A terahertz-based enzyme-free detection method and kit for peripheral blood free microRNAs

Abstract

The present invention discloses a terahertz-based, enzyme-free detection method and kit for peripheral blood free microRNAs. By designing a rational size and utilizing nanoparticles and magnetic nanoparticles to assemble into a 3D nanomagnetic network, the mismatch between the terahertz wavelength and the physical diffraction scale of the miRNAs to be detected is resolved. The surface plasmon resonance caused by the high refractive index of the nanoparticles and the electromagnetic response mechanism of the magnetic nanoparticles achieve a dual amplification effect of the THz metamaterial's resonance peak frequency shift signal, thus resolving the sensitivity issues of enzyme-free, amplification-free detection of miRNAs in biological samples. The biosensor of the present invention has high sensitivity, strong specificity, and can achieve high-throughput detection, providing a technical platform with greater clinical application prospects for highly reproducible and high-throughput detection of miRNAs.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to a method for detecting free microRNAs in peripheral blood without enzymes based on terahertz, and also to a kit for detecting free microRNAs in peripheral blood without enzymes based on terahertz. Background Art

[0002] MicroRNAs (microRNAs) are endogenous, single-stranded, short, non-coding RNA molecules, approximately 21-25 nt in length, that regulate cell growth, apoptosis, and signaling through gene regulation. Numerous studies have demonstrated that abnormal expression of various microRNA molecules can be detected in both tumor tissue and peripheral blood of cancer patients, demonstrating their potential as highly sensitive and specific tumor markers, playing an important role in predicting tumor development, progression, and drug sensitivity. Currently, the most established methods for detecting microRNA in peripheral blood include cloning sequencing, real-time PCR, Northern blot hybridization, and microarray technology. Detection of trace amounts of microRNA in peripheral blood typically requires amplification, but the short length of microRNA sequences makes conventional PCR difficult. While real-time PCR methods based on stem-loop and poly A tailing are available, these specialized amplification techniques are difficult to implement and widely adopted.

[0003] The frequency of terahertz (THz) waves is 0.1-10THz (1THz=10 12 THz (terahertz) is a range of electromagnetic waves that spans the high-frequency microwave region and the low-frequency far-infrared region. Numerous studies have shown that the weak interactions within or between various biomacromolecules within cells, such as amino acids, DNA, RNA, proteins, and peptides, as well as the vibrational and rotational energy levels between their skeletons, lie precisely within the THz band. Therefore, THz waves can be used to characterize various biomacromolecules, making THz technology a promising new, rapid, label-free, and non-invasive spectroscopic detection method in biomedical testing. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a method for detecting free microRNAs in peripheral blood without enzymes based on terahertz; a second objective of the present invention is to provide a kit for detecting free microRNAs in peripheral blood without enzymes based on terahertz.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A terahertz-based method for enzyme-free detection of free microRNAs in peripheral blood. This method uses nanomagnetic beads modified with a three-ring linked hairpin probe to identify the target microRNA. This is then combined with a gold nanoparticle-modified bridge probe to form a 3D nanomagnetic network of nanomagnetic beads and gold nanoparticles, which is then detected using a terahertz metamaterial nanobiosensor.

[0007] The terahertz metamaterial nanobiosensor contains a THz metamaterial chip. The THz metamaterial chip uses high-resistance silicon as the substrate and photolithography technology to deposit metal on the substrate surface to form a metal square open resonant ring. The inner ring of the resonant ring is symmetrically open on the four sides, and the outer ring is symmetrically open at the four corners.

[0008] Preferably, the nanomagnetic bead modified three-ring linked hairpin probe is combined with a three-ring linked hairpin probe and a nanomagnetic bead, and the three-ring linked hairpin probe is composed of four probe sequences, respectively recorded as Seq A, Seq B, Seq C and Seq D, wherein Seq A, Seq B and Seq C are hairpin structures and the 3' end of one of the sequences is modified with an amino group, and the hairpin structure includes a stem-loop region, a complementary region and a hybridization region, the stem-loop region is complementary to the target microRNA molecule to be detected, the complementary region is located at both ends of the stem-loop region, and is reverse-complemented to each other after annealing to form a double-stranded structure, the hybridization region is located at the 3' end of the hairpin structure sequence, and is divided into hybridization region I and hybridization region II; Seq D includes hybridization region I and hybridization region II, and the hybridization region I and hybridization region II of Seq A, Seq B, Seq C and Seq D are cross-complementary, and hybridization forms a three-ring linked hairpin probe modified nanomagnetic bead.

[0009] Preferably, the gold nanoparticle-modified bridge probe is composed of a thiol nucleic acid probe, a bridge probe, and gold nanoparticles; the 3' end of the thiol nucleic acid probe is modified with a thiol group, and the thiol nucleic acid probe is complementary to one end of the bridge probe; the free end of the bridge probe is complementary to the inverted repeat sequence of the three-ring linked hairpin probe.

[0010] Preferably, in the present invention, the resonant rings are arranged in an array to form a detection area.

[0011] Preferably, the thickness of the metal deposition is 200 nm.

[0012] Preferably, in the present invention, the unit area of the detection area is 1×1 cm.

[0013] Preferably, the metal is a high refractive index metal particle, and the metal particle is selected from Au, Ag or Fe.

[0014] Preferably, the terahertz metamaterial nanobiosensor further comprises a fixture for the THz metamaterial nanobiosensor, the fixture comprising a circular base and a chip carrier, the circular base comprising a central hole for THz radiation, an XY directional V-groove, and a hole for fixing to a bracket, the chip carrier having a positioning boss that can slide on the XY directional V-groove to align each independent metal resonant ring sample detection area with the central hole of the circular base.

[0015] 2. A terahertz-based, enzyme-free kit for detecting free microRNAs in peripheral blood, comprising a terahertz metamaterial nanobiosensor, a nanomagnetic bead-modified three-ring linked hairpin probe, and a gold nanoparticle-modified bridge probe;

[0016] The terahertz metamaterial nanobiosensor comprises a THz metamaterial chip. The THz metamaterial chip uses high-resistance silicon as a substrate and uses photolithography technology to deposit metal on the surface of the substrate to form a metal square open resonant ring. The inner ring of the resonant ring is symmetrically open on the four sides, and the outer ring is symmetrically open at the four corners.

[0017] The nanomagnetic bead modified three-ring linked hairpin probe is combined with a three-ring linked hairpin probe and a nanomagnetic bead. The three-ring linked hairpin probe is composed of four probe sequences, respectively recorded as Seq A, Seq B, Seq C and Seq D. The Seq A, Seq B and Seq C are hairpin structures and the 3' end of one of the sequences is modified with an amino group. The hairpin structure includes a stem-loop region, a complementary region and a hybridization region. The stem-loop region is complementary to the target microRNA molecule to be detected. The complementary region is located at both ends of the stem-loop region and forms a double-stranded structure after reverse complementation annealing. The hybridization region is located at the 3' end of the hairpin structure sequence and is divided into hybridization region I and hybridization region II. The Seq D includes hybridization region I and hybridization region II. The hybridization region I and hybridization region II of Seq A, Seq B, Seq C and Seq D are cross-complementary and hybridize to form a three-ring linked hairpin probe modified nanomagnetic bead.

[0018] The gold nanoparticle-modified bridge probe consists of a thiol nucleic acid probe, a bridge probe, and gold nanoparticles. The 3' end of the thiol nucleic acid probe is modified with a thiol group, and the thiol nucleic acid probe is complementary to one end of the bridge probe; the free end of the bridge probe is complementary to the inverted repeat sequence of the three-ring linked hairpin probe.

[0019] Preferably, the microRNAs are miRNA-1290, miRNA-21, and miRNA-320a; the sequence of Seq A is shown in SEQ ID NO.1; the sequence of Seq B is shown in SEQ ID NO.2, and the sequence of Seq C is shown in SEQ ID NO.3; the sequence of Seq D is shown in SEQ ID NO.4; the sequence of the thiol nucleic acid probe is shown in SEQ ID NO.5; and the nucleotide sequence of the bridging probe is shown in SEQ ID NOs.6 to 8.

[0020] The present invention provides a terahertz-based, enzyme-free method for detecting free microRNAs in peripheral blood. This method can detect miRNAs in peripheral blood with high sensitivity and specificity, resolving the problem of conventional amplification detection, which is technically difficult and hinders widespread application. Array chip technology effectively addresses the low-throughput technical challenges of transmission detection using existing THz time-domain spectrometers, enabling high-throughput detection of nine different miRNAs simultaneously. This provides a technical platform for highly reproducible, high-throughput detection of miRNAs with promising clinical applications.

[0021] More importantly, the present invention assembles nanoparticles (such as Au, Ag, Fe, etc.) and magnetic nanoparticles into a 3D nanomagnetic network. The size of the assembled structure can be at the micron level, which perfectly solves the mismatch problem between the terahertz wavelength and the physical diffraction scale of the miRNAs to be detected; the surface plasmon resonance caused by the high refractive index of the formed nanoparticles and the electromagnetic response mechanism of the magnetic nanoparticles achieve a dual amplification effect of the THz metamaterial resonance peak frequency shift signal, solving the sensitivity problem of enzyme-free and amplification-free detection of miRNA in biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0023] Figure 1 This is a structural diagram of the array-type THz metamaterial chip;

[0024] Figure 2 This is the fixture structure diagram;

[0025] Figure 3 Schematic diagram of the detection principle of enzyme-free terahertz metamaterial biosensor based on 3D nanomagnetic network and THz metamaterial sensing technology;

[0026] Figure 4Terahertz spectrum detection diagram of miRNA-1290 (Bare meta is the THz spectrum curve of the blank metamaterial chip; meta+N is the THz spectrum curve of the blank control sample on the metamaterial chip; meta+net is the THz spectrum curve of the 3D nanomagnetic network sample on the metamaterial chip);

[0027] Figure 5 This is the result diagram of miRNA detection sensitivity validation. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0029] Example 1: Construction of a terahertz metamaterial nanobiosensor

[0030] The construction of terahertz (THz) metamaterial nanobiosensor, the specific structure is as follows Figure 1 As shown, the specific structure is:

[0031] The THz metamaterial chip uses a high-resistance silicon substrate. Photolithography is used to deposit metal (such as chromium) onto the substrate surface, forming a 200nm thick metal layer with a 1x1cm square open-ring resonant ring area. The inner ring of the resonant ring is symmetrically open on the four sides, and the outer ring is symmetrically open at the four corners. The resonant rings are arranged in an array to form detection zones. Nine independent arrayed square detection zones enable high-throughput THz spectroscopy detection of nine microRNAs.

[0032] The present invention also includes a fixture for the THz metamaterial nanobiosensor, which is a special fixture made based on the detection module of the THz time-domain spectrometer and the above-mentioned THz metamaterial nanobiosensor structure, and can directionally move the position of the above-mentioned metamaterial chip. The fixture is an aluminum structure and includes a circular base and a chip carrier. Figure 2 As shown, the circular base includes a central hole for THz radiation, an XY directional V-groove, and four small holes for fixing to the bracket. The chip carrier slides on the XY directional V-groove via its positioning bosses, aligning the detection area of each independent metal resonant ring sample with the central hole of the circular base.

[0033] Example 2: High-throughput detection method for detecting free miRNAs in peripheral blood

[0034] The THz metamaterial nanobiosensor designed in Example 1 is used to detect free miRNAs in peripheral blood in a high-throughput detection method without enzyme. The principle is as follows: Figure 3 As shown, the following steps are included:

[0035] (1) Three-loop linked hairpin probes (TH-probes) were designed to specifically capture three or more miRNAs (miRNA-1290, miRNA-21, and miRNA-320a). A single hairpin probe consisted of four probe sequences, which were designated as Seq A, Seq B, Seq C, and Seq D. Among them, Seq A, Seq B and Seq C are hairpin structures, including a stem-loop region, a complementary region and a hybridization region, wherein the stem-loop region is complementary to the target microRNA molecule to be detected and is used to identify the target microRNA to be detected, and the complementary region is located at both ends of the stem-loop region, and is reverse complementary to each other to form a double-stranded structure after annealing. The hybridization region is located at the 3' end of the sequence and is divided into hybridization region I and hybridization region II; wherein Seq D includes hybridization region I and hybridization region II, and the hybridization region I and hybridization region II of Seq A, Seq B, Seq C and Seq D are cross-complementary to form a three-ring linked hairpin probe, and the 3' end of the sequence hybridized with the 5' end of Seq A, Seq B and Seq C is modified with an amino group, which can form a covalent coupling with the abundant carboxyl groups on the surface of the nanomagnetic beads, and after hybridization, a three-ring linked hairpin probe modified nanomagnetic beads is formed;

[0036] (2) designing a thiol nucleic acid probe and a bridge probe, wherein one end of the bridge probe is complementary to the thiol nucleic acid probe, and the other end of the bridge probe is complementary to the complementary region of the hairpin probe, and the thiol nucleic acid is combined with gold nanoparticles via an Au-S bond to form a nanoprobe;

[0037] (3) Seq A, Seq B, Seq C, Seq D in step (1) are mixed with nanomagnetic beads, and covalent coupling is formed through amino and carboxyl groups. The four chains are hybridized with the nanomagnetic beads to form three-ring linked hairpin probe modified nanomagnetic beads. The reaction conditions are 95°C for 10 minutes, and the temperature is quickly reduced to 0°C. Then, a solution containing the target microRNA to be detected and the nanoprobe of step (2) is added. Under the triggering of the target microRNA to be detected, the free end of the bridge probe of the nanoprobe is connected to the complementary region of the three-ring linked hairpin probe modified nanomagnetic beads to form a 3D nanomagnetic network of nanomagnetic beads and gold nanoparticles cross-stacked. The reaction conditions are oscillating reaction at 35°C for 2 hours.

[0038] (4) adding the reaction solution after the reaction in step (3) to the detection area of the array metamaterial chip, and drying the sample at 50° C. for 5-10 minutes to remove moisture from the sample;

[0039] (5) Place the THz metamaterial chip processed in step (4) on the chip fixture, and realize THz spectrum detection by moving the chip on the XY directional V groove. At the same time, use a blank metamaterial chip for comparison. The detection results are as follows: Figure 4As shown in the figure, bare meta is the THz spectrum curve of a blank metamaterial chip; meta+N is the THz spectrum curve of a blank control sample on the metamaterial chip; and meta+net is the THz spectrum curve of a 3D nanomagnetic network sample on the metamaterial chip. The transmission resonance peak of the blank THz metamaterial is located at 0.857 THz. The transmission resonance peak of the 3D nanomagnetic network reaction system formed after the addition of the target miRNA-1290 is shifted to the left by 31.4 GHz.

[0040] In the present invention, the above probes were designed by taking the detection of miRNA-1290 as an example, and the probe sequences are shown in Table 1.

[0041] Table 1. Probe sequences

[0042]

[0043] In the present invention, the biological sample to be tested can be at least one of a patient's peripheral blood miRNAs extraction sample, a healthy person's blood miRNAs extraction sample, an artificially synthesized miRNA target sequence, and an artificially synthesized single-stranded NC sequence.

[0044] Example 3: Sensitivity Verification of Enzyme-Free THz Metamaterial Nanobiosensor for Detecting miRNAs

[0045] Using gradient diluted miRNA-1290 as a template, the sensitivity of the enzyme-free THz metamaterial nanobiosensor and its detection method for detecting miRNAs was verified. The specific steps are as follows:

[0046] (1) Oligo miRNA-1290 (synthesized by TaKaRa) was serially diluted to final concentrations of 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 100 aM, and 10 aM as samples for sensitivity verification experiments;

[0047] (2) Using the THz metamaterial nanobiosensor in Example 1 for detection, Seq A, Seq B, Seq C, Seq D and nanomagnetic beads were mixed, and covalent coupling was formed through amino and carboxyl groups. The four chains were hybridized to form a three-ring linked hairpin probe modified nanomagnetic beads. The reaction conditions were 95°C for 10 min and the temperature was quickly reduced to 0°C;

[0048] (3) Add the gradient diluted miRNA-1290 sample in (1) to the three-ring linked hairpin probe modified nanomagnetic bead solution, and then add the bridge probe Sup-probe solution in sequence. After the reaction solution is placed on a vortex mixer and mixed, it is shaken at 35°C for 2 hours. Under the triggering of the target microRNA to be tested, the bridge probe is connected to the three-ring linked hairpin probe modified nanomagnetic bead to form a 3D nanomagnetic network of nanomagnetic beads and gold nanoparticles;

[0049] (4) adding the reaction solution after the reaction in step (3) to the detection area of the THz metamaterial chip, and drying the sample at 50° C. for 5-10 minutes to remove moisture from the sample;

[0050] (5) Place the THz metamaterial chip processed in step (4) on the chip fixture, and realize THz spectrum detection by moving the chip on the XY directional V groove. At the same time, use a blank metamaterial chip for comparison. The detection results are as follows: Figure 5 Figure 2: Quantitative detection of a synthetic miRNA-1290 sequence using a metamaterial structure. Results show a good linear relationship between the THz frequency shift and the logarithm of the target concentration, Log(C), over a range of 0.1 fM to 1 pM. The linear fit equation is Δf = 5.99Log(C) + 12.44, with a correlation coefficient R² of 0.9962 and a LOD of 64 aM. These studies demonstrate that incorporating a 3D nanomagnetic network onto a metamaterial surface can effectively enhance detection sensitivity.

[0051] The enzyme-free terahertz metamaterial nanobiosensor and detection kit for detecting free microRNAs in peripheral blood, described in this invention, utilizes a uniquely designed "modified 3D nanonetwork" and arrayed metamaterial chip device to effectively address the sensitivity challenges of enzyme-free, non-amplification-based microRNA detection in biological samples, as well as the low-throughput technical challenges of THz time-domain spectroscopy transmission detection. The technical principle is to utilize DNA self-assembly technology to integrate metal and magnetic nanoparticles to construct a "modified 3D nanonetwork," forming a highly controllable composite nanostructure in liquid samples. This composite nanostructure is suitable for the quantitative analysis of nucleic acid markers in liquid biological samples. When this structure exists within the highly dielectrically sensitive and locally plasmon-enhanced electromagnetic environment of the metamaterial surface, the introduction of nanomagnetic beads (Fe₃O₄) significantly enhances the refractive index of the 3D nanonetwork composite structure, compared to commonly used single metal nanoparticle aggregates (such as gold and silver nanoclusters), thereby increasing the metamaterial's THz resonant response. Furthermore, the 3D nanonetwork structure integrates nucleic acid-based biomolecules with a variety of heterogeneous metal nanoparticles through self-assembly technology, achieving micron-scale dimensions after assembly, perfectly resolving the mismatch between terahertz wavelengths and the physical diffraction scales of biomolecules. More importantly, under electromagnetic wave excitation on the metamaterial surface, while single noble metal nanoparticles can only support electric dipole resonance modes, the 3D nanonetwork constructed in this study can provide well-tuned electric and magnetic dipole dual resonance modes, thereby exhibiting richer scattering properties than single-component particles. This represents a novel THz signal-enhancing structure with a larger surface area. It not only generates a high-intensity and high-sensitivity THz metamaterial response signal, but also significantly improves signal reproducibility.

[0052] Its uniqueness lies in the fact that, while ensuring detection sensitivity, the metamaterial chip constructed in this invention, with multiple independent, arrayed detection areas, can be directionally moved on a V-groove fixture using a chip carrier. Multi-sample detection can be performed by aligning the individual detection areas, the carrier's central hole, and the entrance aperture. This method eliminates the tedious cleaning and drying processes of the metamaterial chip and centrally integrates the detection of multiple microRNA targets on a single metamaterial chip, achieving miniaturized, high-throughput detection of biological samples.

[0053] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A kit for detecting free microRNAs in peripheral blood based on terahertz without enzyme, characterized by: The kit includes a terahertz metamaterial nanobiosensor, a nanomagnetic bead-modified three-ring linked hairpin probe, and a gold nanoparticle-modified bridge probe; The terahertz metamaterial nanobiosensor contains a THz metamaterial chip. The THz metamaterial chip is formed by depositing metal on the surface of a high-resistance silicon substrate using photolithography technology to form a metal square open resonant ring. The inner ring of the resonant ring is symmetrically open on the four sides, and the outer ring is symmetrically open at the four corners. The nanomagnetic bead modified three-ring linked hairpin probe is combined with a three-ring linked hairpin probe and a nanomagnetic bead, and the three-ring linked hairpin probe is composed of four probe sequences, respectively recorded as Seq A, Seq B, Seq C and Seq D, wherein Seq A, Seq B and Seq C are hairpin structures and the 3' end of one of the sequences is modified with an amino group, the hairpin structure includes a stem-loop region, a complementary region and a hybridization region, the stem-loop region is complementary to the target microRNA molecule to be detected, the complementary region is located at both ends of the stem-loop region, and forms a double-stranded structure after reverse complementation annealing, the hybridization region is located at the 3' end of the hairpin structure sequence, and is divided into hybridization region I and hybridization region II; the Seq D includes hybridization region I and hybridization region II, and the hybridization region I and hybridization region II of Seq A, Seq B, Seq C and Seq D are cross-complementary, and hybridization forms a three-ring linked hairpin probe modified nanomagnetic bead; the sequence of Seq A is shown in SEQ ID NO.1; the sequence of Seq B is shown in SEQ ID NO.2, Seq The sequence of C is shown in SEQ ID NO.3; the sequence of Seq D is shown in SEQ ID NO.4; the microRNAs are miRNA-1290, miRNA-21, and miRNA-320a; the bridge probe modified with gold nanoparticles is composed of a thiol nucleic acid probe, a bridge probe, and gold nanoparticles, the 3' end of the thiol nucleic acid probe is modified with a thiol group, and the thiol nucleic acid probe is complementary to one end of the bridge probe; the free end of the bridge probe is complementary to the inverted repeat sequence of the three-ring linked hairpin probe; the sequence of the thiol nucleic acid probe is shown in SEQ ID NO.5; the nucleotide sequence of the bridge probe is shown in SEQ ID NOs.6~8.

2. The kit for detecting free microRNAs in peripheral blood based on terahertz without enzyme according to claim 1, characterized in that: The resonant rings are arranged in an array to form a detection area.

3. The kit for detecting free microRNAs in peripheral blood based on terahertz without enzyme according to claim 1, characterized in that: The thickness of the metal deposition is 200 nm.

4. The kit for detecting free microRNAs in peripheral blood based on terahertz without enzyme according to claim 1, characterized in that: The unit area of the detection area is 1×1 cm.

5. The kit for detecting free microRNAs in peripheral blood based on terahertz without enzyme according to claim 1, characterized in that: The metal is a high refractive index metal particle, and the metal particle is selected from Au, Ag or Fe.

6. The kit for detecting free microRNAs in peripheral blood based on terahertz without enzyme according to claim 1, characterized in that: The terahertz metamaterial nanobiosensor also includes a THz metamaterial nanobiosensor fixture, which includes a circular base and a chip carrier. The circular base includes a central hole for THz radiation, an XY directional V-groove, and a hole for fixing to a bracket. The chip carrier has a positioning boss that can slide on the XY directional V-groove to align each independent metal resonant ring sample detection area with the central hole of the circular base.

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