Terahertz probe with target identification and cascade signal amplification functions

Abstract

The invention provides a terahertz probe with target recognition and cascade signal amplification functions, and relates to the field of terahertz detection.The terahertz probe comprises an RCA product part which comprises a Padlock probe and template DNA, the Padlock probe and the template DNA are subjected to cyclization, enzyme digestion and in-situ amplification to form long-chain DNA, and the long-chain DNA serves as a probe framework and is used for achieving first-stage signal amplification; the cascade amplification part is used for forming a secondary signal amplification unit through covalent coupling of gold nanoparticles and the thiolated modified oligonucleotide chain I, and the secondary signal amplification unit is combined with the probe skeleton and is used for realizing secondary signal amplification; the antibody part, an antibody targeting a specific membrane protein and an oligonucleotide chain II modified by sulfhydrylation are covalently coupled to form a recognition unit, and the recognition unit is combined with a probe skeleton to realize specific recognition of the membrane protein. The probe greatly improves the detection sensitivity and reliability of low-abundance biomarkers.

Description

Technical Field

[0001] This invention relates to the field of terahertz detection technology, and more specifically, to a terahertz probe with target identification and cascaded signal amplification functions. Background Technology

[0002] In the fields of biomedical detection and advanced materials, highly sensitive and specific biomolecular detection technologies are crucial for accurate diagnosis and basic research. Existing technologies, such as enzyme-linked immunosorbent assay (ELISA) or traditional fluorescent labeling methods, often face challenges such as insufficient sensitivity, cumbersome operation, or signal quenching when detecting low-abundance biomarkers (e.g., specific membrane proteins). Terahertz waves, due to their unique physical properties, show great potential in non-destructive testing; however, how to convert weak biomolecular interactions into efficient and stable terahertz signal responses is the core bottleneck for their practical application. In developing this invention, the applicant discovered that traditional terahertz probes often lack effective signal amplification mechanisms, resulting in high limits of detection (LODs), making it difficult to meet the stringent requirements of ultra-early diagnosis or single-molecule level detection. Therefore, improving the detection sensitivity and reliability of terahertz probes has become a pressing problem in this technological field. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art or related technologies, and provides a terahertz probe with target recognition and cascaded signal amplification functions, which greatly improves the sensitivity and reliability of the probe.

[0004] This invention is achieved through the following technical solution: a terahertz probe with target recognition and cascade signal amplification functions, comprising: an RCA product portion, including a Padlock probe and template DNA, wherein the Padlock probe and template DNA are circularized, digested with enzymes, and amplified in situ to form a long-chain DNA, which serves as the probe backbone for achieving first-stage signal amplification; a cascade amplification portion, wherein gold nanoparticles are covalently coupled with a thiol-modified oligonucleotide chain I to form a second-stage signal amplification unit, which binds to the probe backbone for achieving second-stage signal amplification; and an antibody portion, wherein an antibody targeting a specific membrane protein is covalently coupled with a thiol-modified oligonucleotide chain II to form a recognition unit, which binds to the probe backbone for achieving specific recognition of the membrane protein.

[0005] This technical solution provides a terahertz probe that combines target recognition and cascaded signal amplification. The core innovation of this probe lies in the construction of a ternary composite structure: antibody-RCA product-gold nanoparticles. It achieves specific recognition through an antibody targeting a specific membrane protein; utilizes rolling circle amplification (RCA) products as a backbone, displaying hundreds of repeating functional units to achieve primary signal amplification; and further couples with gold nanoparticles (AuNPs), leveraging their superior physical properties to generate a secondary signal enhancement effect. This cascaded amplification strategy significantly amplifies the detection signal corresponding to the initial recognition event, thereby greatly improving the probe's sensitivity and reliability.

[0006] The terahertz probe with target recognition and cascaded signal amplification functions provided by the present invention preferably involves incubating the probe skeleton, the recognition unit and the secondary signal amplification unit so that the recognition unit and the secondary signal amplification unit are respectively combined with different positions on the probe skeleton.

[0007] The terahertz probe with target recognition and cascade signal amplification functions provided by the present invention preferably includes the following method for preparing the RCA product portion: after annealing the Padlock probe and template DNA, a closed circular structure is formed by T4 DNA ligase catalysis; and then the closed circular structure is amplified by RCA using Phi29 DNA polymerase to form long-chain DNA.

[0008] The terahertz probe with target recognition and cascaded signal amplification functions provided by the present invention preferably includes the following method for preparing the RCA product portion: First, a cyclization reaction system was prepared, comprising 38.5 μl of nuclease-free water, 5 μl of 10×Phi29 DNA polymerase buffer, 5 μl of 0.01 U / μl T4 DNA ligase, 0.5 μl of 100 mM ATP, 0.5 μl of 100 nM template DNA, and 0.5 μl of 100 nM Padlock oligonucleotide (Padlock probe). The Padlock oligonucleotide is an oligonucleotide formed by 5′ phosphorylation of Padlock as shown in SEQ ID NO.1, and the template DNA is the template DNA shown in SEQ ID NO.2. The ligation reaction was completed by incubating the mixture at 37°C for 30 minutes. To purify the cyclization product, exonuclease I and exonuclease III were added to achieve final concentrations of 0.2 U / μl and 2 U / μl, respectively. The mixture was reacted at 37°C for 30 minutes and then heated at 85°C for 20 minutes to inactivate the enzymes. The RCA reaction was then performed: primers were added to the system at a final concentration of 100 nM, and annealed at 37°C for 20 minutes to allow them to bind to the circular template; dNTPs were added at a final concentration of 1 mM, and phi29 DNA polymerase was added at a concentration of 0.1 U / μl to initiate amplification, and the reaction was carried out at 37°C for 5, 10, 20, or 30 minutes, respectively; finally, the reaction was terminated by heating at 65°C for 10 minutes, and the resulting RCA product was stored at -20°C for later use.

[0009] The terahertz probe with target recognition and cascade signal amplification functions provided by the present invention preferably includes the following preparation method for the antibody portion: Synthesis of antibody-oligonucleotide conjugates via maleimide-thiol chemical reaction: Activation was achieved by incubating 20 μg of antibody with 1 μL of 4 mM Sulfo-SMCC at room temperature for 2 hours; simultaneously, 3 μL of 100 μM thiol-modified oligonucleotide chain II and 12 μL of 100 mM DTT were reduced in PBS containing 5 mM EDTA at 37°C for 1 hour; wherein, the thiol-modified oligonucleotide chain II is an oligonucleotide formed by 3′ thiolization of Olig-II as shown in SEQ ID NO.4. The activated antibody and the reduced thiol-modified oligonucleotide chain II were purified by Zeba desalting column to remove excess reagents. After the purified components were mixed at a preset molar ratio, they were transferred to a Slide-A-Lyzer MINI dialysis device and dialyzed overnight at 4°C against 5 L of PBS buffer containing 0.1% BSA to complete the coupling. Finally, it was stored in PBS containing 0.1% BSA at a concentration of 1 μM and kept at 4°C for later use.

[0010] The terahertz probe with target recognition and cascaded signal amplification functions provided by the present invention preferably includes the following specific methods for preparing the cascaded amplification section: Activation was achieved by co-incubating 100 μl of thiol-modified oligonucleotide chain I with 8 μl of 20 mM TCEP at room temperature for 1 hour; wherein, thiol-modified oligonucleotide chain I is an oligonucleotide formed by 3′ thiolization of Olig-I as shown in SEQ ID NO.3. The activated oligonucleotides were added to 1 ml of a gold nanoparticle solution with a concentration of 2×10 particles / ml, and 5 μl of 4% sodium dodecyl sulfate was added. The mixture was incubated at room temperature for 16 hours. To maximize the DNA loading density, a stepwise salt aging protocol was adopted: 25 μl, 27 μl, 58 μl and 65 μl of 2M sodium chloride solution were added at four time points: 0 hours, 8 hours, 12 hours and 16 hours, respectively.12 Finally, the conjugate was purified by centrifugation at 13,000 rpm for 20 minutes. After discarding the supernatant, the precipitate was washed twice with DEPC water and resuspended in an appropriate amount of DEPC water for later use.

[0011] The terahertz probe with target recognition and cascade signal amplification functions provided by the present invention preferably comprises a long DNA containing hundreds of repeating fragments of Padlock probe and template DNA assembly structures, each repeating fragment containing two functional domains, which are used to bind antibody-conjugated sequences and gold nanoparticle-conjugated sequences, respectively.

[0012] The beneficial effects achieved by this invention include at least the following: A ternary composite structure of "antibody-RCA product-AuNPs" is proposed and constructed for the first time. The antibody achieves specific recognition by targeting a specific membrane protein, improving reliability. The rolling circle amplification (RCA) product serves as a backbone, displaying hundreds of repeating functional units for primary signal amplification. Further coupling with gold nanoparticles (AuNPs) utilizes the excellent physical properties of the gold nanoparticles to generate a secondary signal enhancement effect. Through these two stages of signal amplification, the detection sensitivity is significantly improved. This cascaded amplification strategy significantly amplifies the detection signal corresponding to the initial recognition event, thereby greatly improving the sensitivity and reliability of the probe. This invention provides a powerful tool for the ultrasensitive detection of low-abundance biomarkers and has broad application prospects in early disease diagnosis, drug screening, and biosensing. Attached Figure Description

[0013] Figure 1 A schematic diagram of the overall structure of a terahertz probe with target recognition and cascaded signal amplification functions according to an embodiment of the present invention is shown.

[0014] Figure 2 A schematic diagram illustrating the fabrication process of the RCA product portion of a terahertz probe with target recognition and cascaded signal amplification functions according to an embodiment of the present invention is shown.

[0015] Figure 3 A schematic diagram of an antibody covalently coupled structure of a terahertz probe with target recognition and cascaded signal amplification functions according to an embodiment of the present invention is shown.

[0016] Figure 4 A schematic diagram of a gold nanoparticle covalently coupled structure of a terahertz probe with target recognition and cascaded signal amplification functions according to an embodiment of the present invention is shown.

[0017] Figure 5 A schematic diagram of the unit structure of a terahertz probe with target recognition and cascaded signal amplification functions according to an embodiment of the present invention is shown. Detailed Implementation

[0018] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] like Figure 1 As shown, one embodiment of the present invention discloses a terahertz probe with target recognition and cascade signal amplification functions, comprising: an RCA product portion 1, comprising a Padlock probe and template DNA, wherein the Padlock probe and template DNA are circularized, digested with enzymes and amplified in situ to form a long-chain DNA, which serves as a probe backbone for achieving first-stage signal amplification; a cascade amplification portion 3, wherein gold nanoparticles are covalently coupled with a thiol-modified oligonucleotide chain I to form a second-stage signal amplification unit, which binds to the probe backbone for achieving second-stage signal amplification; and an antibody portion 2, wherein an antibody targeting a specific membrane protein is covalently coupled with a thiol-modified oligonucleotide chain II to form a recognition unit, which binds to the probe backbone for achieving membrane protein-specific recognition.

[0020] like Figure 2 As shown, based on the terahertz probe with target recognition and cascaded signal amplification functions provided in the above embodiments, the RCA product is further constructed through the following steps: First, a cyclization reaction system was prepared, comprising 38.5 μl nuclease-free water (DEPC treated), 5 μl 10× Phi29 buffer, 5 μl T4 DNA ligase (0.01 U / μl), 0.5 μl ATP (100 mM), 0.5 μl template DNA (100 nM), and 0.5 μl Padlock oligonucleotide (100 nM). The mixture was incubated at 37°C for 30 minutes to complete the ligation reaction. To purify the cyclization product, exonucleases I and III were added to a final concentration of 0.2 U / μl and 2 U / μl, respectively. After reacting at 37°C for 30 minutes, the enzymes were inactivated by heating at 85°C for 20 minutes. The RCA reaction was then performed: primers were added to the system at a final concentration of 100 nM, and annealed at 37°C for 20 minutes to bind to the circular template; dNTPs (final concentration 1 mM) and phi29 DNA polymerase (0.1 U / μl) were added to initiate amplification, and the reaction was carried out at 37°C for 5, 10, 20, or 30 minutes, respectively; finally, the reaction was terminated by heating at 65°C for 10 minutes. The resulting RCA product was stored at -20°C for later use. Padlock oligonucleotides are oligonucleotides formed by 5′ phosphorylation of the Padlock sequence in the sequence listing, and the template DNA is the template DNA in the sequence listing.

[0021] like Figure 3As shown, according to the terahertz probe with target recognition and cascade signal amplification functions provided in the above embodiments, the preparation method of the antibody portion specifically includes: Synthesis of antibody-oligonucleotide conjugates via maleimide-thiol chemical reaction: Activation was achieved by incubating 20 μg of antibody with 1 μL of 4 mM Sulfo-SMCC at room temperature for 2 hours; simultaneously, 3′ thiol-modified oligonucleotide (Olig-II; 3 μL 100 μM) and 12 μL 100 mM DTT were reduced in PBS containing 5 mM EDTA at 37°C for 1 hour.

[0022] The activated antibody and reduced Olig-II were purified separately using a Zeba desalting column to remove excess reagents. The purified components were mixed at a predetermined molar ratio and transferred to a Slide-A-Lyzer MINI dialysis unit. Coupling was completed by dialyzing overnight at 4°C against 5 μL of PBS buffer containing 0.1% BSA. The final antibody concentration was 1 μM and stored at 4°C for later use in PBS containing 0.1% BSA.

[0023] In this embodiment, the antibody is the target recognition antibody. The probe of the present invention has antibody flexibility and can achieve the binding ability of the probe to different targets by switching antibody composition. For example, the probe synthesized from CD63 antibody can be used to specifically recognize and amplify the CD63 protein signal.

[0024] like Figure 4 As shown, according to the terahertz probe with target recognition and cascaded signal amplification functions provided in the above embodiments, the preparation method of the cascaded amplification section specifically includes: Activation was achieved by co-incubating 100 μl of 3′-thiolized Olig-I with 8 μl of 20 mM TCEP at room temperature for 1 hour. The activated Olig-I was then added to 1 ml of gold nanoparticle solution (2 × 10⁻⁶ particles / mL), followed by 5 μl of 4% sodium dodecyl sulfate, and incubated at room temperature for 16 hours. To maximize DNA loading density, a stepwise salt aging protocol was used: 25 μl, 27 μl, 58 μl, and 65 μl of 2 M sodium chloride solution were added sequentially at 0, 8, 12, and 16 hours, respectively. Finally, the conjugate was purified by centrifugation at 13000 rpm for 20 minutes. After discarding the supernatant, the precipitate was washed twice with DEPC water and resuspended in an appropriate amount of DEPC water for later use. 12 The terahertz probe with target recognition and cascaded signal amplification functions provided in the above embodiments further includes the following assembly method: The RCA product, the gold nanoparticle conjugated solution, and the antibody conjugated solution were incubated at 4°C for 12 hours in a ratio of 20 μL: 100 μL: 1 mL. After synthesizing the probe, the free gold nanoparticles and antibody were removed by size exclusion chromatography to obtain the purified probe.

[0025] like Figure 5 As shown, in this embodiment, the RCA product includes two sequences having hundreds of copies, one that can bind to an antibody and the other that can bind to gold nanoparticles.

[0026] According to another embodiment of the present invention, a verification process for the fabrication process of the terahertz probe with target recognition and cascaded signal amplification functions provided in the above embodiments is also disclosed: The RCA product exhibits a clustered structure, with its average size gradually increasing with incubation time. The zeta potential of the antibody significantly decreased after conjugation with the targeting antibody, due to the negative charge of the nucleic acid chain, confirming successful antibody-nucleic acid conjugation. Nanoparticle tracking analysis (NTA) further compared the hydrodynamic diameter changes of gold nanoparticles before and after Olig-I conjugation, showing an increase in particle size from 94 nm to 104 nm, indicating successful oligonucleotide modification of the gold nanoparticle surface. To systematically validate the probe assembly process, the morphology of the RCA product synthesized for 10 minutes was characterized using scanning electron microscopy (SEM). The product exhibited a typical rounded and branched chain structure, indicating that the RCA reaction successfully generated a large DNA molecule with a complex secondary structure. Furthermore, to verify the specific binding of the RCA product to the antibody, 10 μL of the RCA product was co-incubated with 10 μL of Olig-II-conjugated CD63 antibody for 1 hour, followed by incubation with fluorescently labeled secondary antibody for another hour. Confocal fluorescence microscopy revealed uniformly distributed fluorophores approximately 2 μm in size, consistent with the morphology of the RCA product observed under dark-field microscopy. In contrast, the control group (incubated with only secondary antibody and RCA product) showed no obvious fluorophores, confirming a specific hybridization reaction between the RCA product and the target antibody. Finally, to investigate the coupling effect between the RCA product and gold nanoparticles, gold nanoparticles modified with oligonucleotide sequence II were co-incubated with the RCA product. Transmission electron microscopy revealed a network structure approximately 2 μm in diameter containing a large number of metal particles tens of nanometers in size, indicating that the RCA product successfully combined with the gold nanoparticles through multivalent hybridization, forming a probe assembly with signal amplification capabilities. Its application in terahertz sensing can verify its superior target specificity and signal amplification ability.

[0027] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A terahertz probe with target recognition and cascade signal amplification function, characterized in that, Comprise: RCA product part, comprising padlock probe and template DNA, the padlock probe and the template DNA are cyclized, enzyme cut and in situ amplified to form long chain DNA as probe skeleton for realizing first level signal amplification; Cascade amplification part, gold nanoparticles and thiol-modified oligonucleotide chain I are covalently coupled to form secondary signal amplification unit, the secondary signal amplification unit is combined with the probe skeleton for realizing second level signal amplification; Antibody part, antibody targeting specific membrane protein and thiol-modified oligonucleotide chain II are covalently coupled to form recognition unit, the recognition unit is combined with the probe skeleton for realizing membrane protein specific recognition. 2.The terahertz probe with target recognition and cascade signal amplification functions according to claim 1, wherein, The probe skeleton, the recognition unit and the secondary signal amplification unit are incubated to make the recognition unit and the secondary signal amplification unit combined with different positions on the probe skeleton respectively. 3.The terahertz probe with target recognition and cascade signal amplification functions according to claim 1, wherein, The preparation method of the RCA product part comprises: After the padlock probe and the template DNA are annealed, a closed ring structure is formed by T4 DNA ligase catalysis, and then the closed ring structure is subjected to RCA amplification by Phi29 DNA polymerase to form the long chain DNA. 4.The terahertz probe with target recognition and cascade signal amplification functions according to claim 3, characterized in that, The preparation method of the RCA product part specifically comprises: First, a cyclization reaction system is prepared, which comprises 38.5 μl of nuclease-free water, 5 μl of 10× Phi29 DNA polymerase buffer, 5 μl of T4 DNA ligase with a concentration of 0.01 U / μl, 0.5 μl of ATP with a concentration of 100 mM, 0.5 μl of template DNA with a concentration of 100 nM and 0.5 μl of padlock oligonucleotide with a concentration of 100 nM, wherein the padlock oligonucleotide is an oligonucleotide formed by 5' phosphorylation modification of the padlock as shown in sequence SEQ ID NO. 1, and the template DNA is a template DNA as shown in sequence SEQ ID NO. 2; The system is incubated at 37°C for 30 minutes to complete the ligation reaction; to purify the cyclization product, exonuclease I and exonuclease III are added to make their final concentrations reach 0.2 U / μl and 2 U / μl respectively, and after 37°C reaction for 30 minutes, enzyme inactivation is performed by heating at 85°C for 20 minutes; Then, RCA reaction is performed: the primer is added to the system at a final concentration of 100 nM, and annealing is performed at 37°C for 20 minutes to make it combined with the circular template; dNTPs are added at a final concentration of 1 mM and phi29 DNA polymerase at a concentration of 0.1 U / μl to start amplification, and reaction is performed at 37°C for 5, 10, 20 or 30 minutes respectively; Finally, the reaction is terminated by heating at 65°C for 10 minutes, and the obtained RCA product is stored at -20°C for standby. 5.The terahertz probe with target recognition and cascade signal amplification functions according to claim 1, wherein, The preparation method of the antibody part specifically comprises: The antibody-oligonucleotide conjugate is synthesized by maleimide-thiol chemical reaction: The activation is completed by incubating 20 μg of the antibody with 1 μL of 4 mM Sulfo-SMCC at room temperature for 2 hours; meanwhile, 3 μL of 100 μM of the thiol-modified oligonucleotide chain II is reduced with 12 μL of 100 mM DTT in PBS containing 5 mM EDTA at 37 °C for 1 hour; wherein the thiol-modified oligonucleotide chain II is an oligonucleotide formed by 3' thiol-modification of Olig-II as shown in SEQ ID NO. 4; The activated antibody and the reduced thiol-modified oligonucleotide chain II are purified by Zeba desalting columns respectively to remove excess reagents; After mixing the purified components in a preset molar ratio, the mixture is transferred to a Slide-A-Lyzer MINI dialysis device and dialyzed against 5 L of PBS containing 0.1% BSA at 4 °C overnight to complete the coupling; Finally, the antibody is stored in PBS containing 0.1% BSA at a concentration of 1 μM at 4 °C for standby. 6.The terahertz probe with target recognition and cascade signal amplification functions according to claim 1, wherein, The preparation method of the cascade amplification part specifically comprises: The activation is completed by incubating 100 μL of the thiol-modified oligonucleotide chain I with 8 μL of 20 mM TCEP at room temperature for 1 hour; wherein the thiol-modified oligonucleotide chain I is an oligonucleotide formed by 3' thiol-modification of Olig-I as shown in SEQ ID NO. 3; The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml of gold nanoparticle solution at a concentration of 2 x 10 12 The activated oligonucleotide was added to 1 ml Finally, the conjugate is purified by centrifugation at 13,000 rpm for 20 minutes, the supernatant is discarded, the precipitate is washed twice with DEPC water, and then resuspended in an appropriate amount of DEPC water for standby.

7. The terahertz probe with target recognition and cascade signal amplification functions according to any one of claims 1 to 6, characterized in that, The long-chain DNA comprises hundreds of repeated fragments of the assembly structure of the Padlock probe and the template DNA, each of which comprises two functional domains for binding to the antibody-coupled sequence and the gold nanoparticle-coupled sequence.

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