Sputum pretreatment method for rapid nucleic acid detection

Abstract

The invention relates to the technical field of biomedical detection, and discloses a sputum pretreatment method for rapidly carrying out nucleic acid detection, and the method comprises the following steps: directly adding a dry intelligent infinitesimal into sputum; applying a low-frequency rotating magnetic field to drive infinitesimal spiral implantation; switching a high-frequency magnetic field to realize in-situ liquefaction and nucleic acid release by utilizing mechanical vibration and piezoelectric potential; adsorbing nucleic acid based on an electrowetting effect, and separating and eluting through a gradient magnetic field; and carrying out isothermal amplification detection on the purified nucleic acid. The intelligent asymmetric magnetoelectric coupling infinitesimal element is used as an execution medium, multiple effects of external dynamic magnetic field frequency modulation, coupling mechanical shearing, acoustic streaming capture and electric field breakdown are matched, physical viscosity reduction of the sample and efficient extraction of nucleic acid are achieved under the condition of no exogenous liquefying agent, dilution-free pretreatment of the high-viscosity sample is achieved, and the detection accuracy is improved. The detection sensitivity of low-abundance pathogens can be improved, and the inhibition of chemical reagents can be avoided.

Description

Technical Field

[0001] This invention relates to the field of biomedical testing, specifically to a sputum pretreatment method for rapid nucleic acid testing. Background Technology

[0002] Sputum, as an important clinical sample for the diagnosis of respiratory diseases, contains a variety of biological components such as exfoliated cells, pathogenic microorganisms, and inflammatory secretions. Nucleic acid extraction and amplification detection of sputum samples is currently the gold standard for diagnosing lower respiratory tract infection pathogens such as Mycobacterium tuberculosis and Streptococcus pneumoniae. Sputum pretreatment is a key step that determines the sensitivity and accuracy of nucleic acid detection.

[0003] Current technologies for sputum sample pretreatment mainly employ a combined strategy of chemical liquefaction and mechanical dispersion. Typically, chemical reagents such as dithiothreitol, N-acetyl-L-cysteine, and proteases are used to disrupt the mucin structure to achieve liquefaction. Subsequently, vortexing or centrifugation is used to separate the precipitate, followed by adsorption and elution of nucleic acids using magnetic beads or centrifuge columns. Some automated equipment also incorporates physical methods such as ultrasonic waves or glass bead grinding to assist in cell lysis, attempting to complete the transformation from high-viscosity sputum to a detectable nucleic acid solution in a shorter time.

[0004] However, due to the significant non-Newtonian fluid properties and extremely high zero-shear viscosity of sputum, existing chemical liquefaction methods often require the addition of an equal or even double volume of liquefying agent or buffer to the sputum to ensure sufficient contact between the reagent and the sample. This introduction of additional liquid media further dilutes the already scarce pathogen concentration in the original sample, greatly increasing the risk of false negatives in the detection of low-abundance pathogens. At the same time, if residual denaturants or proteases in the chemical lysis system are not completely removed, they can easily inhibit the activity of polymerases in the subsequent amplification system. In addition, the movement of traditional magnetic beads is restricted in high-viscosity media that have not been fully liquefied, making it difficult to overcome fluid resistance to achieve active dispersion and target capture, resulting in nucleic acid extraction efficiency that cannot meet the requirements of rapid and highly sensitive detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a sputum pretreatment method for rapid nucleic acid detection, aiming to solve the problems in existing technologies such as sample dilution caused by the introduction of additional liquid media, which reduces detection sensitivity, chemical reagent residues inhibiting amplification reaction activity, and the difficulty of actively dispersing traditional extraction media in high-viscosity samples.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a sputum pretreatment method for rapid nucleic acid detection. This method targets high-viscosity sputum samples and utilizes intelligent asymmetric magnetoelectric coupling micro-elements as the execution medium for multi-physics field responses. Under dynamic magnetic field modulation, it achieves undiluted implantation, in-situ lysis, and nucleic acid extraction of the sample. The method includes the following steps: Constructing an undiluted solid-liquid mixture: Dry, intelligent asymmetric magnetoelectric coupling micro-element was directly added to a high-viscosity sputum sample. Utilizing the low surface energy of the potential-responsive molecular layer modified on the surface of the intelligent asymmetric magnetoelectric coupling micro-element, the micro-element surface exhibited a hydrophobic state without external field excitation. This hydrophobic property was used to block interfacial wetting and non-specific adhesion between the micro-element and the mucin network in the sputum, forming a heterogeneous solid-liquid mixture with the intelligent asymmetric magnetoelectric coupling micro-element dispersed on the sputum surface without adding any liquid medium and thus diluting the sample concentration.

[0007] Deep implantation based on low-frequency precession mechanism: A low-frequency rotating magnetic field is applied to the heterogeneous solid-liquid mixture. The frequency of this magnetic field is set within a range that avoids mechanical resonance of the micro-element. Utilizing the non-centrosymmetry of the geometric configuration of the intelligent asymmetric magnetoelectric coupling micro-element (such as a unilateral cone or tadpole shape), when the micro-element is driven to rotate rigidly by the rotating magnetic field, the fluid resistance on the surface of the micro-element becomes unevenly distributed in all directions.

[0008] Based on fluid dynamics principles, the axial propulsion velocity of the micro-element is positively coupled with its rotational angular velocity and geometric asymmetry factor. Through this dynamic coupling, the magnetic torque is converted into an axial propulsion force along the direction of least fluid resistance, driving the micro-element to actively penetrate and uniformly disperse into the three-dimensional deep network of the high-viscosity sputum sample in a helical propulsion manner.

[0009] In-situ liquefaction and nucleic acid release induced by multi-field coupling: After the micro-element is implanted, the external magnetic field is switched to a high-frequency alternating magnetic field, and the magnetic field frequency is locked to the first-order longitudinal mechanical resonance frequency of the intelligent asymmetric magnetoelectric coupling micro-element.

[0010] Under this frequency excitation, the infinitesimal element produces multiple physical responses: First, the micro-nucleus undergoes high-frequency magnetostrictive deformation, radiating shear waves to the surrounding medium. When the shear stress generated by the shear waves in the sputum exceeds the yield stress of the sputum gel network, it disrupts the cross-linking structure between mucin molecules, causing the sputum to undergo a thixotropic phase transition, changing from a high-viscosity gel state to a low-viscosity sol state, thus achieving in-situ liquefaction.

[0011] Secondly, by utilizing the geometric asymmetry of micro-elements under high-frequency vibration, steady-state acoustic flow micro-vortices are induced in the solid-liquid boundary layer. The fluid drag force generated by the micro-vortices overcomes the Brownian motion of pathogens, actively capturing and pressing suspended pathogens onto the surface of the micro-elements.

[0012] Finally, the micro-element shell, through magnetoelectric-piezoelectric interface coupling, converts the mechanical strain of the core into a transient piezoelectric potential on the surface. This potential establishes a localized high electric field on the micro-element surface, applying a transmembrane potential exceeding the critical breakdown voltage of the cell membrane to the attached pathogen, causing irreversible electroporation and thus releasing intracellular nucleic acid molecules.

[0013] Nucleic acid adsorption and separation recovery are achieved based on surface energy reversal, maintaining a high-frequency alternating magnetic field. The surface wettability is modulated by the continuously existing piezoelectric potential on the micro-element surface. Following the Young-Lippman electrowetting principle, the cosine of the contact angle of the micro-element surface changes proportionally to the square of the surface potential. The high-frequency piezoelectric potential induces the micro-element surface to revert from an initial hydrophobic state to a hydrophilic state, while simultaneously establishing a high effective charge density on the surface.

[0014] The nucleic acid molecules are adsorbed and fixed onto the micro-element surface by utilizing the electrostatic Coulomb attraction between the positive charge on the micro-element surface and the negatively charged nucleic acid molecules free in liquefied sputum. Subsequently, a gradient magnetic field is applied, and the magnetic attraction experienced by the micro-element overcomes the fluid resistance, thus directionally separating the micro-element clusters loaded with nucleic acid molecules from the reaction system and removing the waste liquid.

[0015] Nucleic acid low-salt elution and photoelectric detection involves placing the separated micro-element in an elution buffer, stopping the application of a high-frequency alternating magnetic field, and controlling the mechanical oscillation frequency to be lower than the resonant frequency.

[0016] Under conditions without resonant excitation, the piezoelectric potential of the micro-element surface disappears. Based on the reversibility of surface energy states, the micro-element surface reverts to its initial hydrophobic state. The decrease in surface charge density eliminates electrostatic adsorption, and the hydrophobic surface enhances the repulsion of hydrophilic nucleic acid molecules, prompting rapid desorption and dissolution of the nucleic acid molecules in the buffer solution, resulting in a purified nucleic acid solution.

[0017] Finally, the purified nucleic acid solution was placed in an isothermal amplification system, and the pathogens in the sputum sample were qualitatively identified by real-time monitoring of the changes in photoelectric signals generated by the amplification reaction (such as the rate of change of fluorescence intensity and the endpoint fluorescence value).

[0018] This invention provides a sputum pretreatment method for rapid nucleic acid detection. It has the following beneficial effects: 1. This invention utilizes the non-centrosymmetric geometry and hydrophobic surface modification of intelligent asymmetric magnetoelectric coupling micro-element. Driven by a low-frequency rotating magnetic field, the magnetic torque is converted into an axial propulsive force along the direction of least fluid resistance, enabling the micro-element to actively enter the interior of high-viscosity sputum samples in a helical manner. This method eliminates the need for additional liquefaction agents or buffer solutions, avoiding the reduction in pathogen volume concentration due to sample dilution, and providing a material basis for subsequent detection of low-abundance pathogens.

[0019] 2. This invention employs a high-frequency alternating magnetic field to excite micro-elements to generate magneto-mechanical-electrical multi-field resonance. The shear waves radiated by these micro-elements disrupt the mucin gel network within the sputum, achieving physical liquefaction. Simultaneously, the localized piezoelectric field established on the surface of the micro-elements breaks down the pathogen cell membrane. This physical treatment method achieves sputum viscosity reduction and nucleic acid release without relying on chemical lysis buffers or proteases, reducing the risk of residual chemical reagents inhibiting polymerase activity in subsequent nucleic acid amplification reactions.

[0020] 3. This invention achieves dynamic reversal of the wetting properties of micro-element surfaces by regulating the piezoelectric potential of the micro-element surface to induce an electrowetting effect. Nucleic acid molecules are electrostatically adsorbed by the surface hydrophilic transformation excited by a high-frequency magnetic field, and the desorption of nucleic acids is assisted by the restored hydrophobic properties of the micro-element surface after the magnetic field is removed. This allows for the enrichment and recovery of nucleic acids on a single medium, simplifying the operational process. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the method flow of the present invention; Figure 2 This is a schematic diagram illustrating the structure and multi-physics interaction principle of the intelligent asymmetric magnetoelectric coupling micro-element of the present invention. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] See attached document Figure 1 - Appendix Figure 2 This invention provides a sputum pretreatment method for rapid nucleic acid testing. Addressing the technical challenge of high viscosity and difficulty in lysing sputum samples, which leads to false negatives in nucleic acid testing, this method utilizes an intelligent micro-element with a non-centrosymmetric geometric configuration and multi-physics response characteristics (magnetic, mechanical, and electric fields) as the execution medium. Through frequency modulation of an external dynamic magnetic field, a complete pretreatment process, from in-situ implantation to lysis and extraction, is achieved within the sputum sample.

[0024] The method of this invention mainly includes the following steps performed in sequence: Step S1: Constructing an initial sputum and micro-element pretreatment system for nucleic acid detection. A high-viscosity sputum sample to be tested for nucleic acid is placed in a reaction vessel. Based on the hydrophobic and anti-adhesion properties of the potential-responsive molecular layer pre-modified on the micro-element surface in the absence of a magnetic field, a dried intelligent asymmetric magnetoelectric coupling micro-element is directly placed into the sputum sample. This process utilizes the low surface energy of the micro-element surface to prevent non-specific aggregation with the mucin network in the sputum, thereby establishing a solid-liquid mixed initial state for subsequent efficient pretreatment without diluting the sputum.

[0025] Step S2 achieves deep active implantation and dispersion of sputum based on a low-frequency vortexing mechanism. A low-frequency rotating magnetic field is applied to the mixture formed in step S1. Based on the anisotropy of rotational inertia caused by the geometric asymmetry of the micro-element itself, the micro-element is driven to convert magnetic torque into hydrodynamic propulsion. This allows the micro-element to actively overcome the huge resistance generated by sputum as a non-Newtonian fluid in a helical advance manner. Without relying on external liquid dilution, the pretreatment medium is uniformly implanted into the three-dimensional deep network of sputum, preparing the space for the release of nucleic acids from the whole sample.

[0026] Step S3 utilizes the coupling of high-frequency magnetic field, mechanical field, and electric field to induce in-situ liquefaction of sputum and release of nucleic acids. After the micro-element is deeply implanted in the sputum, the external magnetic field is switched to a high-frequency alternating magnetic field that matches the mechanical resonance frequency of the micro-element, simultaneously stimulating multiple physical responses of the micro-element. First, the high-frequency magnetostrictive vibration of the micro-element core generates mechanical shear force, cutting the long chains of sputum mucoprotein to achieve in-situ liquefaction and eliminate the interference of sputum viscosity on detection. Second, the asymmetric vibration of the micro-element induces steady-state acoustic flow micro-vortices, actively capturing pathogens suspended in the liquefied sputum and pressing them onto the surface of the micro-element. Finally, the localized high potential generated by the piezoelectric effect of the micro-element shell performs contact electroporation lysis on the captured pathogens, thereby fully releasing the intracellular nucleic acids to be detected into the system.

[0027] Step S4 achieves nucleic acid target enrichment and protection based on surface polarity reversal. Maintaining a high-frequency alternating magnetic field, the piezoelectric effect continuously generates a surface potential that induces a conformational change in the potential-responsive molecular layer of the micro-element surface. This causes the micro-element surface to flip from a hydrophobic state in step S1 to a positively charged hydrophilic state, thereby enabling in-situ electrostatic adsorption and enrichment of the negatively charged nucleic acids released in step S3. Simultaneously, the surface reactive oxygen species layer generated by piezoelectric catalysis inhibits the activity of endogenous nucleases in sputum, protecting the enriched nucleic acid targets from degradation.

[0028] Step S5 involves magnetic field gradient separation and recovery of the pretreatment products. The alternating magnetic field is removed and a constant gradient magnetic field is applied. Utilizing the ferromagnetism of the micro-nucleus, micro-clusters loaded with high concentrations of nucleic acid on their surface are directionally separated from liquefied sputum waste. After washing and elution, a high-purity and high-concentration nucleic acid solution is obtained. This solution can be directly used as a template for subsequent nucleic acid amplification and detection.

[0029] The present invention will be further described in detail below, taking into account the specific micro-element structure design and the multiphysics mechanism in each of the above steps.

[0030] Step S1 involves constructing an initial pretreatment system for sputum and micro-elements for nucleic acid detection. The aim is to establish a heterogeneous solid-liquid mixture system through the structural design and interface modification of specially designed micro-elements, without introducing additional liquid media and thus diluting the sample concentration. This provides a material basis for the subsequent motion and work of micro-elements in non-Newtonian fluids.

[0031] The specific implementation of step S1 includes sub-steps S1.1, S1.2, and S1.3.

[0032] S1.1 Fabrication and Selection of Intelligent Asymmetric Magnetoelectric Coupled Micro-elements To achieve anisotropy in fluid dynamics and energy conversion across multiple physics fields, this step first involves fabricating a smart asymmetric magnetoelectric coupling micro-element with a non-centrosymmetric core-shell structure.

[0033] Specifically, the geometric configuration of the intelligent asymmetric magnetoelectric coupling micro-element is selected from one of the following: a unilateral cone shape, an eccentric dumbbell shape, or a tadpole-shaped structure with a blunt head and a sharp tail. To quantify the structural asymmetry, a geometric asymmetry factor is defined for the intelligent asymmetric magnetoelectric coupling micro-element. Geometric asymmetry factor The calculation formula is as follows: ; In the formula, It is a geometric asymmetry factor; The geometric center position vector of the intelligent asymmetric magnetoelectric coupling micro-element; The position vector of the mass center of the intelligent asymmetric magnetoelectric coupling micro-element; The characteristic length of the intelligent asymmetric magnetoelectric coupling micro-element along its major axis is given.

[0034] In this embodiment, the geometric parameters of the intelligent asymmetric magnetoelectric coupling micro-element are controlled through mold design, thereby enabling the geometric asymmetry factor to be adjusted. .

[0035] The core driving phase of the intelligent asymmetric magnetoelectric coupling micro-element is a terbium-dysprosium-iron alloy (Terfenol-D), with the chemical formula […]. The shell functional phase of the intelligent asymmetric magnetoelectric coupling micro-element is made of barium titanate piezoelectric ceramic. The shell functional phase is tightly coated onto the surface of the core driving phase using physical vapor deposition, forming a heterogeneous core-shell structure with a thickness of 0.5 micrometers to 2 micrometers.

[0036] After coating, the intelligent asymmetric magnetoelectric coupling micro-element is annealed in an argon inert gas environment at a temperature range of 600℃ to 800℃, with an annealing holding time of 1 to 3 hours. This annealing process is used to eliminate lattice defects at the core-shell interface, ensuring that the mechanical strain transfer efficiency between the core and shell is greater than 90%.

[0037] After obtaining the intelligent asymmetric magnetoelectric coupling micro-element, in order to endow the surface of the intelligent asymmetric magnetoelectric coupling micro-element with dual properties of anti-adhesion and dynamic adsorption, this step grafts a potential-responsive self-assembled molecular layer onto the outermost layer of the intelligent asymmetric magnetoelectric coupling micro-element.

[0038] Specifically, octadecyltrichlorosilane was selected as the modifier. The intelligent asymmetric magnetoelectric coupling micro-elements prepared in step S1.1 were dispersed in an anhydrous ethanol solution containing 1% to 5% (v / v) octadecyltrichlorosilane. The pH of the solution was adjusted to between 4.5 and 5.5, and the mixture was continuously stirred at room temperature for 12 to 24 hours. The intelligent asymmetric magnetoelectric coupling micro-element was then removed and ultrasonically cleaned with anhydrous ethanol. Finally, it was cured at a constant temperature of 80°C for 1 to 2 hours to form a dense monolayer on the surface of the intelligent asymmetric magnetoelectric coupling micro-element.

[0039] The wettability of the modified intelligent asymmetric magnetoelectric coupling micro-element surface varies with surface potential according to a variant of the Young-Lippmann equation. The relationship between contact angle and surface potential is expressed as follows: ; In the formula, The contact angle of the surface of the intelligent asymmetric magnetoelectric coupling micro-element; The initial contact angle of the surface of the intelligent asymmetric magnetoelectric coupling micro-element under zero potential; It is a double-layer capacitor at the solid-liquid interface; The interfacial tension between liquid and gas; The surface potential of the intelligent asymmetric magnetoelectric coupling micro-element.

[0040] In the absence of an external high-frequency magnetic field during step S1, the surface potential of the intelligent asymmetric magnetoelectric coupling micro-element is... According to the above formula, the surface of the intelligent asymmetric magnetoelectric coupling micro-element exhibits a contact angle. It exhibits a strongly hydrophobic state.

[0041] Construction of the S1.3 Zero-Dilution Solid-Liquid Mixture System: After material preparation and modification, high-viscosity sputum samples to be tested for nucleic acid were collected in reaction tubes. The dried intelligent asymmetric magnetoelectric coupling micro-element was added directly to the sputum sample at a ratio of 50 mg to 200 mg per milliliter of sputum.

[0042] During this process, no additional liquefaction agents, buffer solutions, or saline are added to maintain the original volume concentration of pathogens and nucleic acids in the sputum sample. Due to the hydrophobic properties of the surface of the intelligent asymmetric magnetoelectric coupling microelement in step S1.2, the intelligent asymmetric magnetoelectric coupling microelement does not induce interfacial wetting upon contact with sputum. Instead, it remains suspended as independent particles on the sputum surface or dispersed in local voids, forming a heterogeneous solid-liquid mixed initial system.

[0043] In this embodiment, a sputum-microelement pretreatment initial system for nucleic acid detection was successfully constructed by preparing core-shell structured microelements with specific asymmetry factors and implementing strict surface hydrophobic modification. This system utilizes the low surface energy of the microelement surface to effectively block the non-specific adhesion of mucin in high-viscosity sputum to the microelement surface, preventing early aggregation of the microelements. This creates the necessary material conditions for the subsequent step S2, which utilizes a rotating magnetic field to achieve active deep implantation and dispersion of the microelements in a non-Newtonian fluid.

[0044] Step S2 involves the active implantation and dispersion of sputum deep within the sputum layer based on a low-frequency vortex mechanism. This is achieved by controlling the motion mode of the intelligent asymmetric magnetoelectric coupling micro-element using an external magnetic field, driving it from a static suspension state to a dynamic propulsion state. This allows for the uniform delivery of the intelligent asymmetric magnetoelectric coupling micro-element into the high-viscosity sputum without disrupting the overall sputum sample concentration. Step S2 is specifically implemented through sub-steps S2.1, S2.2, and S2.3.

[0045] S2.1 Construction and parameter setting of low-frequency rotating magnetic field: In order to drive the intelligent asymmetric magnetoelectric coupling micro-element to rotate rigidly, this step uses a magnetic field generator to construct a low-frequency rotating magnetic field environment.

[0046] Specifically, the magnetic field generating device employs a triaxial orthogonal Helmholtz coil array. This is achieved by introducing magnetic fields with a 90-degree phase difference into each of the orthogonal coils. A standard sinusoidal alternating current is used to synthesize a uniform magnetic field in the central region of the reaction vessel, whose vector direction rotates periodically with time. The frequency of the rotating magnetic field is set. The frequency range is between 10Hz and 100Hz, and the magnetic induction intensity of the rotating magnetic field is set between 10mT and 50mT. The above frequency range is set to avoid the mechanical resonance frequency range of the intelligent asymmetric magnetoelectric coupling micro-element core material, ensuring that the intelligent asymmetric magnetoelectric coupling micro-element core does not undergo high-frequency magnetostrictive deformation during this stage, but only acts as a magnetic moment force-bearing unit to synchronously rotate at low frequency following the external rotating magnetic field vector.

[0047] S2.2 The spiral propulsion motion of the asymmetric micro-element is excited under the action of the rotating magnetic field. Based on the geometric asymmetry defined in step S1.1, the intelligent asymmetric magnetoelectric coupling micro-element transforms the simple rotational motion into translational propulsion motion along the direction of minimum fluid resistance.

[0048] According to drag theory, the anisotropic fluid drag experienced by the intelligent asymmetric magnetoelectric coupled micro-element during rotation generates a net axial propulsion force. The dynamic relationship between the axial propulsion velocity, rotational angular velocity, and geometric asymmetry factor of the intelligent asymmetric magnetoelectric coupled micro-element can be expressed as follows: ; In the formula, The axial propulsion velocity of the intelligent asymmetric magnetoelectric coupling micro-element; The rotational drag coefficient of the intelligent asymmetric magnetoelectric coupling micro-element; The translational resistance coefficient of the intelligent asymmetric magnetoelectric coupling micro-element; It is the rotational angular velocity; For depends on the geometric asymmetry factor Dimensionless coupling function.

[0049] In this embodiment, since the geometric asymmetry factor has been set in step S1.1 Dimensionless coupling function A positive value drives the intelligent asymmetric magnetoelectric coupling micro-element to generate continuous propulsion motion along its long axis.

[0050] S2.3 Non-Newtonian Fluid Shear Thinning and Deep Implantation: The rotational motion of intelligent asymmetric magnetoelectric coupling elements actively penetrates the high-viscosity sputum network, utilizing the shear thinning properties of sputum to reduce penetration resistance. The apparent viscosity of sputum decreases with increasing local shear rate. When the intelligent asymmetric magnetoelectric coupling element rotates within the sputum, a shear field is generated at the interface between the intelligent asymmetric magnetoelectric coupling element surface and the sputum. Based on the Carlo-Jasoda rheological model, the formula for calculating the local apparent viscosity around the intelligent asymmetric magnetoelectric coupling element is as follows: ; In the formula, The local apparent viscosity around the intelligent asymmetric magnetoelectric coupling micro-element; The viscosity is zero shear viscosity for sputum; The infinite shear viscosity of sputum; The relaxation time constant; Local shear rate; The liquidity index; For Yasouda parameters.

[0051] In this embodiment, the frequency of the rotating magnetic field is controlled. The local shear rate generated on the surface of the intelligent asymmetric magnetoelectric coupling micro-element Greater than 10s −1 At this shear rate, the local apparent viscosity of sputum is... Significantly lower than far-field sputum viscosity, a low-viscosity fluid channel is formed in front of the intelligent asymmetric magnetoelectric coupling micro-element, enabling the intelligent asymmetric magnetoelectric coupling micro-element to penetrate the dense gel network composed of mucin cross-linking and drill from the sputum surface to the bottom of the sputum within 10 to 60 seconds.

[0052] In this embodiment, by constructing a low-frequency rotating magnetic field with precise phase control and utilizing the dynamic coupling between the asymmetric geometry of the micro-element and fluid resistance, a helical propulsion mode of the micro-element was successfully excited. This mode utilizes the shear-thinning rheological properties of sputum itself to achieve active deep implantation and three-dimensional uniform dispersion of the treatment medium in ultra-high viscosity samples without adding any chemical liquefaction agents or performing physical stirring. This helps to solve the problem that traditional magnetic beads cannot be actively dispersed in viscous samples.

[0053] Step S3 involves using high-frequency magnetic field mechanical field and electric field coupling to induce in situ liquefaction of sputum and release of nucleic acid. The aim is to use high-frequency alternating magnetic field to excite the multi-physics field resonance response of intelligent asymmetric magnetoelectric coupling micro-element. Through the combined effects of mechanical shearing, fluid capture and electric field breakdown, the physical degradation of sputum matrix and in situ release of intracellular nucleic acid of pathogens can be achieved without the participation of chemical lysis solution.

[0054] The specific implementation of step S3 includes sub-steps S3.1, S3.2, and S3.3.

[0055] S3.1 Excitation of high-frequency alternating magnetic field and in-situ thixotropic liquefaction of sputum To trigger the magnetostrictive and piezoelectric effects of the intelligent asymmetric magnetoelectric coupling micro-element, this step switches the low-frequency rotating magnetic field in step S2 to a high-frequency alternating magnetic field.

[0056] Specifically, the output frequency of the magnetic field generator is controlled to be... The sinusoidal alternating magnetic field locks the magnetic field frequency to the first-order longitudinal mechanical resonance frequency of the intelligent asymmetric magnetoelectric coupling element. The axial length and equivalent longitudinal wave velocity of the intelligent asymmetric magnetoelectric coupling micro-element are determined by the following formula: ; In the formula, Resonant frequency; The axial length of the intelligent asymmetric magnetoelectric coupling micro-element; This is the average Young's modulus; This represents the average density.

[0057] In this embodiment, the resonant frequency is calculated and set based on the geometric dimensions of the intelligent asymmetric magnetoelectric coupling micro-element. Set the magnetic field strength between 20kHz and 100kHz. It is between 10kA / m and 30kA / m.

[0058] Under the drive of a resonant magnetic field, the supermagnetostrictive material of the intelligent asymmetric magnetoelectric coupled micro-element core undergoes high-frequency axial stretching deformation. Based on the magnetomechanical coupling effect, the relationship between the mechanical strain generated by the intelligent asymmetric magnetoelectric coupled micro-element and the applied magnetic field is expressed as follows: ; In the formula, The axial strain varies with time; The dynamic piezomagnetic coefficient of the intelligent asymmetric magnetoelectric coupling micro-core material; This represents the amplitude of the alternating magnetic field. It is the resonant frequency; It is a time variable.

[0059] The high-frequency mechanical vibration of the intelligent asymmetric magnetoelectric coupling micro-element radiates shear waves to the surrounding sputum medium. When the shear stress generated by the shear wave exceeds the yield stress of the sputum gel network (typically 10 Pa to 100 Pa), the three-dimensional gel network structure maintained by disulfide bonds and hydrophobic interactions between mucin molecules within the sputum breaks or unwinds. This process transforms the sputum from a gel state to a sol state, achieving in-situ liquefaction of the sputum, reducing the viscosity of the medium, and providing a low-flow-resistance environment for the subsequent migration and capture of pathogens.

[0060] S3.2 Active Pathogen Capture Induced by Asymmetric Acoustic Microvortices: Simultaneously with sputum liquefaction, the acoustic flow effect induced by the geometric asymmetry of intelligent asymmetric magnetoelectric coupling micro-element under high-frequency vibration is utilized to establish a steady-state microvortex flow field pointing towards the surface of the intelligent asymmetric magnetoelectric coupling micro-element. Due to the non-centrosymmetric structure defined in step S1.1, the amplitude distribution in different regions of the surface of the intelligent asymmetric magnetoelectric coupling micro-element is non-uniform. When the intelligent asymmetric magnetoelectric coupling micro-element vibrates in the fluid, a time-averaged acoustic flow field perpendicular to the vibration direction of the intelligent asymmetric magnetoelectric coupling micro-element is generated outside the boundary layer of the intelligent asymmetric magnetoelectric coupling micro-element. The vibration velocity amplitude on the surface of the intelligent asymmetric magnetoelectric coupling micro-element... The axial strain amplitude of step S3.1 The relationship is represented as: .

[0061] In the formula, The amplitude of the vibration velocity on the surface of the intelligent asymmetric magnetoelectric coupling micro-element; It is the resonant frequency; The axial length of the intelligent asymmetric magnetoelectric coupling micro-element; This represents the maximum axial strain amplitude.

[0062] According to Nyquist acoustic flow theory and geometric corrections, the acoustic flow velocity field generated by micro-vortices can be approximately expressed as: ; In the formula, The velocity of the acoustic flow; The density of liquefied sputum; Angular frequency; The equivalent radius of the intelligent asymmetric magnetoelectric coupling micro-element; For intelligent asymmetric magnetoelectric coupling micro-element asymmetric factor Positively correlated dimensionless shape coefficient.

[0063] This acoustic micro-vortex generates a fluid drag force on pathogens suspended near the intelligent asymmetric magnetoelectric coupling micro-element. According to Stokes' law, the formula for calculating the fluid drag force is as follows: ; In the formula, For fluid drag force; The dynamic viscosity of liquefied sputum; It is the equivalent radius of pathogens (such as Mycobacterium tuberculosis).

[0064] In this embodiment, the magnetic field strength is adjusted. This causes the vibration velocity amplitude Greater than 0.1 m / s, thus generating fluid drag force. The Brownian motion force, greater than that of the pathogen, draws the pathogens, which are dispersed in the range of 10 to 50 micrometers around the intelligent asymmetric magnetoelectric coupling micro-element, into the micro-vortex and presses the pathogens onto the outer shell surface of the intelligent asymmetric magnetoelectric coupling micro-element.

[0065] S3.3 Contact electroporation lysis achieved by piezoelectric interface coupling effect: After pathogens are captured by acoustic vortex and attached to the surface of intelligent asymmetric magnetoelectric coupling micro-element, cell membrane breakdown is achieved by using the high electric field generated by the piezoelectric effect of the shell of the intelligent asymmetric magnetoelectric coupling micro-element.

[0066] The high-frequency mechanical strain of the core of the intelligent asymmetric magnetoelectric coupling micro-element is transmitted to the piezoelectric shell through the interface, causing the piezoelectric shell to become polarized and accumulate bound charges on its surface. Under open-circuit conditions, the transient piezoelectric potential generated on the surface of the intelligent asymmetric magnetoelectric coupling micro-element can be expressed as: ; In the formula, It is the piezoelectric potential; The piezoelectric strain constant of the outer shell material; The Young's modulus of the outer shell material; The thickness of the piezoelectric housing; It is the dielectric constant under constant stress.

[0067] Because the pathogen adheres closely to the surface of the intelligent asymmetric magnetoelectric coupling micro-element, the piezoelectric potential acts directly on both sides of the pathogen's cell membrane. Based on the relationship between the pathogen's size and the local electric field of the micro-element surface, a transmembrane potential is established. When the transmembrane potential exceeds the critical breakdown voltage of the pathogen's cell membrane (for Mycobacterium tuberculosis, this threshold is set to 0.2 volts to 1 volt), irreversible electrical breakdown occurs in the cell membrane, forming nanoscale hydrophilic channels.

[0068] As the electric field continues to act, the pores expand and cause the cell membrane to disintegrate, releasing intracellular material, including nucleic acid molecules, into the fluid boundary layer on the surface of the intelligent asymmetric magnetoelectric coupling micro-element.

[0069] In this embodiment, through high-frequency magnetic field modulation in step S3, the magnetostrictive effect of the intelligent asymmetric magnetoelectric coupling micro-element core provides the mechanical energy source. The asymmetric structure is used to convert the mechanical energy into directional fluid kinetic energy to achieve pathogen capture. Core-shell coupling is used to convert the mechanical energy into local electrical energy to achieve contact electroporation lysis. This multi-field coupling of magnetic field, mechanical field, flow field, and electric field enables continuous processing from macroscopic liquefaction to microscopic lysis on a single intelligent asymmetric magnetoelectric coupling micro-element entity.

[0070] Step S4 involves the electrostatic adsorption and magnetic separation recovery of nucleic acids based on the electrowetting surface energy reversal. This aims to utilize the electrowetting effect induced by the piezoelectric potential generated by the intelligent asymmetric magnetoelectric coupling micro-element under high-frequency vibration, causing the surface of the micro-element to flip from a hydrophobic, anti-adhesive state to a hydrophilic adsorption state. Released nucleic acid molecules are captured through electrostatic interactions, and a gradient magnetic field is used to separate the nucleic acid-loaded intelligent asymmetric magnetoelectric coupling micro-element from liquefied sputum. The specific implementation of step S4 includes sub-steps S4.1, S4.2, and S4.3.

[0071] S4.1 Piezoelectric electrowetting-induced surface energy flipping and nucleic acid adsorption During and after the implementation of step S3, the excitation of the high-frequency alternating magnetic field is maintained, and the piezoelectric potential that exists continuously on the surface of the intelligent asymmetric magnetoelectric coupling micro-element is used to change the wettability of the surface, so as to achieve the adsorption of negatively charged nucleic acid molecules.

[0072] According to Young's Lippmann equation, the contact angle of the surface of the intelligent asymmetric magnetoelectric coupling micro-element decreases with increasing surface potential. The high-frequency piezoelectric potential in step S3... Under the influence of the action, the dynamic contact angle of the surface of the intelligent asymmetric magnetoelectric coupling micro-element The changes follow the formula below: ; In the formula, This is the dynamic contact angle when a piezoelectric potential exists; The initial contact angle at zero potential; It is the vacuum permittivity; The relative permittivity of the self-assembled molecular layer; The thickness of the self-assembled molecular layer; The surface tension of liquefied sputum; The piezoelectric potential of the surface of the intelligent asymmetric magnetoelectric coupling micro-element is defined in step S3.3.

[0073] In this embodiment, due to the piezoelectric potential Due to the square effect of the term, the surface of the intelligent asymmetric magnetoelectric coupling micro-element changes from an initial strongly hydrophobic state to a hydrophilic state. At this point, an electrostatic Coulomb attraction is generated between the induced charge on the surface of the intelligent asymmetric magnetoelectric coupling micro-element and the negatively charged nucleic acid molecules ionized in the liquefied sputum. The electrostatic adsorption force between the nucleic acid molecules and the surface of the intelligent asymmetric magnetoelectric coupling micro-element... The approximate calculation formula is as follows: ; In the formula, The electrostatic adsorption force between nucleic acid molecules and the surface of intelligent asymmetric magnetoelectric coupling micro-element; The effective charge density on the surface of a smart asymmetric magnetoelectric coupling micro-element; The effective charge of a nucleic acid molecule; The dielectric constant of the solution; The distance between the nucleic acid molecule and the surface of the intelligent asymmetric magnetoelectric coupling micro-element is denoted as denoted as denoted as denoted as denoted as denoted as denoted as denoted as denoted by ...

[0074] By maintaining a high-frequency alternating magnetic field for 30 to 60 seconds, the intelligent asymmetric magnetoelectric coupling micro-element utilizes electrostatic adsorption force to enrich the nucleic acid molecules released in step S3 onto the surface of the intelligent asymmetric magnetoelectric coupling micro-element.

[0075] S4.2 Gradient Magnetic Field Driven Solid-Liquid Separation and Micro-Element Recovery After nucleic acid adsorption is completed, an external gradient magnetic field is used to overcome fluid resistance and separate the nucleic acid-loaded intelligent asymmetric magnetoelectric coupling micro-element from the reaction tube.

[0076] Specifically, the high-frequency alternating magnetic field in step S3 is removed, and a constant gradient magnetic field is applied to the sidewall or bottom of the reaction tube. The gradient magnetic field generator is a neodymium iron boron permanent magnet assembly or a DC electromagnet, and the magnetic field gradient within the reaction tube region is set. The magnetic field strength ranges from 5 T / m to 15 T / m, with the gradient pointing towards the bottom of the reaction tube. Under the influence of the gradient magnetic field, the intelligent asymmetric magnetoelectric coupling micro-element experiences magnetic attraction. Magnetic attraction The calculation formula is as follows: ; In the formula, The magnetic attraction force experienced by the intelligent asymmetric magnetoelectric coupling micro-element; The volume of the intelligent asymmetric magnetoelectric coupling micro-element core; The magnetization vector of the intelligent asymmetric magnetoelectric coupling micro-element core material; denoted as the gradient of magnetic flux density.

[0077] Since the viscosity of the sputum has been reduced by shearing in step S3.1, the magnetic attraction force is now... The force, exceeding the resistance of fluid viscosity and gravity, drives the intelligent asymmetric magnetoelectric coupling micro-element to move directionally towards the bottom of the reaction tube and aggregate. After standing for 1 to 2 minutes, the supernatant waste liquid in the reaction tube is aspirated, retaining the clusters of intelligent asymmetric magnetoelectric coupling micro-element adsorbed with nucleic acids.

[0078] S4.3 Low-salt elution and micro-element regeneration of nucleic acids To obtain purified nucleic acid solution and recover intelligent asymmetric magnetoelectric coupling elements, an elution operation is performed in this step. Ethylenediaminetetraacetic acid (EDTA) elution buffer with a pH of 8.0 to 8.5 is added to the reaction tube containing the intelligent asymmetric magnetoelectric coupling elements. The applied magnetic field is stopped, and the intelligent asymmetric magnetoelectric coupling elements are dispersed using a low-frequency mechanical oscillation device or by pipetting, with the oscillation frequency controlled below 1 kHz to avoid triggering mechanical resonance in the intelligent asymmetric magnetoelectric coupling elements.

[0079] Under conditions where no external high-frequency magnetic field excitation is applied and the mechanical oscillation frequency is far from the resonant frequency, the intelligent asymmetric magnetoelectric coupling micro-element does not generate a piezoelectric potential. Approximately equal to 0. According to the Young's Lippmann equation in step S4.1, the surface of the intelligent asymmetric magnetoelectric coupling micro-element is restored to its initial hydrophobic state, and the effective charge density on the surface tends to zero.

[0080] At this point, the electrostatic adsorption force between the intelligent asymmetric magnetoelectric coupling element and the nucleic acid molecules disappears, and the repulsive effect of the hydrophobic intelligent asymmetric magnetoelectric coupling element surface on the hydrophilic nucleic acid molecules is enhanced, causing the nucleic acid molecules to desorb from the surface of the intelligent asymmetric magnetoelectric coupling element and dissolve in the elution buffer. The gradient magnetic field of step S4.2 is applied again to adsorb and fix the deloaded intelligent asymmetric magnetoelectric coupling element onto the tube wall. The supernatant is then aspirated to obtain the purified nucleic acid solution for subsequent detection.

[0081] In this embodiment, the electrowetting effect of intelligent asymmetric magnetoelectric coupling micro-element under high-frequency vibration is used to realize the dynamic reversal of surface properties. The piezoelectric potential generated by physical cleavage is reused for the electrostatic capture of nucleic acids without the need for additional binding buffer or centrifugation steps. Combined with gradient magnetic field separation technology, the entire process of in-situ release, in-situ capture and magnetic recovery of trace pathogen nucleic acids from high-viscosity sputum is completed, improving the efficiency and purity of nucleic acid extraction.

[0082] Step S5 involves isothermal nucleic acid amplification and in-situ detection of photoelectric signals. It aims to utilize loop-mediated isothermal amplification (LAMP) technology to exponentially amplify the pathogen target genes in the purified nucleic acid solution obtained in step S4, converting the change in nucleic acid concentration into observable optical or electrical signals, thereby achieving qualitative or quantitative detection of pathogens in sputum samples. Step S5 is specifically implemented through sub-steps S5.1, S5.2, and S5.3.

[0083] Construction of S5.1 amplification reaction system and specific primer annealing The purified nucleic acid solution obtained in step S4.3 is transferred to an optical detection tube, and pre-prepared lyophilized amplification reagent powder is added. The amplification reagent powder contains strand-displacement deoxyribonucleic acid polymerase, deoxyribonucleoside triphosphate, reaction buffer, intercalation fluorescent dye, and a specific primer set designed for a specific pathogen. The reaction buffer contains magnesium sulfate or magnesium chloride, with a magnesium ion concentration higher than 6 mmol / L, to counteract the inhibitory effect of residual ethylenediaminetetraacetic acid (EDTA) from step S4 on polymerase activity.

[0084] For the detection of Mycobacterium tuberculosis, the specific primer set is designed based on the conserved region of the IS6110 insertion sequence in the Mycobacterium tuberculosis genome, and includes forward and reverse inner primers, forward and reverse outer primers, and forward and reverse loop primers.

[0085] The optical detection tube was placed in a constant-temperature heating module, and the temperature was raised to 60-65 degrees Celsius. At this temperature, the specific primer set specifically bound to the single-stranded template deoxyribonucleic acid in the purified nucleic acid solution. Since protein and polysaccharide impurities in the sputum were removed in step S4, the template deoxyribonucleic acid in the purified nucleic acid solution could efficiently renature with the primers, initiating the strand displacement synthesis reaction.

[0086] S5.2 Exponential Amplification Reaction Kinetics and Product Formation Under isothermal conditions, the strand displacement activity of strand-displacement deoxyribonucleic acid polymerase is utilized to form stem-loop deoxyribonucleic acid (DNA) structures within the reaction system, which then serve as starting sites for cyclic extension. As the amplification reaction proceeds, the double-stranded DNA byproducts and magnesium pyrophosphate precipitate within the reaction system exhibit an S-shaped growth curve over time. The real-time fluorescence intensity of the reaction system is also measured. Over time The change in follows the Logistic growth model, and its calculation formula is as follows: ; In the formula, The amplification reaction proceeded to the specified time. Real-time fluorescence intensity; The baseline fluorescence intensity at the start of the amplification reaction; The maximum saturation fluorescence intensity at the end of the amplification reaction; The apparent rate constant of the amplification reaction; Reaction time; The half-saturation time point is when the fluorescence intensity reaches half of the maximum growth rate.

[0087] In this embodiment, the total reaction time for isothermal amplification is set to 45 to 60 minutes. During the reaction, the intercalating fluorescent dye binds to the generated double-stranded deoxyribonucleic acid, resulting in a change in real-time fluorescence intensity. Significantly enhanced.

[0088] S5.3 Photoelectric signal acquisition and detection result interpretation utilizes a photodiode or fluorescence sensor integrated below the constant temperature heating module to measure the real-time fluorescence intensity within the optical detection tube at time intervals of 30 to 60 seconds. Perform continuous data collection.

[0089] The acquired fluorescence signal data is transmitted to a microprocessor for analysis. A threshold time is defined to determine whether a sample is positive. The threshold time is determined when the real-time fluorescence intensity... The fluorescence threshold was exceeded for the first time. The time corresponding to this time is the threshold time. The preset fluorescence threshold... and the following rate thresholds The results are obtained by statistically analyzing the average background signal from at least three negative control experiments, typically set to be at least three times the standard deviation of the negative control background signal. The interpretation logic for the test results is based on the following discriminant function. : ; In the formula, The values ​​are used to determine the test results, where 1 represents a positive result and 0 represents a negative result. The maximum first derivative of the real-time fluorescence intensity change over time is the maximum amplification rate. The preset rate threshold is used to exclude background signal drift caused by nonspecific amplification; The endpoint fluorescence intensity at the end of the reaction; This is the preset positive fluorescence threshold.

[0090] If the calculated judgment value of the test result If the result equals 1, the target pathogen is determined to be present in the original sputum sample; if the test result judgment value is 1, the target pathogen is determined to be present in the original sputum sample. If the value is 0, it is determined that the target pathogen is not present in the original sputum sample.

[0091] In this embodiment, step S5 connects to the high-purity nucleic acid output from step S4. The isothermal amplification technology avoids the dependence on precise temperature control of traditional variable temperature equipment. At the same time, the combination of the dual interpretation criteria of the maximum amplification rate of kinetic parameters and the endpoint fluorescence intensity effectively reduces the false positive rate and realizes rapid qualitative screening of low-abundance pathogens in high-viscosity sputum samples.

Claims

1. A sputum pre-treatment method for rapid nucleic acid testing, characterized by, The method comprises the following steps: adding the intelligent asymmetric magneto-electric coupling micro-element in dry state into a high-viscosity sputum sample directly, forming a heterogeneous solid-liquid mixture without adding liquid medium by using the hydrophobic property of the surface of the intelligent asymmetric magneto-electric coupling micro-element; applying a low-frequency rotating magnetic field to the heterogeneous solid-liquid mixture to drive the intelligent asymmetric magneto-electric coupling micro-element to disperse into the high-viscosity sputum sample; switching the low-frequency rotating magnetic field to a high-frequency alternating magnetic field to excite the intelligent asymmetric magneto-electric coupling micro-element to generate high-frequency mechanical vibration and surface piezoelectric potential, realizing in-situ liquefaction of the high-viscosity sputum sample and release of nucleic acid molecules; maintaining the high-frequency alternating magnetic field, using the surface piezoelectric potential to induce electrowetting effect, making the surface of the intelligent asymmetric magneto-electric coupling micro-element flip from hydrophobic state to hydrophilic state and enriching the nucleic acid molecules, then applying a gradient magnetic field to separate the intelligent asymmetric magneto-electric coupling micro-element loaded with the nucleic acid molecules, and obtaining a purified nucleic acid solution through elution operation; transferring the purified nucleic acid solution to an amplification reaction system for constant-temperature nucleic acid amplification, converting the concentration change of the nucleic acid molecules into an optoelectronic signal, and determining whether the target pathogen exists in the high-viscosity sputum sample according to the optoelectronic signal.

2. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized in that, The geometric configuration of the intelligent asymmetric magneto-electric coupling micro-element is selected from one of a single-side cone, an eccentric dumbbell, and a tadpole structure with a blunt head and a sharp tail; the core driving phase of the intelligent asymmetric magneto-electric coupling micro-element is a terbium-dysprosium-iron alloy; the shell functional phase of the intelligent asymmetric magneto-electric coupling micro-element is barium titanate piezoelectric ceramic; and the outermost layer of the intelligent asymmetric magneto-electric coupling micro-element is grafted with a potential-responsive self-assembled molecular layer composed of octadecyltrichlorosilane.

3. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized in that, The specific process of driving the intelligent asymmetric magneto-electric coupling micro-element to disperse into the high-viscosity sputum sample includes: using a three-axis orthogonal Helmholtz coil array to synthesize the low-frequency rotating magnetic field with a frequency of 10-100 Hz, and setting the frequency of the low-frequency rotating magnetic field to avoid the mechanical resonance frequency range of the intelligent asymmetric magneto-electric coupling micro-element; using the low-frequency rotating magnetic field to drive the intelligent asymmetric magneto-electric coupling micro-element with geometric asymmetry to generate rigid body rotation; using the anisotropic fluid resistance received by the intelligent asymmetric magneto-electric coupling micro-element during rotation to generate translational propulsion motion in the direction of minimum fluid resistance, and actively implanting deep in a spiral manner.

4. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized in that, The specific process of realizing in-situ liquefaction of the high-viscosity sputum sample includes: locking the frequency of the high-frequency alternating magnetic field to be the first-order longitudinal mechanical resonance frequency of the intelligent asymmetric magneto-electric coupling micro-element; driving the intelligent asymmetric magneto-electric coupling micro-element core to occur high-frequency magnetostrictive deformation, and based on the magneto-mechanical coupling effect, using the intelligent asymmetric magneto-electric coupling micro-element to radiate shear waves to the surrounding medium; when the shear stress generated by the shear waves exceeds the yield stress of the high-viscosity sputum sample, the mucin gel network inside the high-viscosity sputum sample is broken, and the high-viscosity sputum sample is transformed from a gel state to a sol state.

5. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized in that, The specific process for realizing the release of the nucleic acid molecules includes: The geometric asymmetry of the intelligent asymmetric magnetoelectric coupling micro-element under high-frequency vibration is used to establish acoustic flow micro-vortex outside the boundary layer of the intelligent asymmetric magnetoelectric coupling micro-element, and the fluid drag force is used to roll and press the pathogens into the surface of the intelligent asymmetric magnetoelectric coupling micro-element; The core-shell interface coupling of the intelligent asymmetric magnetoelectric coupling micro-element is used to convert the high-frequency mechanical strain of the inner core into the transient piezoelectric potential of the outer shell surface; The transient piezoelectric potential is used to establish a local electric field on the surface of the intelligent asymmetric magnetoelectric coupling micro-element, and a transmembrane potential exceeding the critical breakdown voltage is applied to the pathogens attached to the surface of the intelligent asymmetric magnetoelectric coupling micro-element to form irreversible electroporation and release the intracellular nucleic acid molecules.

6. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized in that, The specific process for enriching the nucleic acid molecules includes: Based on the Young-Lippmann equation, the square term of the piezoelectric potential generated by the intelligent asymmetric magnetoelectric coupling micro-element under the high-frequency alternating magnetic field is used to reduce the contact angle of the surface of the intelligent asymmetric magnetoelectric coupling micro-element, so that the surface of the intelligent asymmetric magnetoelectric coupling micro-element changes from a hydrophobic state to a hydrophilic state; The electrostatic Coulomb attraction between the effective charge density of the surface of the intelligent asymmetric magnetoelectric coupling micro-element and the negatively charged nucleic acid molecules is used to adsorb and fix the nucleic acid molecules on the surface of the intelligent asymmetric magnetoelectric coupling micro-element.

7. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized in that, The specific process for applying a gradient magnetic field to separate the intelligent asymmetric magnetoelectric coupling micro-element loaded with the nucleic acid molecules includes: The magnetic field gradient of the gradient magnetic field is set to 5 tesla per meter to 15 tesla per meter, and the magnetic field gradient direction is set to point to the bottom of the reaction container; The magnetic attraction force received by the intelligent asymmetric magnetoelectric coupling micro-element in the gradient magnetic field is used to overcome the fluid viscous resistance and drive the intelligent asymmetric magnetoelectric coupling micro-element loaded with the nucleic acid molecules to directionally gather to the bottom of the reaction container; The supernatant waste liquid in the reaction container is sucked out, and the intelligent asymmetric magnetoelectric coupling micro-element cluster is retained.

8. The sputum pre-treatment method for rapid nucleic acid detection according to claim 1, characterized by, The specific process of the elution operation includes: The intelligent asymmetric magnetoelectric coupling micro-element is resuspended using an ethylenediaminetetraacetic acid elution buffer with a pH value of 8.0 to 8.5; The mechanical oscillation frequency is controlled to be lower than 1000 Hz, so that the intelligent asymmetric magnetoelectric coupling micro-element does not occur mechanical resonance, and the piezoelectric potential on the surface of the intelligent asymmetric magnetoelectric coupling micro-element tends to zero; Based on the disappearance of the piezoelectric potential, the surface of the intelligent asymmetric magnetoelectric coupling micro-element returns to the initial hydrophobic state, the electrostatic adsorption force is eliminated, and the repulsion to the hydrophilic nucleic acid molecules is enhanced, so that the nucleic acid molecules are desorbed and dissolved in the elution buffer.

9. The sputum pre-treatment method for rapid nucleic acid testing according to claim 8, wherein, The specific process of the isothermal nucleic acid amplification includes: The purified nucleic acid solution is added to the amplification reaction system, and the amplification reaction system includes a strand displacement deoxyribonucleic acid polymerase, a deoxyribonucleoside triphosphate, an intercalating fluorescent dye, a specific primer set designed for a specific pathogen, and a reaction buffer containing magnesium ions; The concentration of magnesium ions in the reaction buffer is set to be higher than 6 mM / L, and the magnesium ions are used to offset the inhibition of the chain displacement DNA polymerase activity by the residual EDTA in the purified nucleic acid solution; The loop-mediated isothermal amplification reaction is carried out under constant temperature conditions of 60-65 DEG C, and the fluorescence intensity signals of the amplification reaction system are collected in real time.

10. The sputum pre-treatment method for rapid nucleic acid testing according to claim 9, wherein, The specific process of determining whether the target pathogen exists in the high-viscosity sputum sample includes: The background signal of the negative control experiment is counted, and a rate threshold and a fluorescence threshold are set; The first derivative of the real-time collected fluorescence intensity with respect to time is calculated to obtain the maximum amplification rate; The end-point fluorescence intensity at the end of the amplification reaction is obtained; If the maximum amplification rate is greater than the rate threshold, and the end-point fluorescence intensity is greater than the fluorescence threshold, it is determined that the target pathogen exists in the high-viscosity sputum sample.

Images