An electrochemical sensor fusing local hairpin self-assembly and metal nanoparticle synergistic amplification strategy

Abstract

The application discloses an electrochemical sensor fusing local hairpin self-assembly and metal nanoparticle cooperative amplification strategies, which utilizes the characteristics that nano metal and thiol are easy to combine to fix DNA base column probe S1 on the electrode surface. Then, S1 and HP1, two kinds of nucleic acid probes, are modified on the nano metal particles, and auxiliary probe HP2 and nucleic acid TS to be detected are added, local hairpin self-assembly reaction occurs among the three, a plurality of metal nanoparticles are connected with each other through nucleic acid probes S1 and HP1. The multistage structure is connected with S1 on the electrode surface, which promotes a large number of oxidation-reduction signal molecules to be gathered on the electrode surface in a short time, and multistage amplification is realized. Finally, the differential pulse voltammetry in the electrochemical technology is utilized to convert the substance amount of the oxidation-reduction signal molecules into the current signal in proportion, and high-sensitivity and large-range nucleic acid detection sensing is completed. In actual detection, the lowest detectable amount of some microRNA is in the order of fM.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to an electrochemical sensor that integrates local hairpin self-assembly with a synergistic amplification strategy of metal nanoparticles. Background Technology

[0002] Highly sensitive nucleic acid detection is an important tool in biomedicine, disease diagnosis, and environmental monitoring. It provides accurate, rapid, and reliable results, which is of great significance for scientific research and social development. In disease diagnosis, the detection of circulating tumor DNA in blood enables non-invasive detection of early-stage cancer, and the detection of multiple microRNAs in various body fluids allows for real-time monitoring of disease conditions. Similarly, in infectious disease diagnosis, such as virus detection, highly sensitive nucleic acid detection ensures early detection and isolation of viruses. In biomedical research, highly sensitive nucleic acid detection helps scientists gain a deeper understanding of phenomena such as gene expression, gene editing, and gene mutation. Furthermore, it can be used to study the diversity and function of microorganisms. In environmental science, highly sensitive nucleic acid detection can be used to detect and monitor microorganisms in the environment. By detecting nucleic acids in microorganisms in water or soil samples, the degree and type of environmental pollution can be assessed. In the food industry, highly sensitive nucleic acid detection can be used to detect microbial contamination in food or for the identification of food sources. For example, by detecting nucleic acids in food samples, it can be determined whether the food contains illegally added ingredients or whether fraudulent activities have occurred.

[0003] Hairpin self-assembly is a nucleic acid-based biosensing and amplification technique. It utilizes two hairpin-structured nucleic acid molecules to self-assemble into a larger, more stable double-stranded DNA structure via a specific catalyst or trigger. Its main advantage is that it can amplify signals without enzymes, making it excellent for detecting low-abundance biomarkers. This method was initially proposed by the California Institute of Technology in 2004. Initially, hairpin self-assembly was mainly used for DNA computing and logic gate construction. However, in the last decade or so, it has begun to be applied to biosensing and disease diagnosis, detecting biomolecules including DNA, RNA, and proteins. It has now become an important tool in the fields of biosensing and molecular diagnostics.

[0004] Metal nanoparticles, especially gold nanoparticles, also have many applications in biosensing. By utilizing their surface chemical properties, such as the presence of numerous binding sites for nucleic acids and their strong conductivity that promotes electron transfer on the electrode surface and thus increases the intensity of electrochemical signals, many applications in the field of nucleic acid detection can be derived. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an electrochemical sensor that integrates local hairpin self-assembly with a synergistic amplification strategy of metal nanoparticles.

[0006] The objective of this invention is achieved through the following technical solution: an electrochemical sensor integrating local hairpin self-assembly and metal nanoparticle synergistic amplification strategy. In this sensor, firstly, an electrode to be detected is prepared, and finally, the base probe S1 is fixed on the electrode. Then, metal nanoparticles modified with functional hairpin probes HP1 and S1 are prepared. After mixing the metal nanoparticles, auxiliary probe HP2, and nucleic acid to be detected, they are incubated on the electrode to trigger the local nucleic acid hairpin self-assembly and metal nanoparticle cascade amplification reaction. The redox signal molecules on the functional hairpin probe HP1 and auxiliary hairpin probe HP2 are restrained on the electrode surface, and a current signal is output through the reaction.

[0007] The specific sequence is shown below:

[0008] DNA column probe S1 information: 5'-GTC TGG ACT CAT TTT TTT TTT-3'-SH-HS C6;

[0009] Functional hairpin probe HP1: SH-HS C6-5'-TCA ACA TCA GTC TGA TAA GCT ATG ATG TTGA-3'-Ferrocene;

[0010] Auxiliary hairpin probe HP2: Ferrocene-5'-GAG TTC GAG CAG ACT GAT GTT GAC ATT GCTCGA ACT CAA CAT CAG-3'.

[0011] Furthermore, the electrode body is a commercially available screen-printed metal or carbon electrode product. It undergoes electrochemical cyclic voltammetry and isopotential bonding to reduce metal ions in the solution and form metal nanoparticles, which are densely arranged on the electrode surface. Thiol-modified S1 is then fixed onto the metal nanoparticle layer of the electrode via drop-coating. Next, 20 mM 6-mercapto-1-hexanol (MCH) and 1% bovine serum albumin (BSA) are sequentially drop-coated onto the S1-fixed electrode to form an anti-specific binding isolation layer. Finally, the electrode, after the above steps, is immersed in a TE buffer solution prepared with Tris and EDTA to clean non-specific binding impurities from the electrode, ensuring the consistency of the sensing results.

[0012] Furthermore, HP1 and S1, which were modified with disulfide bonds and ferrocene probes at both ends respectively, were simultaneously added to the metal nanoparticle solution and then quickly placed in an environment of -20°C for 2 hours. The concentration of HP1 and S1 was 3:1, and the ratio of the total nucleic acid content of HP1 and S1 to the concentration of nanoparticles was 50-300:1. The larger the size of the metal nanoparticles, the greater the total nucleic acid content.

[0013] Furthermore, HP2 and the target nucleic acid TS were simultaneously added to a solution of metal nanoparticles modified with HP1 and S1, and incubated at room temperature for 1 hour. During incubation, the hairpins of HP1 were gradually opened from the hairline by TS, and then the hairpins of HP2 were gradually opened from the hairline by the unfolded HP1 chain. At the same time, HP2 replaced TS from the HP1 base chain. TS would repeat the above operation at other unopened HP1 sites, which is the hairpin self-assembly amplification process. Furthermore, HP1 and HP2 conjugates were generated on the surface of the metal nanoparticles. Through nucleic acid sequence design, a free single strand of HP1 was exposed at the outermost end of the entire metal nanosphere. The base of this single strand bound to S1 modified on the surface of another nanosphere, thereby connecting the two nanoparticles. This process occurred between pairs of nanospheres, forming a complex multi-level structure. At the same time, the exposed fragment of HP1 could not only bind to S1 modified on the nanosphere surface, but also to S1 fixed on the electrode surface. Thus, the multi-level structure amplified in this cascade was attached to the electrode surface.

[0014] Ferrocene is modified on a large number of HP1 and HP2 structures that appear near the electrodes in proportion to the multi-level structure. Finally, the ferrocene outputs a current signal through a redox reaction.

[0015] Furthermore, the sensor's detection equipment includes two lithium batteries, a low-ripple power module, an integrated Bluetooth MCU unit minimum control system, and an electrochemical detection signal circuit; the device housing is made by 3D printing and is equipped with a power switch and an electrochemical three-electrode SMA interface.

[0016] The MCU unit with integrated Bluetooth uses Nordic's nRF52832 chip. In its SDK software program, custom Bluetooth service content and configuration for the electrochemical detection device are written, including content actively written by the PC and mobile phone to the detection device, current signal data content periodically updated by the detection device and sent to the PC and mobile phone, as well as communication frequency band and interaction period parameters.

[0017] The beneficial effects of this invention are as follows: The detection principle of this invention differs from amplification methods such as PCR and LAMP, requiring no temperature control, involving no proteases, and thus avoiding excessive environmental influences. Furthermore, this sensor, as a low-cost, small-sized portable device, is suitable for a wide range of personal and customized scenarios, and can be widely used in disease detection, monitoring, and initial screening in medical experiments in the future. The detection effect of this principle is good; in the preliminary detection experiment of miRNA-21, it exhibits good linearity over a wide range of 10 fM to 100 μM, while the detection limit can reach the 1 fM level. Attached Figure Description

[0018] Figure 1 This is a schematic flowchart illustrating the principle of the detection method of the present invention;

[0019] Figure 2 This is a block diagram of the hardware composition of the detection device of the present invention;

[0020] Figure 3 This is a nucleic acid electrophoresis diagram of the hairpin self-assembly amplification of HP1, HP2, and miRNA-21;

[0021] Figure 4 This is a graph showing the results of miRNA-21 amplification detection. Detailed Implementation

[0022] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0024] The nucleic acid sequences designed in this example are shown below. The synthesis and purification of DNA and RNA were completed by Shanghai Sangon Biotech Co., Ltd. They were dissolved in TE buffer and prepared to 20 μM, then stored at -20℃ for later use.

[0025] DNA column probe S1 information (SEQ ID No. 1): 5'-GTC TGG ACT CAT TTT TTT TTT-3'-SH-HS C6;

[0026] Functional probe hairpin HP1 (SEQ ID No. 2): SH-HS C6-5'-TCA ACA TCA GTC TGA TAA GCTATG ATG TTG A-3'-Ferrocene;

[0027] Auxiliary probe hairpin HP2 (SEQ ID No. 3): Ferrocene-5'-GAG TTC GAG CAG ACT GAT GTTGAC ATT GCT CGAACT CAA CAT CAG-3';

[0028] The 3' end of S1 and the 5' end of HP1 are modified with disulfide bonds (SH-HS), and the 3' end of HP1 and the 5' end of HP2 are modified with ferrocene.

[0029] The target nucleic acid for detection is microRNA-21 (SEQ ID No. 4): 5'-UCA ACA UCA GUC UGA UAA GCUAUU U-3'.

[0030] The present invention provides an experimental example of an electrochemical sensor that integrates local hairpin self-assembly with a synergistic amplification strategy of metal nanoparticles. Figure 1 As shown, the details are as follows:

[0031] (1) Prepare the electrode to be detected, the steps are as follows:

[0032] The electrode body is a commercially available screen-printed metal or carbon electrode product. It undergoes sequential electrochemical cyclic voltammetry and isopotential bonding to reduce metal ions in solution and form nanoparticles, which are then densely arranged on the electrode surface. Specifically, the working electrode is modified with metal nanoparticles by immersing a screen-printed carbon electrode in 10 mL of 1 mM NaOH solution. The three electrodes are then connected to an electrochemical workstation (CHI660E). Cyclic voltammetry is used to activate and clean the electrode, with a scan range of -0.2 to 0 V, a scan speed of 0.005 V / s, and 12 scan cycles. A mixed solution of 4 mL of 5 mM chloroauric acid (HAuCl4) and 0.5 M H2SO4 was placed in a 5 mL centrifuge tube. The screen-printed working electrode was immersed in this solution, and the three screen-printed electrodes were simultaneously connected to an electrochemical workstation. First, isopotentiometric voltammetry was used at -0.2 V for 20 s, followed by cyclic voltammetry with a scan range of -0.2 to 0.5 V, a scan rate of 0.05 V / s, a step voltage of 0.004 V, and 5 scan cycles. After sequentially undergoing cyclic voltammetry and isopotentiometric voltammetry, the gold-containing anionic groups in the solution were reduced to gold, and metal nanoparticles were gradually formed during the cyclic redox process, densely arranging on the electrode surface.

[0033] The working electrode was modified with an S1-based probe. Specifically, 10 μL of 5 μM S1 probe was added in excess to the working electrode surface and incubated at 37°C for 2 hours. Then, 10 μL of 1 mM MCH was added in excess to the working electrode surface and incubated at 37°C for 0.5 hours. Next, 10 μL of 1 wt% BSA was added in excess to the working electrode surface and incubated at 37°C for 0.5 hours. Finally, 10 μL of TE buffer was added to the working electrode surface three times consecutively, and each time the electrode was immersed at room temperature for 0.5 hours to form an isolation layer against specific binding. After each incubation step, the electrode was gently rinsed 3–5 times with TE buffer. The purpose of each step is to fix the S1 probe to the electrode surface with Au-S bonds; MCH and BSA can block the remaining empty gold sites of unbound DNA probes to eliminate non-specific adsorption as much as possible; and finally, the electrode is immersed in buffer to wash away non-specific binding impurities and ensure the consistency of sensing results.

[0034] (2) Preparation of metal nanoparticles modified with HP1 and S1 probes, the steps are as follows:

[0035] The commercially available metal nanoparticles were approximately 5 nM in concentration and 10–15 nm in size, with an ion concentration of approximately 10 mM in the solution. 8 μL of 20 μM HP1 probe and 2 μL of 20 μM S1 probe (both nucleic acid probes were prepared with TE buffer) were simultaneously added to 290 μL of gold nanoparticle solution. After thorough mixing, the solution was immediately incubated at -20°C for 2 hours. This freezing process allows the negatively charged metal nanoparticles to approach the DNA strands sufficiently without charge repulsion, facilitating contact and binding. After returning to room temperature, the solution was centrifuged three times at 15,000 rpm. The supernatant was collected each time, and the precipitate was dissolved to remove unbound free S1 and HP1 probes. Finally, the solution was brought to a final volume of 100 μL using TE buffer. At this point, the majority of the solute in the solution consisted of metal nanoparticles modified with a specific ratio of S1 and HP1. The Mg2+ concentration in both the nucleic acid solution and the metal nanoparticle solution was [not specified in the original text]. 2+ Na + The plasma concentration must be kept consistent.

[0036] Specific testing procedures:

[0037] The detection process uses a small-sized portable electrochemical detection device, and the hardware block diagram is as follows: Figure 2As shown, the device includes two lithium batteries, a low-ripple power module, a minimum control system for an integrated Bluetooth MCU unit, and an electrochemical detection signal circuit. The device housing is 3D printed and includes a power switch and an electrochemical three-electrode SMA interface. The sensor measures 80×60×40mm. The integrated Bluetooth MCU unit uses Nordic's nRF52832 chip. Its SDK software program includes custom Bluetooth service content and configuration for the electrochemical detection device, including write operations initiated by the PC or mobile phone, and periodic updates of current signal data sent by the device to the PC or mobile phone, as well as configuration parameters such as communication frequency band and interaction period.

[0038] The detection procedure is as follows: First, ensure that S1-based pillars are uniformly distributed on the electrode to be tested. Take 8.8 μL of a metal nanoparticle solution modified with HP1 and S1 probes, add 0.2 μL of 20 μM HP2 auxiliary probe and 1 μL of the nucleic acid solution to be tested, mix well, and wait for the reaction to proceed at room temperature for 1 hour. Then, drop the reaction system onto the surface of the working electrode and incubate at room temperature for 1–2 hours to fully form a richer hierarchical structure. During the incubation process, the hairpins of HP1 are gradually opened from the hairline end by TS, and then the hairpins of HP2 are gradually opened from the hairline end by the unfolded HP1 chain. At the same time, HP2 replaces TS from the HP1 base chain. TS will repeat the above operation at other HP1 sites where the hairpins have not been opened. This is the amplification process of hairpin self-assembly. Furthermore, HP1 and HP2 conjugates are generated on the surface of the metal nanoparticles. Through nucleic acid sequence design, a free single-stranded segment of HP1 is exposed at the outermost end of the entire metal nanosphere. The base of this single-stranded segment binds to S1 modified on the surface of another nanosphere, thereby connecting the two nanoparticles. This process occurs between pairs of nanospheres, forming a complex hierarchical structure. Simultaneously, the exposed HP1 fragment can bind not only to S1 modified on the nanosphere surface but also to S1 fixed on the electrode surface. This cascaded amplification of the hierarchical structure is then attached to the electrode surface. A large number of HP1 and HP2 particles, proportional to the hierarchical structure, are modified with ferrocene near the electrode. Finally, the ferrocene outputs a current signal through a redox reaction. After the incubation period, the working electrode surface was cleaned 3-5 times with TE buffer. The three electrodes were then connected to a portable detection device, and the current signal was acquired using a custom differential pulse voltammetry method. The parameters were set as follows: scan voltage range 0.1-0.5V, step voltage 0.004V, pulse voltage 0.05V, and step period 0.2s.

[0039] In the test results, the current baseline slowly increased in the range of 0.1V to 0.5V, and there was a clear ferrocene oxidation peak at a scanning potential of about 0.3V.

[0040] Since hairpin-catalyzed self-assembly is a crucial step in the amplification reaction of this invention, the electrophoretic results of the amplification reactions of the nucleic acid sequences HP1, HP2, and miRNA-21 involved in the amplification are attached. Figure 3 As shown, samples in lanes 1, 2, and 3 contain HP1, HP2, and miRNA-21, respectively. Samples in lanes 4 and 5, after annealing, yielded hybridization products of HP1 and miRNA-21, and HP1 and HP2, respectively. Lanes 6, 7, and 8 contained samples mixed at room temperature for 1 hour. Comparing lanes 5, 7, and 8, lane 8, containing HP1, HP2, and miRNA-21, also showed hybridization products at the same positions as in lane 5 at room temperature. This indicates that miRNA-21 is indeed a necessary "catalyst" for the amplification reaction, and the catalytic hairpin self-assembly reaction was also verified.

[0041] As attached Figure 4 As shown, the detection effect of this principle is good. In the preliminary detection experiment of miRNA-21, it has good linearity in a wide range of 10fM to 100μM, while the detection limit can reach the order of 1fM.

[0042] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. An electrochemical sensor integrating local hairpin self-assembly and a synergistic amplification strategy of metal nanoparticles, characterized in that, In this sensor, the electrode to be detected is first prepared, and then metal nanoparticles are modified on the electrode to fix the base probe S1 onto the electrode via Au-S bonds. Metal nanoparticles modified with functional hairpin probes HP1 and S1 are then prepared. Specifically, HP1 and S1 are simultaneously added to a metal nanoparticle solution, which is then rapidly placed in an environment at -20°C for 2 hours. The metal nanoparticles, auxiliary probe HP2, and the nucleic acid to be detected are mixed and then incubated on the electrode, triggering local nucleic acid hairpin self-assembly and a cascade amplification reaction of the metal nanoparticles. Redox signal molecules on the functional hairpin probe HP1 and auxiliary hairpin probe HP2 are trapped on the electrode surface, and a current signal is output through the reaction. The metal nanoparticles are gold nanoparticles. The specific sequence is shown below: DNA column probe S1 information: 5'-GTC TGG ACT CAT TTT TTT TTT-3'-SH-HS C6; Functional hairpin probe HP1: SH-HS C6-5'-TCA ACA TCA GTC TGA TAA GCT ATG ATG TTG A-3'-Ferrocene; Auxiliary hairpin probe HP2: Ferrocene-5'-GAG TTC GAG CAG ACT GAT GTT GAC ATT GCT CGAACT CAA CAT CAG-3'.

2. The electrochemical sensor according to claim 1, which integrates local hairpin self-assembly and a synergistic amplification strategy of metal nanoparticles, is characterized in that... The electrode body is a commercially available screen-printed metal or carbon electrode product. It undergoes electrochemical cyclic voltammetry and isopotential bonding to reduce metal ions in solution and form metal nanoparticles, which are then densely arranged on the electrode surface. Thiol-modified S1 is then fixed onto the metal nanoparticle layer of the electrode via drop-coating. Next, 20 mM 6-mercapto-1-hexanol (MCH) and 1% bovine serum albumin (BSA) are sequentially drop-coated onto the S1-fixed electrode to form an anti-specific binding isolation layer. Finally, the electrode, after these steps, is immersed in a TE buffer solution prepared with Tris and EDTA to clean non-specific binding impurities and ensure consistent sensing results.

3. An electrochemical sensor according to claim 1, which integrates local hairpin self-assembly and a synergistic amplification strategy of metal nanoparticles, characterized in that, The concentrations of HP1 and S1 are 3:

1. The ratio of the total amount of nucleic acid in HP1 and S1 to the concentration of nanoparticles is 50~300:

1. The larger the size of the metal nanoparticles, the greater the total amount of nucleic acid.

4. The electrochemical sensor according to claim 1, which integrates local hairpin self-assembly and a synergistic amplification strategy of metal nanoparticles, is characterized in that... HP2 and the target nucleic acid TS were simultaneously added to a solution of metal nanoparticles modified with HP1 and S1, and incubated at room temperature for 1 hour. During incubation, the hairpins of HP1 were gradually opened from the hairline by TS, and then the hairpins of HP2 were gradually opened from the hairline by the unfolded HP1 chain. At the same time, HP2 replaced TS from the base chain of HP1. TS would repeat the above operation at other unopened hairpins of HP1, which is the hairpin self-assembly amplification process. Further, HP1 and HP2 complexes were generated on the surface of the metal nanoparticles. Through nucleic acid sequence design, a free single strand of HP1 was exposed at the outermost end of the entire metal nanosphere. The base of this single strand bound to S1 modified on the surface of another nanosphere, thereby connecting the two nanoparticles. This process occurred between pairs of nanospheres, forming a complex multi-level structure. At the same time, the exposed fragment of HP1 could not only bind to S1 modified on the surface of the nanospheres, but also to S1 fixed on the electrode surface. The multi-level structure amplified in this cascade was then attached to the electrode surface. Ferrocene is modified on a large number of HP1 and HP2 structures that appear near the electrodes in proportion to the multi-level structure. Finally, the ferrocene outputs a current signal through a redox reaction.

5. The electrochemical sensor according to claim 1, which integrates local hairpin self-assembly and a synergistic amplification strategy of metal nanoparticles, is characterized in that... The sensor's detection equipment includes two lithium batteries, a low-ripple power module, an integrated Bluetooth MCU unit minimum control system, and an electrochemical detection signal circuit; the device housing is made by 3D printing and is equipped with a power switch and an electrochemical three-electrode SMA interface. The MCU unit with integrated Bluetooth uses Nordic's nRF52832 chip. In its SDK software program, custom Bluetooth service content and configuration for the electrochemical detection device are written, including content actively written by the PC and mobile phone to the detection device, current signal data content periodically updated by the detection device and sent to the PC and mobile phone, as well as communication frequency band and interaction period parameters.

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