preferably, in the step (3), the electrodeposition time is 10 to 60 s.

Preferably, in the step (3), the electrodeposition time is 30 s.

Preferably, in the step (3), after the TB standard solution is dripped on the surface of the electrode, the incubation time is more than or equal to 10 min.

Preferably, in the step (3), after the TB standard solution is dripped on the surface of the electrode, the incubation time is 10-60 min.

Preferably, in the step (3), after the TB standard solution is dripped on the surface of the electrode, the incubation time is 30-60 min.

Preferably, in the step (3), after the TB standard solution is dripped on the surface of the electrode, the incubation time is 40-60 min.

In a second aspect, the present invention provides an electrochemical aptamer sensor prepared by the above method.

In a third aspect, the present invention provides the use of the electrochemical aptamer sensor as defined above in the detection of thrombin.

As mentioned above, the octahedral-based Cu of the present invention₂The electrochemical aptamer sensor of O-Au and the preparation method thereof have the following beneficial effects: the electrochemical aptamer sensor provided by the invention has good sensitivity (the detection limit is as low as 23fM), high specificity and acceptable repeatability, and can be used for detection in human serum samples. Furthermore, Cu₂The O-Au nano composite material also has excellent photocatalytic activity, Cu₂The O-Au nano composite material has wide application prospect in the field of electroluminescent sensors.

Drawings

FIG. 1 shows Cu in an example of the present invention₂FE-SEM image (A) of O nanocrystals, Cu₂FE-SEM image (B) of O-Au nanocomposite, Cu₂EDS (EDC), Cu of O-Au nanocomposites₂XPS spectrum (D) of O-Au nanocomposite, Cu₂Cu2P3(E) and Cu in O-Au nanocomposite₂Au4F (F), Cu in O-Au nanocomposite₂Spectrogram of O1s (G) in O-Au nanocomposite.

FIG. 2 shows 5mM [ Fe (CN) ] containing 0.1M KCl in the examples of the present invention₆]^3-/4-CV (A) and EIS (B) response plots for different electrodes in solution.

FIG. 3 shows AuNPs (A) in the embodiment of the present invention、NH₂-TBA/AuNPs(B)、BSA/NH₂-TBA/AuNPs(C)、TB/BSA/NH₂AFM three-dimensional images of TBA/AuNPs/GCE (D).

FIG. 4 shows an embodiment of the present invention without H₂O₂(a) And contains 15mM H₂O₂(b) CV plots of different material-modified electrodes in 0.1M PBS): (A) octahedron Cu₂O nanocrystals; (B) cu₂O-Au nanocomposites.

FIG. 5 shows the i-t curves for aptamer sensors incubated with different signal labels in an example of the invention: (A) AuNPs-TBA-BSA (a-Curve) and Cu₂O-Au-TBA-BSA (b-curve); (B) cu₂O-Au-TBA-BSA (a-Curve) and hemin/G-quatrefox labeled Cu₂O-Au-BSA (b-curve); (C) hemin/G-quadreplex labeled Cu₂O-Au-BSA (a-Curve) and Tb and hemin/G-quatrefox labeled Cu₂O-Au-BSA (b-curve).

FIG. 6 shows the electrodeposition time (A) of AuNPs, the concentration (B) of TBA, the incubation time (C) of TB, the pH (D) of the working buffer, HAuCl₄Concentration (E) of (C), Cu₂Graph of the effect of the concentration (F) of O-Au on the electrochemical signal of the aptamer sensor.

Fig. 7 shows the current response (a) of the aptamer sensor to different concentrations TB in the embodiment of the present invention, from a to h: 0, 100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 20 nM; and a standard curve (B) for the adaptive sensor for different concentrations TB (n-3).

FIG. 8 shows the target TB (1nM) and other interfering substances in an example of the invention: electrochemical signal response plot (A) for electrochemically adapted sensors of BSA (10nM), Hb (10nM) and CEA (10nM), and their mixtures compared to 1nM TB; and aptamer sensors tested electrochemical signal response plots (B) for 10nM TB after storage for different weeks (n-3).

FIG. 9 shows a schematic diagram of an electrochemical aptamer sensor assembly and a schematic diagram of a signal tag manufacturing process according to an embodiment of the invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It should be understood that the processing equipment or devices not specifically mentioned in the following examples are conventional in the art; all pressure values and ranges refer to absolute pressures.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

1. Overview

The nano material has unique properties, such as large ratio of surface area to volume, good biocompatibility, high conductivity, high molecular load of a receptor and strong catalytic capability. Today, nanomaterials have been widely used as signal amplifying molecules and nanocarriers to improve the sensitivity of sensors. Gold nanoparticles (AuNPs) are the most commonly used material in nanomaterials due to their large surface area, good redox activity, and good biocompatibility. In recent years, cuprous oxide (Cu)₂O) nanomaterials have attracted the researchers' extensive attention because of their excellent catalytic H₂O₂Capacity and lower cost. Wei Chen's group reportsCu encapsulated with graphene₂The O nanotube is used for a non-enzymatic electrochemical sensor for detecting glucose and hydrogen peroxide. Huimin Wu research group reported Cu₂The O-Pt core-shell nano-particle is used as a substrate for sensitive detection of dopamine. As shown in the study, octahedral Cu₂O nanocrystals exhibit Cu in contrast to other shapes₂The O nanocrystal has more excellent electro-catalytic performance. In addition, octahedral Cu₂O nanocrystals have a large surface area and have a general structural unit of reversible redox activity. Compared with single metal oxide, the nano composite material doped with noble metal has more obvious unique characteristics compared with the single metal thereof. Thus, Cu was synthesized by a simple in situ method₂O-Au nanocomposites. AuNPs coating on octahedral Cu₂Surface of O nanocrystals, further increasing surface area to fix more recognition elements and electroactive species, while Cu₂The O-Au nanocomposite shows excellent catalytic H₂O₂Capability.

Aptamers have been widely used in disease diagnosis and bioassay since the first report in 1990. Compared with conventional recognition element antibodies, aptamers have many unique properties, including low cost, mass production, no limitations on detection targets, etc. In recent years, a large number of aptamer-based sensors have emerged, such as fluorescent aptamer sensors, electrochemical aptamer sensors, colorimetric aptamer sensors, and surface plasmon resonance aptamer sensors. Among these sensors, electrochemical aptamer sensors are widely used because of their simplicity of operation, miniaturization, fast response time, and relatively low cost. In addition, thrombin aptamers (TBAs) can fold into G-quadruplex structures and their conformational switches are triggered by binding to thrombin. Hemin can be inserted into TBA as a cofactor to form heme/G-quadruplexes and catalyze H₂O₂Mediated oxidation reactions. Therefore, hemin/G-quadruplex (a known horseradish peroxidase mimic DNase) can be widely used in the signal amplification strategy of electrochemical aptamer sensors.

The invention researches a work based on AuNPsOctahedral Cu capable of being transformed₂The O nanocrystal is used as a signal amplification molecule and an enzyme-free electrochemical aptamer sensor of a nano carrier to detect the thrombin. First, AuNPs grow directly on octahedral Cu by simple in situ reaction₂On the surface of the O nanocrystals. And octahedral Cu₂Synthetic Cu compared to O nanocrystals₂The O-Au nano composite material has larger surface area, more remarkable catalytic capability and good stability, and can fix more electroactive substances and identification probes, thereby improving electrochemical signals and improving the sensitivity of the sensor. Toluidine blue (Tb) generally serves as an electron transfer mediator to provide an electrochemical signal. Tb with ammonia and amino terminated TBA attached to Cu by Au-N bond₂On the surface of the O-Au nanocomposite. With the introduction of hemin, a large amount of hemin/G-quadruplex mimic DNase is formed, and the sensitivity of the aptamer sensor is further improved. AuNPs were immobilized on the surface of a Glassy Carbon Electrode (GCE) by electrodeposition to capture TBA. Captured TBA, target proteins Thrombin, Tb and henmin/G-quadruplex labeled Cu₂The O-Au nano composite materials form a sandwich type electrochemical adaptive sensor together. H is to be₂O₂After introduction into the working buffer, Cu₂O, hemin/G-quatruplex co-catalyst H₂O₂And the electron transfer of Tb is promoted, and excellent electrochemical signal response is obtained. The enzyme-free aptamer sensor provided by the invention has higher sensitivity for quantitative determination of thrombin in human serum, and has great application potential in clinical and diagnostic aspects.

2. Experimental methods

2.1 reagents and chemicals

Thrombin (TB), chloroauric acid (HAuCl)₄·4H₂O), hemin (hemin) and Bovine Serum Albumin (BSA) were all purchased from Sigma-Aldrich Chemical (st. louis, USA, www.sigmaaldrich.com). Hydrazine hydrate (N)₂H₄·H₂O98%), polyvinylpyrrolidone (PVP K)_29-32) And toluidine blue (Tb) were purchased from Aladdin reagent, Inc. (Shanghai, China), anhydrous copper (II) chloride (CuCl)₂) And thrombin aptamer (TBA) was purchased from Sangon biotechco, Ltd. (Shanghai, China)) The oligonucleotide sequences are as follows:

TBA：5'-NH₂-(CH₂)₆-GGT TGG TGTGGT TGG-3'。

to contain 1mM MgCl₂、1mM CaCl₂20mM Tris-HCl buffer (pH 7.4) containing 5mM KCl and 140mM NaCl was used as the aptamer buffer. So as to contain 0.1M Na₂HPO₄、0.1M KH₂PO₄And 0.1M KCl phosphate buffered saline (PBS, pH 6.8) as the working buffer for all electrochemical measurements. All chemicals were analytical reagent grade. Ultrapure distilled water and deionized water (18.2M Ω) were used for all solution formulations.

2.2 instrumentation

Electrochemical measurements including chronoamperometry (i-t), Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) were performed on an AUTOLAB PGSTAT302N electrochemical workstation (Vantone technologies, Inc., Switzerland). Field emission scanning electron microscope FE-SEM) images were obtained using Hitachi S4800(Hitachi Limited, Japan). X-ray photoelectron spectroscopy (XPS) was performed using a VG Scientific ESCALAB 250 spectrometer (thermoelectricity instruments, USA) with Al Ka X-rays (1486.6eV) as the light source. Atomic Force Microscope (AFM) images were monitored by BrukerDimension icon (usa). Energy dispersive X-ray spectroscopy (EDS) was calibrated using a JEOL JSM-6700F microscope (japan). A conventional three-electrode system was used for all electrochemical measurements, with a platinum wire electrode as the counter electrode, Ag/AgCl (containing 3M KCl) as the reference electrode, and a modified glassy carbon electrode (GCE, 4mm diameter) as the working electrode. PBS was used as the working buffer for all electrochemical measurements and was purged with nitrogen for 30 minutes prior to use to remove dissolved oxygen from PBS. All experiments were performed at room temperature (25. + -. 1 ℃ C.).

2.3 octahedral Cu₂Synthesis of O nanocrystals

First, 1mL of 0.1M CuCl was added to the beaker with vigorous stirring at room temperature₂Solution and 28mL deionized water. Then, 1mL of 1.0M NaOH solution was added. The resulting solution turned blue immediately, indicating the formation of Cu (OH)₂. In Cu (OH)₂After the precipitate had completely formed, 120. mu.m were pipetted in 3 secondsL1M N₂H₄·H₂O was quickly injected into the beaker. The solution in the beaker was kept stirring at room temperature for 10 minutes and then centrifuged at 5000rpm for 5 minutes. Centrifuging the obtained Cu₂O was redispersed in 30mL of deionized water, then 10. mu.L of 1M N was added₂H₄·H₂And O, and stirring is continued for 60 minutes to perform nanocrystal growth. Cu to be prepared₂The O nanocrystals were centrifuged at 5000rpm for 5 minutes and washed 3 times with 10mL deionized water to remove unreacted chemicals. The final wash step used 10mL ethanol, and the precipitate was dispersed in 4mL ethanol for storage and analysis.

2.4Cu₂Synthesis of O-Au nanocomposite

To synthesize Cu₂O-Au nanocomposite, 7.2mg of Cu were first added₂The O octahedral nanocrystals were dissolved in 26mL of deionized water. 3mL of 1% PVP solution was added dropwise to Cu₂O octahedral solution, then 1mL of 5mM HAuCl was added to the sample₄An aqueous solution. HAuCl₄Typical yellow and Cu of aqueous solution₂The brick-red color of O immediately disappeared to form a black product. The solution was placed on a magnetic stirrer at room temperature and stirred vigorously for 3 hours. After the reaction is finished, the prepared Cu₂The O-Au nanocomposite was centrifuged at 5000rpm for 5 minutes and washed 3 times with 20mL of deionized water to remove unreacted chemicals. The final washing step used 10mL of ethanol and the nanocomposite was dried in vacuo overnight. The reaction formula is as follows:

3Cu₂O+2AuCl₄ ^-+6H⁺＝6Cu²⁺+2Au+3H₂O+8Cl^-

2.5 preparation of Tb and hemin/G-quadruplex-labeled Cu₂O-Au nanocomposite (Signal tag)

To fix NH₂TBA, 1mL of prepared Cu₂O-Au (2mg/mL) nanocomposite with 100 μ LNH₂TBA (2.0. mu.M) and 50. mu.LTb were mixed, followed by gentle stirring at 4 ℃ for 12 hours. Thus, NH₂TBA and Tb by AuNPs and-NH₂The interaction between the groups being attached to Cu₂O-Au nano composite material. Then, the user can use the device to perform the operation,mu.L of heme (0.5 mg/mL) was added to the mixture and stirred at 4 ℃ for 2 hours to form hemin/G-quadruplex. Next, 50 μ LBSA (w/w, 1%) was introduced into the mixture and held for 30 minutes to block the remaining active sites. The final product was centrifuged, resuspended in 1mL of water, and stored at 4 ℃ for further use.

2.6 preparation of aptamer Sensors

The process for preparing the electrochemical adaptive sensor is shown in fig. 9. The GCE was mirror polished repeatedly with 0.3 μm and 0.05 μm alumina slurries and rinsed thoroughly with deionized water. Then, GCE was ultrasonically washed in absolute ethanol for 5 minutes, followed by ultrasonic washing in water for 5 minutes. GCE was dried at room temperature and then soaked in HAuCl₄(w/w, 1%) and electrodeposited at-0.2V for 30 seconds, gold nanoparticles (AuNPs) were successfully immobilized on the GCE surface. Next, 20 μ LNH₂TBA (2.0. mu.M) was added dropwise to the AuNPs-modified GCE surface and incubated for 16 h at room temperature. To prevent non-specific binding to the electrode surface, 10. mu.L of BSA solution (w/w, 1%) was added dropwise to the electrode surface and incubated for 40 min. Then 20 μ L of TB standard solutions of different concentrations (as shown in fig. 7B) were added dropwise to the modified electrode at room temperature for 40 minutes of incubation, followed by washing. Finally, 15. mu. LTb and hemin/G-quadruplex labeled Cu were added₂O-Au nanocomposites (signal labels) were added dropwise to the resulting modified electrodes and incubated for an additional 40 min. After extensive washing to remove unbound signal labels, the electrodes are ready for measurement.

2.7 electrochemical testing

All electrochemical tests were performed in a conventional electrochemical cell containing a three-electrode arrangement. CV and EIS measurements at 5mM [ Fe (CN) ] containing 0.1M KCl₆]^3-/4-Gradually going through the solution. CV measurements were performed at a scan rate of 100mV/s and a voltage range of-0.2 to 0.6V, EIS parameters included 10mV amplitude and from 0.1 to 10 at room temperature⁵Frequency sweep in Hz. For electrochemical measurements, current-time curves were recorded in 8mL of working buffer (pH 6.8) at room temperature and-0.4V, and after the background current had stabilized, 10 μ L of 5M H was added to the solution₂O₂Recording electrificationThe optical signal changes.

3. Results and discussion

3.1 octahedral Cu₂O nanocrystals and Cu₂Characterization of O-Au nanocomposites

As can be seen from the FE-SEM image (FIG. 1A), most of the synthesized Cu₂O nanocrystals exhibited a clear octahedral structure with an average diameter of about 250 nM. FIG. 1B shows uniform coating on octahedral Cu₂AuNPs of approximately 30-50nM diameter on the surface of O nanocrystals, indicating Cu₂O-Au nanocomposites have been successfully synthesized. In addition, application of EDS and XPS further confirmed Cu₂And (3) synthesizing the O-Au nano composite material. As shown, in fig. 1C, there are corresponding O, Cu and Au peaks in the EDS image. Meanwhile, Cu in XPS image₂In the O — Au nanocomposite, characteristic peaks of the core-level regions of Cu2p3, O1s, C1s and Au4f were clearly observed (fig. 1D). The spectra of Cu2p3, Au4F, and O1s are shown in fig. 1E, fig. 1F, fig. 1G, respectively, consistent with previous reports (Zhu, h., Du, m.l., Yu, d.l., Wang, y., Wang, l.n., zuo, m.l., Zhang, m.fu, y.q.,2012, a new structure for the surface-ee-energy-distribution induced selection and controlled formation of Cu 2O-Au-rear specific structures with a server of organic evolution. journal of materials Chemistry A1 (3); 9199). The above experimental results prove that Cu₂O-Au nanocomposites have been successfully synthesized.

3.2 electrochemical characterization of stepwise modified electrodes

To characterize the interfacial properties of the modified electrode, the assembly step of the sensing interface was studied after each step by EIS and CV measurements. CV spectra of electrodes prepared in different steps are shown in fig. 2A. The reaction conditions are as follows: 2.0. mu.M NH₂TBA and 40min TB incubation time. A clear redox wave was recorded with a reversible redox reaction of ferricyanide ions on the glassy carbon electrode surface (curve a). With the AuNPs immobilized on the electrode (curve b), the redox peak current increased significantly as the AuNPs promoted electron transfer. Next, the peak current decreased significantly after AuNPs/GCE capture of TBA (curve c). The reason is the non-electroactive TBA can severely impede the electron transfer of the redox probe. Similarly, when non-specific sites were blocked using non-conductive BSA, the peak current was further reduced (curve d). Finally, when the aptamer sensor was incubated with 10nM thrombin, the thrombin-aptamer complex formed with TBA capturing thrombin resulted in a further reduction of the redox peak current, since it severely hampered the transport of electrons (curve e).

Furthermore, EIS is a well established technique that is commonly used to verify the stepwise modification process of electrodes. In the nyquist plot, the semi-circular diameter is equal to the electron transfer resistance, and the linear portion of the low frequency curve represents the diffusion process. As shown in fig. 2B. The bare GCE showed a smaller resistance (curve a). Due to the high electrical transport properties of AuNPs, the semicircular diameter decreases when AuNPs are electrodeposited onto the electrode surface (curve b). When TBA was added dropwise to the electrode surface, the resistance increased significantly (curve c), demonstrating that TBA passed AuNPs and-NH₂The interaction between the groups is attached to the electrode. Non-conductive BSA was then added as a blocker, again causing an increase in resistance. After incubation of thrombin (curve d), the resistance further increased (curve e), meaning that thrombin had been successfully captured by TBA.

To confirm the preparation of the electrodes, we performed AFM image characterization, and three-dimensional images of AFM are shown in fig. 3A to 3D. Fig. 3A shows an AuNPs layer formed by electrodeposition. After TBA was coated on the electrode, the surface became relatively smooth due to the adsorption of DNA to the electrode (fig. 3B). When the electrode was blocked by BSA, the surface roughness increased a little (fig. 3C). After incubation with TB, the electrode surface roughness increased slightly (fig. 3D), indicating that TBA successfully captured TB. All results obtained further support successful manufacture of the sensor and are consistent with the results of previous studies.

3.3 comparative octahedral Cu₂O nanocrystals and Cu₂Properties of O-Au nanocomposites

By pairs H₂O₂The current response of the catalytic reaction of (2) evaluated octahedral Cu₂O nanocrystal modified electrode (FIG. 4A) and Cu₂The O-Au nanocomposite modified the performance of the electrode (FIG. 4B). Curve a showsIs free of H₂O₂In 0.1M PBS. Curve b is shown as containing 15mM H₂O₂0.1M PBS. Thus, we have found octahedral Cu₂The current response of the O nanocrystal modified electrode is far smaller than that of Cu₂And the O-Au nano composite material modifies the response of the electrode. The reason for this phenomenon may be that AuNPs are in octahedral Cu by in situ methods₂The surface growth of O nanocrystals, which significantly improves the catalytic ability and electrical conductivity of the nanocomposite.

3.4 comparing different Signal amplification strategies

To verify the signal amplification performance of the proposed aptamer sensor, we prepared four signal tags to detect the same analyte concentration. The four signal labels comprise (a) AuNPs-TBA-BSA, (b) Cu₂O-Au-TBA-BSA, (c) hemin/G-quadruplex-labeled Cu₂O-Au-BSA, and (d) Tb and hemin/G-quadruplex-labeled Cu₂O-Au-BSA. As shown in FIG. 5A, Cu compares to the aptamer sensor using AuNPs-TBA-BSA as signal tag₂The electrochemical signal is obviously increased when O-Au-TBA-BSA is used as a signal label because of Cu₂O has excellent catalytic activity on H₂O₂The ability of the cell to perform. When aptamer sensors were contacted with hemin/G-quadreplex labeled Cu₂hemin/G-quadruplex mimic peroxidase can catalyze H when O-Au-BSA is incubated together₂O₂Resulting in an increase in electrochemical signal (fig. 5B). Notably, when Tb and hemin/G-quattreplex labeled Cu₂O-Au-BSA has a larger electrochemical signal response as a signal tag than other signal tags (FIG. 5C). The possible reason for this phenomenon is Cu₂O, AuNPs and hemin/G-quatruplex co-catalyst H₂O₂The electron transfer of Tb is promoted to enhance the electrochemical signal. Therefore, the signal amplification method proposed by the present invention (Tb and hemin/G-quatrefox labeled Cu)₂O-Au-BSA as a signal tag) is a suitable choice for the aptamer sensor.

3.5 optimization of the Experimental conditions

In the following optimization experiments, except for different optimization parameters, other steps and parameters are consistent with the steps 2.1-2.6.

The electrochemical signal of an aptamer sensor is affected by many factors. To ensure optimal performance of aptamer sensors, we studied several key factors. The AuNPs electrodeposition time determines the thickness of AuNPs layers and the number of AuNPs, and further influences the fixation and electron transfer of TBA. Fig. 6A shows the effect of different electrodeposition times for AuNPs. The electrochemical signal change increases with increasing electrodeposition time, with a maximum electrochemical signal observed at30 s, followed by a decrease. The reason for this phenomenon is that long electrodeposition times form excessive amounts of AuNPs, which hinder electron transfer. Therefore, the AuNPs electrodeposition time of the aptamer sensor is preferably 30 s.

The concentration of TBA is another important factor in capturing thrombin and affecting the detection range. As shown in fig. 6B, it can be seen that the electrochemical signal gradually increases with increasing concentration of TBA, and the electrochemical signal is maximal at 2.0 μ M, and then remains substantially stable. Therefore, 2.0 μ M was selected as the optimum concentration of TBA.

The incubation time of the reaction between TB and TBA is also an important parameter affecting the performance of the aptamer sensor assay. As shown in fig. 6C, as the incubation time increased, the electrochemical signal change increased first and remained stable after about 40 min. This finding indicates that aptamer binding to TB is saturated. Therefore, 40min was chosen as the optimal incubation time.

The pH value mainly affects the catalytic ability of the signal tag and the activity of the biological protein. Working buffers were prepared at various pH values to investigate the performance of the aptamer sensor. As shown in fig. 6D, the optimal electrochemical signal response was found at pH 6.8. The reason is that Cu₂O has the best performance under weak acidity condition, and Cu is treated under weak alkalinity condition₂The performance of O has an impact. When the pH is higher>7.0 time, Cu₂The enhancement of O is reduced, resulting in a reduction of the electrochemical signal. Therefore, the optimal electrochemical signal response was obtained when the pH of 6.8 was chosen as the working buffer.

In Cu₂HAuCl in the process of synthesizing O-Au nano composite material₄·4H₂The concentration of O determines Cu₂In O-Au nanocompositesThe content of AuNPs, which further influences the fixed amounts of Tb and hemin/G-quadreplex. Thus, different concentrations of HAuCl were used₄·4H₂O-synthesized Cu₂The O-Au nano composite material is used as a signal label to research the performance of the aptamer sensor. As shown in FIG. 6E, with HAuCl₄·4H₂The electrochemical signal increases when the concentration of O increases from 1.25mM to 5.0mM, but decreases when the concentration increases from 5.0mM to 7.5 mM. The reason for this phenomenon may be an excess of HAuCl₄·4H₂O consumes more Cu₂O, causing a decrease in electrochemical signal. Therefore, in Cu₂During the synthesis of O-Au nano composite material, 5mM is selected as HAuCl₄·4H₂Optimum concentration of O, the aptamer sensor has optimum performance.

Cu₂The concentration of the O-Au nano composite material is also an important factor influencing the catalytic efficacy of the aptamer sensor. Cu of synthetic signal tag₂The electrochemical signal response is shown in FIG. 6F for different concentrations of O-Au nanocomposite. With Cu₂The increase in O-Au nanocomposite concentration from 0.5mg/mL to 2.0mg/mL rapidly increased the change in electrochemical signal response followed by a subsequent decrease. Supposing that Cu₂An increase in the thickness of the O-Au nanocomposite film may lead to an increase in interfacial electron transfer resistance; in this case, electron transfer becomes more difficult. Therefore, 2.0mg/ml was selected as Cu₂Optimum concentration of O-Au nanocomposite.

3.6 Performance of aptamer sensors

Under optimal experimental conditions (AuNPs electrodeposition time preferably 30s, TBA concentration 2.0. mu.M, incubation time for reaction between TB and TBA 40min, working buffer pH 6.8, HAuCl₄·4H₂O concentration 5mM, Cu₂Concentration of O-Au nanocomposite 2.0mg/mL), Cu labeled with Tb and hemin/G-quattreplex₂O-Au-Apt-BSA as a signal tag prepared aptamer sensor, TB was quantitatively detected at different concentrations by chronoamperometry in 8mL PBS (pH 6.8) at-0.4V. The relationship between electrochemical signal and TB concentration is shown in FIG. 7A. As shown in FIG. 7BA linear relationship between the electrochemical signal and the log of TB concentration was observed in the range of 100fM to 20nM, with a calculated detection limit of 23fM (based on S/N-3). The regression equation is that Y is 25.44logC_TB+553.24, correlation coefficient R is 0.9989. The lower detection limit may be due to several factors. On one hand, AuNPs have good biocompatibility as a substrate, and can capture more TBA to amplify the detection range. On the other hand, Cu₂The O-Au nano composite material not only has good catalytic capability and high conductivity, but also has large specific surface area, and can fix a large amount of Tb and hemin/G-quatrefox horseradish peroxidase analog DNase, thereby further enhancing electrochemical signals and obtaining higher sensitivity. Comparison with the previous method for detecting TB is shown in table 1, and it can be seen from the data in the table that the aptamer sensor proposed by the present invention has a lower detection limit than the existing sensor.

TABLE 1 comparison of Thrombin aptamer sensors

3.8 specificity, stability and repeatability of electrochemical aptamer sensors

The specificity of aptamer sensors plays a very important role in isolating biological samples. Some non-target substances, such as BSA, Hb, and CEA, were used as interfering substances to test the specificity of aptamer sensors. As shown in FIG. 8A, substantially no change in electrochemical signal was observed for the aptamer sensor incubated with BSA (10nM), Hb (10nM) and CEA (10nM) compared to the blank signal. However, the electrochemical signal increase was evident after incubation with TB (1nM) and its mixture with the three non-targets mentioned above (10 nM). These results show the high specificity of the proposed adaptive sensor.

To investigate the stability of the aptamer sensors, the prepared aptamer sensors were stored at 4 ℃ and removed for use. As shown in fig. 8B, after 1 week, the electrochemical signal of the aptamer sensor was 98.07% of the initial electrochemical signal with only a small change. After 4 weeks, the electrochemical signal retained 87.88% of its initial electrochemical signal, indicating that the proposed aptamer sensor had acceptable stability. In addition, we also tested the reproducibility of the aptamer sensors by recording the current response at three different concentrations (1nM, 100pM, 1 pM). Five electrodes were prepared for each concentration. The Relative Standard Deviation (RSD) ranged from 0.49% to 1.29%, as shown in table 2. The results show that the proposed compliant sensor has acceptable repeatability.

TABLE 2 reproducibility of five different electrodes for three concentrations of analyte

3.9 serum sample analysis applications

To evaluate the practical applicability and accuracy of the proposed aptamer sensor, TB (as shown in table 3) was added at various concentrations to human serum samples (obtained at city hospital, university subsidiary of Chongqing medical university) diluted 10 times, followed by detection using the proposed electrochemical sensor, and the current curves of the resulting samples were recorded. As shown in table 3, the Relative Standard Deviation (RSD) of the assay results ranged from 1.24% to 3.77%, with recovery rates ranging from 91.75% to 107.2%. The result shows that the aptamer sensor prepared by the invention is feasible for detecting thrombin and can meet the requirement of practical analysis.

Table 3 measurement of thrombin in human serum using aptamer sensor prepared according to the present invention (n ═ 3)

4. Conclusion

In the invention, a sensitive sandwich type electrochemical aptamer sensor is prepared, and Cu marked by Tb and hemin/G-quadruplex is adopted₂The O-Au nano composite material is used as a signal label, and AuNPs are used as a substrate for detecting thrombin. Due to Cu₂Octahedral surface area of O-Au nanocomposites and excellent biocompatibility it is used as an ideal nanocarrier for immobilizing large amounts of electroactive Tb and hemin/G-quadruplex horseradish peroxidase mimics DNAzyme. Further, AuNPs, Cu₂The co-catalytic ability of O and hemin/G-quadruplex can amplify electrochemical signals and improve the sensitivity of the aptamer sensor. In combination with these advantages, the proposed electrochemical aptamer sensor has good sensitivity (detection limit of 23fM), high specificity and acceptable reproducibility, and can also be used for detection in human serum samples. Furthermore, Cu₂The O-Au nano composite material also has excellent photocatalytic activity, so that the Cu prepared by the method₂The O-Au nano composite material has wide application prospect in the field of electroluminescent sensors.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. Based on octahedron Cu₂The preparation method of the O-Au electrochemical aptamer sensor is characterized by comprising the following steps:

(2) marking of Cu₂O-Au nanocomposite: mixing Cu₂O-Au nanocomposite, NH₂Mixing and reacting-TBA and toluidine blue, adding hemin, and after the reaction is finished, adding BSA aqueous solution for reaction to prepare marked Cu₂An O-Au nanocomposite; wherein TBA is a thrombin aptamer;

2. The production method according to claim 1, wherein in the step (1), Cu₂The preparation method of O comprises the following steps: will contain Cu²⁺Is mixed with an alkaline solution and reacted to obtain Cu (OH)₂Adding hydrazine hydrate into the mixture to react to obtain Cu₂O。

3. The method of claim 1, wherein: in the step (1), Cu is firstly added₂O is prepared into an aqueous solution, and then Cu is added₂Adding PVP and HAuCl into O aqueous solution₄Aqueous solution, reaction to obtain Cu₂O-Au nanocomposites.

4. The method of claim 1, wherein: in the step (1), HAuCl is contained in the mixed solution₄·4H₂The concentration of O is 1.25-7.50 mM;

and/or, in the step (2), Cu in the mixed solution₂The concentration of O-Au is 0.5-3.5 mg/mL;

and/or in the step (2), the concentration of TBA in the mixed solution is 0.5-3.0 mu M;

and/or, in the step (3), the electrode is a modified glassy carbon electrode.

5. The method of claim 1, wherein: in the step (3), the electrode is immersed in HAuCl₄In aqueous solution, electrodepositing until gold nano-particles are fixed on the surface of an electrode, and then adding NH₂And (3) dropwise adding a TBA solution onto the surface of the gold nanoparticle modified electrode, incubating, dropwise adding a BSA solution onto the surface of the electrode, incubating, dropwise adding a thrombin standard solution onto the surface of the electrode, and incubating to obtain the modified electrode.

6. The method of claim 5, wherein: in the step (3), the electrodeposition time is 10-60 s;

and/or in the step (3), after the thrombin standard solution is dripped on the surface of the electrode, the incubation time is 10-60 min.

7. An electrochemical aptamer sensor manufactured according to the manufacturing method of any one of claims 1 to 6.

8. Use of the electrochemical aptamer sensor according to claim 7 in thrombin detection.