Free fetus DNA enrichment and quantitative analysis method based on nano magnetic beads

Abstract

The invention relates to a free fetal DNA enrichment and quantitative analysis method based on a nano magnetic bead. The method comprises the following steps: preparing the nano magnetic bead of which the surface is modified with a polyethylene glycol (PEG) chain brush, regulating and controlling the density and spatial arrangement of the PEG chain, forming a structure screening layer adaptive to free DNA with the length of 140-160bp on the surface of the magnetic bead, and capturing short fragment DNA to finish selective enrichment; adding the functionalized nano magnetic beads into a pregnant woman plasma reaction system, and applying a directional electric field to guide negatively charged DNA to migrate to the surfaces of the magnetic beads to form a local focusing area of free fetal DNA; after adsorption and enrichment are completed, the pH value or temperature of a reaction system is adjusted, the structural tension gradient of a PEG chain brush area is excited, target fetal DNA with medium binding force is released from the surfaces of the magnetic beads, and specific desorption is completed; and introducing the released DNA solution into a microcavity structure provided with a micro-resistance flow channel, and only collecting DNA components which are relatively high in diffusion speed and high in leading edge centrality to carry out subsequent fluorescent quantitative PCR or digital PCR analysis so as to finish high-purity quantitative detection of the target fetal DNA.

Description

TECHNICAL FIELD

[0001] The present application relates to a free DNA enrichment and quantitative analysis method, in particular to a free fetal DNA enrichment and quantitative analysis method based on nano-magnetic beads. BACKGROUND

[0002] In the prior art, such as the fetal free DNA enrichment method disclosed in Chinese patent CN110846382A, the method uses truncated Y-shaped adapters to connect total free DNA to form library fragments, and then uses magnetic beads to purify and recover DNA in the length range of 50-150 bp to achieve fetal fragment enrichment. Although this strategy solves the problem of the biological characteristics of fetal cfDNA short fragments to some extent, it still has many limitations and deficiencies in terms of enrichment mechanism, physical behavior control precision, specific screening mechanism, and system response control. First, the core steps of this method mainly rely on adapter ligation and fragment length selection in the construction of second-generation sequencing library. The principle is essentially a sequence post-selection mechanism, that is, only the double-stranded DNA of the library is enriched by magnetic beads, and the original state of the DNA is not selected in the physical structure layer. Therefore, it has a high risk of background residue for maternal DNA with a length close to the boundary (such as 130-160 bp) and overlapping sources, especially in samples with low fetal DNA concentration, which will significantly affect the sensitivity and specificity of subsequent quantitative analysis.

[0003] Secondly, the magnetic bead purification method used in this patent is the traditional length selection type purification, which relies on the PEG / NaCl sedimentation system and the adsorption capacity of the magnetic bead coating layer surface to distinguish the length of the fragments. This physical selection method usually has a resolution that is disturbed by many factors such as molecular chain flexibility, non-specific adsorption, and viscous layer disturbance, and cannot achieve true structural adaptability selection. Thirdly, this method does not set the behavior control of the DNA migration path, binding area or release direction, and the enrichment process is passive adsorption and centrifugal elution, which lacks physical field response regulation ability. In addition, this method does not involve any desorption selectivity or re-separation operation, and the DNA enrichment obtained still contains a large amount of maternal residual fragments with strong binding force, which is easy to cause false positive amplification or Ct value drift.

[0004] Furthermore, this patent does not integrate a microfluidic screening module, and the subsequent analysis still needs to rely on external systems for PCR amplification, which has a long sample processing path and complicated operation steps, and has the risk of sample loss and cross contamination. Finally, this method does not provide a mathematical model or behavior function expression, and lacks the ability of physical modeling and parameter feedback of the DNA migration-selection-enrichment process, so it is difficult to realize the system optimization and process re-development guided by the algorithm.

[0005] In summary, as an early-stage technical route, the method still stays in the level of passive length filtering and structure modification regulation, and has the problems of rough screening mechanism, weak selectivity, uncontrollable process, lack of dynamic response and micro-flow integration, and is difficult to adapt to the requirement of high-sensitivity detection of extremely low concentration of fetal DNA in early pregnancy. SUMMARY

[0006] The purpose of the present application is to provide a free fetal DNA enrichment and quantitative analysis method based on nano-magnetic beads, so as to solve some of the problems and deficiencies pointed out in the background art.

[0007] The technical scheme adopted by the present application to solve the above-mentioned technical problems is as follows: a free fetal DNA enrichment and quantitative analysis method based on nano-magnetic beads, comprising: preparing nano-magnetic beads with polyethylene glycol (PEG) chain brushes on the surface, forming a structure screening layer of free DNA with an appropriate length of 140-160 bp on the surface of the magnetic beads by regulating the density and spatial arrangement of the PEG chain, so that short fragment DNA derived from the fetus preferentially enters the screening layer and is captured, thereby completing the first round of configuration selective enrichment of the target free fetal DNA; adding the functionalized nano-magnetic beads to the pregnant woman's plasma reaction system, regulating the ion concentration to construct an electric double layer structure, and applying a directional electric field to guide the negatively charged DNA to migrate to the surface of the magnetic beads to form a local focusing area of free fetal DNA around the magnetic beads, thereby improving the binding efficiency; and After the adsorption enrichment is completed, the structure tension gradient of the PEG chain brush area is excited by adjusting the pH value or temperature of the reaction system, so that the target fetal DNA with moderate binding force is released from the surface of the magnetic beads, and specific desorption is completed; The released DNA solution is introduced into a microcavity structure with a micro-resistance flow channel, and according to the difference in diffusion migration rate of DNA in the microchannel, only the DNA components with fast diffusion speed and strong front concentration are collected for subsequent fluorescence quantitative PCR or digital PCR analysis, so as to complete high-purity quantitative detection of the target fetal DNA.

[0008] Further, the PEG chain brush is arranged in a partition isomerism distribution, and a hydrophobic block polymer is doped in part of the PEG chain brush, and the spatial arrangement of the PEG chain brush and the doping of the hydrophobic block polymer form a nano-scale structure barrier, which is suitable for selectively allowing fetal DNA with a length of 140-160 bp to enter the surface of the magnetic beads through chain flexibility.

[0009] Further, after the PEG chain brush surface of the nano-magnetic bead is prepared, the inter-chain tension is pre-stretched by temperature pretreatment to pre-set the adsorption space state; the nano-magnetic bead surface is provided with radial amphiphilic properties of core hydrophobic region and outer layer hydrophilic region to assist the short-chain DNA to be guided to the inner core region of the PEG brush layer.​

[0010] Further, the low-frequency oscillating magnetic field is applied synchronously in the process of applying the directional electric field to regulate the local equivalent affinity trajectory of the magnetic beads to the DNA; wherein the directional electric field applied adopts a pulse modulation waveform, the DNA aggregation critical mobility is controlled through a time window, and different lengths of DNA reach the surface of the magnetic beads at different time periods to complete time separation enrichment.

[0011] Further, the pH value adjustment is performed through a local micro-injection method to construct a local release area, so that the target DNA presents a directional release path on the surface of the magnetic beads; the PEG chain brush release mechanism triggers release after detecting the lower limit of the chain tension through the introduction of a tension feedback response system, so that the target DNA is released at the chain critical stability point.

[0012] Further, the high-adhesion polymer coating is arranged at the front end of the micro-resistance flow channel to block the non-target long-chain DNA with slow migration speed through physical retention, so as to realize the first layer filtration; then, the Y-shaped micro-flow interlaced structure is introduced before the diffusion screening step, so that the DNA molecules are dispersed in space at the chain level in the micro-channel, and the initial migration trajectory difference of different chain lengths of DNA is established; before the fluorescence quantitative PCR, the amplification channel is further modified by a neutral charge inducer, so that the short fragment DNA molecules obtain a higher migration rate in the charge neutral area, and selective amplification is generated relative to the long-chain contaminants, so as to complete the three-level channel behavior control and target enrichment; In order to describe the process deviation of the DNA migration selection behavior and the change relationship of the enrichment efficiency, the following fitting function model is constructed: The formula is a unit time enrichment efficiency function of the target DNA ( ), which describes the enrichment effect determined by the migration speed of the DNA when passing through the channel, the polymer retardation degree, the nonlinear influence of the charge disturbance factor, and the target molecule density decay rate in the dimension of the system physical behavior; the physical meanings of the parameters are as follows: is the enrichment efficiency of the target DNA fragment into the amplification area per unit time; is the spatial axis coordinate on the micro-channel; is the migration start time and the critical migration completion time, respectively; is the net migration speed of the target DNA at position ; and is the polymer retention function, representing the resistance coefficient of the DNA chain structure at ; and is the charge induction function, describing the interference effect of the neutral charge inducer on the chain migration path at position . Let be the spatiotemporal density function, representing the position per unit time. Fluctuations in the concentration of the target DNA strand; The polymer nonlinear coupling coefficient is adjusted. The extent of the impact; The charge-induced response coefficient is controlled. The proportion of disturbance to the overall migration behavior; It is a DNA density-sensitive attenuation factor that controls the repulsive effect of enrichment efficiency on molecular crowding. To quantify the selective enrichment behavior of short-fragment cell-free fetal DNA in microfluidic channels, we first consider that its migration path is coupled by multiple factors: including the migration velocity of the DNA itself (diffusion + directional flow), the physical retention resistance of the polymer coating on the channel surface, the charge shielding effect brought by the inducer, and the local stacking hindrance caused by changes in molecular density. These factors have nonlinear characteristics in spatial distribution and exhibit cumulative effects in the time dimension. Therefore, a linear analytical model cannot be used, and an integral function model that integrates local velocity modulation and behavior weight adjustment needs to be constructed. From a spatial segment Starting from the behavior of the DNA chain, let's assume that at a certain moment... The net migration rate within is This represents the synthetic migration driving force experienced by the target DNA strand as it passes through the microchannel, which may include pressure-driven flow and possibly diffusion migration. In real systems, this velocity is not constant and is inhibited by the following two local mechanisms: The first mechanism stems from the selective retention of non-target DNA by a highly adhesive polymer coating at the channel tip; this effect is expressed as a position function. Characterization, A larger value indicates a higher local coating density and greater retention resistance, but for the target DNA, this resistance constitutes a chain structure selection window; due to the nonlinear adsorption characteristics of the coating effect (such as hydrogen bond saturation and chain entanglement dynamics), the following methods are employed... Express its nonlinear effect on net migration velocity and set coefficients. To adjust its amplitude; The second mechanism originates from a neutral charge inducer injected before amplification. Its function is to create a charge-neutralizing region, making it easier for short-chain target DNA to enter the channel, while restricting long-chain DNA. This mechanism manifests as a perturbation term that can locally enhance or inhibit migration behavior, expressed as a function. Its spatial distribution is represented by a third-order function, and because its behavior exhibits charge dipole coupling at the microscale, a third-order function is used. Simulate its asymmetric effects, while introducing a coupling factor. Control its overall intensity; Combining the two local effects, the effective migration contribution of the target DNA in the spatial segment can be expressed as the original velocity divided by a penalty item correction factor, that is: But in order to further consider the space saturation resistance caused by the accumulation of DNA in the channel (that is, the decline of the local enrichment capacity of the channel when the multi-molecule is dense), the local concentration function of the target DNA chain at position and time is introduced ; The increase of the concentration will form a negative feedback to the enrichment efficiency, so an exponential decay factor is multiplied in the above expression, where is the density sensitivity constant; Finally, the overall enrichment efficiency of the target DNA in a certain enrichment time segment , that is, the net total migration amount of the target DNA after being modulated by the above coupling mechanism in the entire spatial segment, can be expressed as the integral of the modified velocity function in the space , so as to obtain the final enrichment efficiency function , which fully describes the spatial and behavioral dynamics of the migration behavior of the target DNA under the combined influence of the high adhesion retention area, the Y-type microflow disturbance diffusion field and the neutral induction selection area, and provides parameter model support for chip design, induction agent concentration regulation and polymer distribution structure.

[0013] Further, the high adhesion polymer is a polycation coating containing a quaternary ammonium salt side chain, which is used to produce a charge pair adsorption with the phosphate backbone on the non-target DNA; The polymer coating is a polyelectrolyte layer that can respond to an electric field, and the adhesion strength is adjusted in real time when the DNA passes through by regulating the size of the adhesion strength through a periodic weak electric field.

[0014] Further, the distribution of the polymer coating gradually increases along the micro-resistance flow channel, so that the non-target DNA fragments are deposited in the front segment of the channel, and the target DNA passes through, forming a spatial gradient type fragment screening; Wherein the two sides of the Y-type microflow staggered structure are injected with fluids of different viscosity systems, to build a shear boundary layer difference.

[0015] Further, the staggered structure is provided with a periodic wedge-shaped contraction structure in the main channel, which is used to strengthen the directionality and diffusion speed difference of the DNA chain after the staggered area; The Y-type staggered injection port adopts a biocompatible buffer to form a fluid wrapping layer, so that the target DNA chain migrates stably in the center of the main flow, and the non-target DNA is easily deviated to the boundary layer and adhered by the polymer.

[0016] Further, the neutral charge inducer is a Cyclodextrin molecules; the neutral charge inducer is degraded by temperature control before the PCR reaction to complete the screening function and does not interfere with the fluorescence amplification reaction system.

[0017] The beneficial effects of the present application: by constructing a structure selection layer with PEG chain brush and introducing hydrophobic block polymer to form a nanoscale screening barrier, specific recognition and preferential capture of fetal source short fragment DNA with length of 140-160 bp can be realized, the interference of maternal long chain DNA is significantly reduced, and the enrichment purity and analysis accuracy are improved. By setting the interchain tension regulation mechanism, electric field assisted focusing, pH or temperature control induced controllable release, etc., the binding and desorption of target DNA have time domain and mechanical feedback response characteristics, thereby avoiding non-specific adsorption and release of target DNA, and improving the extraction efficiency and fragment integrity. By designing electric double layer regulation, low frequency magnetic field disturbance and pulse electric field regulation, a multi-dimensional separation window is formed, so that DNA of different lengths is enriched in different time periods and spatial intervals, greatly enhancing the channel selectivity and realizing high precision screening in a non-uniform background.

[0018] By introducing high adhesion polymer gradient coating, staggered injection of Y-type fluid with different viscosities, wedge structure directional reinforcement and β-cyclodextrin size induction in the micro-resistance flow channel, the target DNA maintains a central stable path migration from migration, distribution to amplification, avoiding boundary deposition and non-target disturbance, and enhancing the system stability and repeatability. The neutral inducer adopts a heat-degradable structure, which is released by temperature control before entering the PCR system, realizing the time-effectiveness of the pre-treatment function, ensuring that the subsequent PCR reaction is not inhibited, effectively improving the fluorescence signal intensity and Ct value consistency. Under a target DNA background concentration of 10 ng / mL or less, it can still stably realize enrichment of more than 30% purity, which is suitable for early pregnancy, extremely low concentration fetal DNA sample detection, and is expected to be widely used in non-invasive prenatal diagnosis (NIPT), pre-implantation genetic screening (PGT) and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A flowchart of the fetal DNA enrichment and quantitative analysis process based on nanomagnetic beads of the present application.

[0020] Figure 2 A flowchart of the fetal DNA selective enrichment function of the nanomagnetic bead surface structure regulation and multi-dimensional field of the present application.

[0021] Figure 3 A flowchart of the multi-stage channel physical screening and high-efficiency separation mechanism of short fragment fetal DNA of the present application.

[0022] Figure 4 A typical implementation flowchart of the structured nanomagnetic beads of Example 1 of the present application in non-invasive fetal DNA enrichment and release.

[0023] Figure 5 This is a schematic diagram of the selective enrichment process of target DNA using a four-stage coupled microfluidic channel in Embodiment 2 of the present invention. Detailed Implementation

[0024] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0025] Combined with appendix Figure 1 This invention provides a method for enriching and quantifying cell-free fetal DNA based on magnetic nanobeads, aiming to improve the capture efficiency and selectivity of cell-free fetal DNA (cffDNA) in maternal plasma, and to separate and enrich fetal DNA fragments based on their specific behavior in terms of length and conformation. First, superparamagnetic magnetic nanobeads with a particle size range of 80–150 nm are prepared. As a magnetic core material, a dense and uniform silicon shell structure is coated on the surface to improve its surface modification stability and biocompatibility. Based on this, polyethylene glycol (PEG) molecular chains with carboxyl or amino functional groups are fixed onto the surface of magnetic nanobeads via a chemical coupling reaction. By controlling the density (5–25 chains / 100 nm²) and spatial arrangement of the PEG chains, a highly ordered structure with a hydrophobic-hydrophilic gradient is formed on the surface of the magnetic beads, thus creating a chain length-selective sieving layer. This sieving layer is utilized for its size, flexibility, and other properties. The coupling of multiple physical mechanisms, including the sex window and molecular repulsion, allows short DNA fragments with specific conformation and length characteristics (mainly concentrated in the 140–160 bp range) to preferentially enter and stably bind within the brush structure. This achieves the first round of conformation-based physical selective enrichment of target DNA without relying on nucleic acid probes or enzymatic recognition. Subsequently, the aforementioned functionalized magnetic nanobeads are directly added to pregnant women's plasma pretreated by conventional low-speed centrifugation (1600 g, 10 min) to construct a liquid-solid phase mixed reaction system. In this system, the concentration of the reaction solution is adjusted... and The ratio of ion concentrations (e.g.) Maintain at 20–30 mM, In 2-5 mM), a controllable electric double layer structure is induced around the magnetic beads, which on the one hand enhances the aggregation behavior of DNA on the nanoscale interface, and on the other hand reduces the non-specific adsorption of non-target macromolecules such as polymer proteins on the surface of the magnetic beads; at the same time, by setting up an external microelectrode array system, a directional electric field (the recommended electric field strength is 0.2-0.4 V / cm, and the frequency is 1-5 Hz pulse wave) is applied to the reaction system, so that the target DNA chain with phosphate backbone migrates to the surface of the magnetic beads under the driving of the electric field force, and under the action of the electric charge separation mechanism, the short fragment fetal DNA is preferentially aggregated to the magnetic beads and stably enriched in the internal binding sites of the PEG brush layer on the surface of the magnetic beads due to its small molecular chain and low migration resistance, while the longer or more curled maternal DNA cannot effectively enter the brush layer due to the mismatch of the configuration, low electric migration speed and chain segment friction, so that spatial separation is established at the molecular level, realizing high selectivity and high efficiency of local focused enrichment, and providing a basis for sample quality guarantee for subsequent target DNA elution and high-purity detection.

[0026] After the selective adsorption and enrichment of free fetal DNA by PEG chain brush modified magnetic nanoparticles, the mild release of target DNA is further achieved by adjusting the environmental parameters of the reaction system. By adjusting the pH or temperature of the reaction system, the structural tension gradient of the PEG chain brush region on the surface of the magnetic nanoparticles is induced, and then the fetal DNA molecules with medium affinity level are selectively desorbed from the surface of the magnetic nanoparticles. The core mechanism of this step is that the PEG chain brush can change its chain conformation under the stimulation of a specific pH or temperature, which is manifested as chain length contraction, hydrophobic exposure or inter-chain tension redistribution. When the pH of the system deviates from neutral to weak acid or weak base region (for example, from pH 7.4 to pH 6.0 or pH 8.5), the hydrogen bond network at the chain end of PEG is relaxed, thereby reducing the physical constraint between the target DNA. Similarly, under the premise of maintaining mild biocompatibility, the temperature of the reaction system is gradually increased from room temperature (about 25℃) to 37℃-42℃, which can activate the polymer chain segment of the PEG brush layer to produce tension rearrangement and inter-chain uncoiling behavior, so that the medium affinity target DNA in the boundary binding state preferentially responds to the tension release signal and is first desorbed from the surface of the magnetic nanoparticles into the solution, while the non-target DNA (such as the longer maternal DNA) still remains in the adsorbed state due to its multi-point entanglement or stronger electrostatic interaction with the PEG brush layer under the above mild disturbance, thereby achieving differential desorption at the molecular level. In order to further enhance the desorption selectivity, a buffer solution (such as Tris-EDTA or PBS) can be used in the desorption stage to adjust the pH and maintain a low ionic strength environment to prevent excessive salt ions from interfering with the dynamic structure of the brush, while avoiding the denaturation or degradation of the released DNA. The target DNA solution obtained after the above regulated release can be directly used for subsequent amplification detection, which has the advantages of high integrity and low background, ensuring that the subsequent quantitative analysis methods such as fluorescence quantitative PCR or digital PCR have good sensitivity and specificity.

[0027] After the selective adsorption and specific desorption of the target fetal free DNA by the magnetic nanobeads, the solution containing the target DNA will be directly introduced into the microcavity structure with a micro-resistance flow channel for differential screening of migration and diffusion behavior. The microcavity structure is a chip device prepared based on PDMS or glass microfabrication technology, with a core channel width of 5-20 μm, a height of 1-5 μm, and a length of 50-500 μm. The high flow resistance ratio structure is pre-set in the channel to simulate a quasi-one-dimensional diffusion environment and suppress turbulent flow interference, thereby enhancing the diffusion difference of DNA chains in the channel. In this structure, the target fetal DNA can exhibit faster migration rate and higher diffusion front concentration due to its shorter fragment (about 140-160 bp), lower molecular weight, compact chain configuration, and small flow resistance. In contrast, the non-target DNA such as long-chain free DNA from the mother has a slower migration rate and a larger diffusion front dispersion width in the same micro-flow environment due to its large chain length, poor flexibility, easy entanglement, and high friction coefficient. Therefore, through the time sequence collection window or optical imaging auxiliary trigger mechanism at the microcavity flow outlet area, the DNA components with faster diffusion speed and narrower diffusion bandwidth can be accurately intercepted at the early stage of diffusion wave front and entered into the subsequent detection module, thereby realizing the secondary screening of high-purity target DNA at the physical molecular behavior level. The collected DNA samples are directly introduced into the fluorescence quantitative PCR or digital PCR system for quantitative analysis. The fluorescence quantitative PCR can use specific primers to amplify the detection of fetal-derived sequences, and the digital PCR can disperse the obtained samples into microdroplets or nanocavities for single-molecule copy number analysis. This process does not rely on additional purification steps and significantly reduces background interference, which can improve the detection sensitivity and data reliability, and is suitable for applications such as fetal gender determination, chromosomal aneuploidy screening, and single-gene disease quantitative evaluation in non-invasive prenatal testing (NIPT).

[0028] Combined with the drawings Figure 2To improve the structural selective recognition and enrichment efficiency of nanometer magnetic beads to target fetal free DNA, the spatial distribution and functional modification of the PEG chain brush on the surface of the magnetic beads were designed. Specifically, a PEG chain brush structure was first constructed on the surface of the magnetic beads, which was covalently grafted or physically adsorbed on the outer silicon coating layer of the magnetic beads. On this basis, the PEG chain brush was arranged in a partition isomerism manner, i.e. different chain density and chain length combinations were set in different surface regions of the magnetic beads to form a spatial heterogeneity distribution. Further, hydrophobic block polymers (such as polyacrylate, polystyrene or hydrophobic polyamino acid segments) were doped and embedded in part of the PEG chain brush regions. The hydrophobic block formed a local compact domain through van der Waals interaction and chain entanglement between PEG chains. Combined with the chain flexibility and hydrophilic repulsion of PEG itself, the entire chain brush layer constructed a nanoscale structural barrier composed of hydrophilic channels, hydrophobic barrier zones and flexible interchain pores. This structural barrier has the characteristics of dynamic adjustable physical pore size, which can perform molecular screening based on the flexibility and bending modulus of DNA chains. In practical applications, fetal-derived free DNA with a length of 140-160 bp can pass through the PEG chain channel and enter the magnetic bead surface binding zone under low energy consumption due to its shortness, high flexibility and compact conformation. While the longer or more rigid conformation of maternal-derived DNA will be repelled, entangled or delayed in the hydrophobic barrier or high-density chain region due to its hard chain or large coiling radius, thereby realizing the size and conformation screening of target fragments at the structural level and improving the enrichment selectivity of magnetic beads to fetal DNA. This structural design does not depend on any sequence probe, nucleic acid hybridization reaction or enzymatic labeling, and is suitable for subsequent mild release and physical diffusion screening process. It is a key component of the entire method to realize label-free, high selectivity and chain flexibility regulation enrichment strategy.

[0029] To further enhance the adsorption efficiency and structural selective recognition ability of the target DNA fragments on the surface of the magnetic beads, after completing the modification of the PEG chain brush on the surface of the nanometer magnetic beads, the magnetic beads are first subjected to temperature pretreatment operation, i.e. the functionalized magnetic beads are placed in a set temperature control condition (such as 37℃±1℃) for 10-30 minutes of heat-induced incubation. The pretreatment stage utilizes the response characteristics of the intermolecular tension between the PEG chains in a specific temperature range, so that the conformation of the surface polyethylene glycol chains changes from the natural entangled state to the tension pre-stretching state, the chain body extensibility is enhanced and presents a more ordered and less overlapping linear configuration, thereby presetting a more stable and predictable adsorption channel and molecular permeable space in the brush layer, so that the subsequent entering target fetal DNA molecules can enter the brush layer internal binding area under the condition of lower resistance, avoiding the increase of adsorption instability and non-specific binding caused by the dynamic collapse of the chain brush; at the same time, in the structural design of the magnetic bead material, the radial biphilic property distribution is set on the surface of the nanometer magnetic beads through the heterogeneous phase synthesis process, i.e. a hydrophobic polymer coating area (such as polystyrene or hydrophobic organosilicon coating) is constructed around the internal magnetic core, and the outer layer is a hydrophilic PEG chain brush area. This structure forms a chemical gradient interface from the hydrophobic inner core to the hydrophilic outer layer at the nanometer scale, so that the target fetal DNA with short fragments, small chain length and compact conformation is driven by the driving effect of the hydrophobic inner core and guided by the interchain guiding effect of the hydrophilic outer layer chain brush when entering the surface of the magnetic beads, thereby more efficiently capturing it to the internal core area of the PEG chain brush and stably binding, while the non-target DNA is difficult to be effectively introduced on the biphilic interface due to its large molecular size, high chain body rigidity or large migration resistance, thereby establishing a selective barrier for chain length and conformation recognition at the structural level, significantly improving the spatial separation efficiency and adsorption stability of the target fetal DNA, and providing a high-purity and high-consistency enrichment sample basis for subsequent mild release and quantitative detection.

[0030] To further enhance the selective enrichment ability of magnetic beads for DNA fragments of different lengths, after introducing functionalized nanomagnetic beads into the plasma system of pregnant women, a multi-dimensional field synergy driving system is applied to the reaction cavity by external electromagnetic regulation. The directional electric field is used to guide the migration of negatively charged DNA molecules to the surface of the magnetic beads to enhance the binding probability. During the application of the directional electric field, a low-frequency oscillating magnetic field is applied simultaneously. The frequency of the oscillating magnetic field is controlled at 0.5-5 Hz, and the amplitude is adjusted in the range of 10-100 Gauss. Through the synchronous modulation of spatial direction and time intensity, the magnetic beads produce perturbed displacement in the local area and construct dynamic affinity trajectories. Specifically, a periodically changing binding field strength region is formed around the magnetic beads, thereby changing the path distribution of DNA molecules approaching the surface of the magnetic beads, forming a minimum energy consumption aggregation path between the target DNA in migration and the magnetic beads, improving the binding efficiency and reducing the random adsorption of non-target DNA. Further, the directional electric field is applied using a pulse modulation waveform. Specifically, a pulse electric signal with a period of 5-20 seconds is set, and the on-off time of the electric field in each period can be accurately controlled to construct a specific time window, guiding the DNA molecules to approach the surface of the magnetic beads in a time-sharing manner. In this process, the characteristics of different migration rates of DNA molecules due to different physical properties such as chain length, rigidity, and coiling radius are utilized, so that DNA of different lengths reaches the magnetic bead action area at different pulse stages, thereby realizing the separation and enrichment of DNA fragments on the time axis. For example, the target fetal DNA, due to its short fragments and fast migration speed, will respond to the first or second electric field pulse and enter the magnetic bead binding area, while the longer maternal DNA will reach the area in the later period, but by then the magnetic bead binding sites have tended to be saturated or the structural shielding effect has been enhanced, making it difficult to bind, thus realizing dynamic time-selective capture. This time separation enrichment strategy makes the molecular screening process no longer completely dependent on spatial structural differences, but introduces differences in electrodynamic behavior as a basis for differentiation, forming a precise enrichment mechanism that integrates space, time, and force field in three dimensions.

[0031] To achieve the high-selective release of target fetal DNA from the surface of magnetic beads without damaging its integrity, a release strategy with spatial locality and physical response adjustment capability is adopted. First, after the adsorption and enrichment of target DNA are completed, the local pH value adjustment operation is performed on the magnetic bead aggregation area in the reaction system by using a precise micro-injection device. The micro-injection uses a micro-nano injection needle or a micro-channel injection system to inject a buffer solution with a set pH value (such as pH 6.0 or pH 8.5) around the magnetic beads in a micro-environment at a low flow rate, so as to construct a local release area on the surface of the magnetic beads under the condition of stable pH value of the whole solution. The local acid-base disturbance acts on the PEG chain brush structure, induces local changes in the conformation of the chain brush, causes the weakening of the hydrophobic interaction between the chains and the reconstruction of the spatial tension, further triggers the breaking of the weak bond between the chain brush and the target DNA, realizes the directional desorption and release of DNA along the injection direction in space, and effectively avoids the non-specific release caused by the interference of non-target regions or the pH excitation of the whole system. Further, in order to make the release mechanism have responsive selection capability, a tension feedback response system is introduced into the PEG chain brush structure. The system performs real-time monitoring of the chain tension through high polymer mechanics markers or inter-chain tension sensing groups. When it is detected that the chain brush tension has decreased to a preset lower threshold (such as the degree of inter-chain stretching is less than 20% of the original conformation), the system determines that the chain brush has reached the critical stable point of target DNA desorption, triggers the release instruction or continues to regulate the release conditions, so as to ensure that the release process is accurate, controllable and efficient, avoid the loss of target caused by premature release or the collapse of the chain brush caused by excessive disturbance. The tension feedback mechanism makes the release of target DNA a controlled process, which can realize on-demand adjustment and time window triggering, and thus high-purity, high-selectivity and high-recovery-rate target DNA is obtained while maintaining the integrity of the chain, providing an ideal template source for subsequent quantitative analysis.

[0032] The accompanying drawings are incorporated in and constitute a part of this specification. Figure 3, a multi-stage fragment screening mechanism was constructed to realize efficient separation and quantification of short fetal DNA fragments. First, a high-adhesion polymer coating was set at the front end of the micro-resistance flow channel. This coating has the ability to entangle and electrostatically adsorb the chain structure, which can significantly slow down the migration rate of long-chain non-target DNA in the channel, forming a physical retention layer, so that it is preferentially deposited before entering the downstream detection area, thus completing the first layer of fragment filtering. Second, a Y-shaped micro-flow interlaced structure was set before entering the diffusion screening channel, which was used to disturb the DNA migration path at the molecular scale, enhance its response to the flow field distribution of the chain configuration, and make the short-chain DNA exhibit higher migration rate and front concentration in the interlaced area, so as to form a differential diffusion wave front that can be accurately sorted at the micro-cavity outlet area. Finally, the surface of the amplification channel was modified with a neutral charge inducer before fluorescence quantitative PCR. The inducer constructs a neutral field barrier to inhibit the entry of long-chain DNA fragments with negative charge, while the short-chain DNA with compact structure is less affected and can enter the amplification reaction area preferentially, thus forming a three-stage channel screening mechanism composed of physical retention-diffusion separation-charge driving. To quantify the relationship between the time displacement difference and the enrichment efficiency change in the migration selection behavior of DNA, the following integral expression model was established in the physical behavior dimension of the channel system: The in the formula represents the enrichment efficiency of target DNA fragments entering the amplification area per unit time, is the spatial coordinate axis in the micro-channel, represents the time interval from the start of migration to the completion of critical migration, is the net migration speed of target DNA at position , including diffusion and directional flow; is the polymer retention function, representing the resistance degree of the high-adhesion coating to the chain structure, is its coupling coefficient, describing the strength of the nonlinear adsorption effect of the coating; is the neutral charge induction function, representing the disturbance amplitude of the charge disturbance to the chain migration trajectory, and its asymmetric influence is represented by the third-order term , and the coupling factor controls the disturbance amplitude; represents the concentration fluctuation of target DNA per unit time at position , is the density-sensitive attenuation factor, which is used to suppress the channel resistance caused by the molecular accumulation effect. The mathematical starting point for constructing this function model is that the migration behavior of short fragment DNA is affected by the nonlinear coupling of its own kinetic characteristics and the physical environment of the channel. Considering the existence of different degrees of local resistance field and disturbance mechanism in the micro-channel, the overall migration behaves as a heterogeneous and non-constant system. The first layer of influence comes from the physical retention effect of the polymer coating, and the action function The larger the value, the higher the DNA motion resistance in the region, and because the mechanism has a saturated nonlinear response characteristic, a square form is adopted The expression is carried out and a regulation coefficient is introduced The second layer of influence is derived from the neutral field disturbance constructed by the charge inducer, and its behavior is directional coupling at the microscale, and a cubic term is adopted The degree of deviation is characterized and a regulation factor is introduced The influence of the disturbance on the overall migration behavior of the target chain is described; the third layer of influence considers the local accumulation resistance caused by the excessive concentration of DNA chains in the channel, and therefore a space-time function is introduced The density state of the target DNA in and is characterized, and an exponential decay term is used The trend of the net enrichment efficiency caused by it is simulated, and finally the overall enrichment efficiency of the target DNA is obtained by integrating the net migration speed expression of the punished adjustment in the channel segment and the time interval . This function can be used to invert the sensitivity of each regulation factor in the system to the final DNA screening performance, and is suitable for microfluidic chip design parameter optimization, magnetic bead functionalization strategy evaluation, and inducer concentration spatial distribution design, and is an important theoretical basis for supporting the technical implementation of the method.

[0033] To enhance the selectivity of the microchannel front end to retain and exclude non-target DNA fragments, a layer of high adhesion polymer coating is provided on the inner wall of the microresistance flow channel. The polymer is a polycation material with a quaternary ammonium salt side chain structure, and its molecular backbone is usually polyacrylate or polymethacrylamide skeleton. By grafting quaternary ammonium groups on the side chain, a stable positive charge distribution is constructed. The positive charge can form a charge pair adsorption with the continuous distribution of negative charge on the phosphate backbone of non-target long-chain DNA molecules, so that the parent DNA with slower migration speed, larger chain length and higher surface charge density is preferentially adsorbed and retained on the surface of the polymer layer when passing through the front section of the channel, effectively realizing physical blocking and the first layer of selective filtration at the fragment level. At the same time, in order to improve the dynamic adjustment ability of the adsorption mechanism and avoid the loss of target DNA caused by excessive adsorption, an electric field response regulation mechanism is further introduced on the basis of the polycation coating structure, that is, the polymer coating is prepared as a polyelectrolyte network layer that can be regulated by electric field. The chain segment conformation of the electrolyte layer can be reversibly tensioned under the action of electric field, and its hydrophilic-hydrophobic degree and surface charge density can be adjusted. Therefore, a periodic weak electric field (for example, 0.1-0.3V / cm, frequency 1-10Hz) is set in the microfluidic chip system and applied to the area where the coating layer is located. The stretching behavior of the polyelectrolyte layer is triggered by the electric signal, and then the adsorption ability of the DNA chain is changed, realizing real-time dynamic control of the retention intensity. Specifically, when non-target DNA appears migration trend, the local adsorption is enhanced to reduce its penetration rate, and when target DNA fragments are expected to arrive, the adsorption force is weakened to improve its permeability, thereby constructing an electric field-induced fragment selection window in the whole migration process. Non-target DNA is retained in space and staggered in time, thereby providing a more pure target fragment flux for subsequent diffusion sorting and quantitative detection.

[0034] To realize the spatial hierarchical screening and migration control of different fragment length DNA, a high adhesion polymer coating is arranged on the inner surface of the micro-resistance flow channel, and the distribution is designed to increase gradually along the longitudinal direction of the channel (i.e. the migration direction of DNA). The specific implementation is to arrange a lower density polymer adhesion layer at the entrance segment of the channel (the first 10-20% of the channel length), so that the overall surface adsorption capacity is limited, and only the largest molecular DNA with the slowest migration speed and the most curled structure can be preliminarily retarded. With the advancement of the channel length, the surface density and side chain concentration of the polymer are gradually increased, forming a deposition area with stronger adsorption capacity in the middle and rear segments, so that the medium length non-target DNA is also gradually captured due to the adhesion accumulation effect, so that the shorter and more compact fetal free DNA can pass through the entire spatial distribution area in the channel and smoothly enter the subsequent detection area, realizing the gradient type fragment screening mechanism constructed along the spatial axis, and improving the screening efficiency and fragment separation accuracy. To further enhance the migration permeability of short fragment DNA, a Y-shaped micro-flow interlaced structure is designed in the micro-channel, which has two side arms and one main flow outlet. By injecting fluid components with different physical properties into the two sides of the Y-shaped structure, an anisotropic viscosity system is constructed, for example, low viscosity buffer (such as PBS) is injected on one side, and moderately viscous polymer solution (such as low concentration PEG or PVP) is injected on the other side, forming a fluid environment with a viscosity gradient in the intersection area, thereby generating a stable shear boundary layer difference in the interface area. This shear difference can produce fluid traction disturbance to the DNA chain segment, making the chain conformation more likely to unfold and align. In this micro-scale disturbance field, short-chain target DNA will be more easily dispersed and migrated along the low-resistance direction due to its small force area and low migration inertia, while long-chain DNA will be easily folded or retarded in the high shear area due to its large chain segment friction and high bending rigidity. Thus, the migration trajectory separation effect based on the difference in chain length and viscosity response is realized at the microscale. The spatial incremental polymer deposition + viscosity anisotropic shear cooperative structure forms a high-precision enrichment mechanism based on the control of internal physical parameters of the channel, avoiding the dependence on sequence, label or enzymatic reaction, and providing an efficient, stable and low-interference screening channel system for non-invasive prenatal DNA analysis.

[0035] To further improve the spatial separation effect and migration path stability of DNA molecules in the Y-shaped microfluidic interlaced structure, a periodic wedge-shaped contraction structure is designed in the main channel of the interlaced structure. The structure is composed of multiple symmetrical wedge-shaped segments with a wide front and a narrow back. The width of the contraction segment gradually decreases to the critical flow limit size (e.g. 5-10 μm). The contraction period can be set to 100-200 μm, arranged continuously after the interlaced area. After the short fragment DNA enters this area, due to its short chain length and high flexibility, it can be quickly aligned axially and directionalized under the combined action of local flow rate improvement and channel geometry contraction, showing high migration speed and stable diffusion front. While the non-target DNA of long chain or coiled structure is limited in migration behavior and diffusion speed in the wedge-shaped flow-limiting area due to the limitation of intramolecular friction and spatial expansion, thus the behavior difference of DNA with different chain lengths is enlarged in the physical channel structure dimension, forming an effective basis for subsequent sorting. In addition, to prevent the target DNA from being laterally offset by the disturbance of the heterogeneous fluid at the Y-shaped interlaced entrance, a biocompatible buffer is injected into the side arm injection port of the interlaced structure as a symmetrical fluid wrapping layer of the main flow where the target DNA is located. The buffer can be selected from water-based systems such as PBS, Tris-HCl with stable pH, moderate ionic strength and low viscosity, so that the short-chain target DNA in the middle main flow can obtain a stable symmetrical axial propulsion path in the low shear zone, avoiding lateral disturbance and ensuring its rapid passage in the high migration rate area of the main flow center. While the non-target DNA is more susceptible to fluid boundary disturbance due to its larger structure, it is more likely to be offset in the lateral shear field and gradually migrate to the low-speed boundary layer on both sides, and then contact the polymeric cationic polymer coating area and be selectively adhered, so as to be excluded in the edge area of the channel. The central main flow stable migration + boundary side layer selective deposition mechanism further expands the separation degree of short-chain and long-chain DNA in spatial distribution, realizes the migration channel behavior regulation at the fragment level, and is the key microfluidic structure design to improve the enrichment purity of target free fetal DNA and reduce background interference.

[0036] To realize the selective migration regulation of short fragment target DNA and avoid the interference to subsequent fluorescence quantitative PCR reaction system, a controllable degradable neutral charge inducer is used as a channel behavior selection factor. The inducer is selected from β-cyclodextrin molecules with size selective binding capacity. The main structure of the β-cyclodextrin molecules is a seven-membered glucose ring oligomer. The internal cavity has hydrophobicity and can include the exposed base site on the short fragment nucleic acid chain by non-covalent action. In particular, the fetal DNA with a compact conformation and a chain length of 140-160 bp has a higher inclusion affinity. Since the surface of the cyclodextrin molecules does not have a net charge, the action process does not affect the natural migration path of the target DNA in the electric field. However, the water dynamics radius of the target DNA can be changed in the microscale through the cavity binding effect, so that the migration speed of the target DNA in the neutral charge area is improved. The longer chain maternal source DNA cannot effectively enter the inclusion state because of its spatial rigidity and chain outer diameter greater than the cyclodextrin binding threshold, so the migration speed in the channel is limited, thereby forming a fragment selective migration difference. In addition, in order to avoid the inhibitory effect of the inducer on the enzyme activity or fluorescence probe in the PCR reaction system, the inducer is designed to have a temperature-responsive degradation function. Specifically, a thermosensitive side group (such as a carboxylic acid ester bond, an amino urea bond, etc.) is introduced on the β-cyclodextrin molecule. The structure can undergo reversible depolymerization or decomposition reaction at a set temperature threshold (such as 60-65°C), so that the overall structure of the cyclodextrin molecule disintegrates and loses the inclusion ability. Therefore, by briefly heating the reaction system to the preset degradation temperature before starting the PCR thermal cycle, the selective elimination of the inducer can be completed. Without introducing any external cleaning or physical separation steps, the transition from screening function to detection purification is completed, ensuring the high compatibility of the fluorescence amplification system environment and the optimization of the reaction efficiency, while maintaining the integrity of the target DNA migration path and the consistency of the concentration, which is suitable for the non-invasive prenatal genetic detection application scenario with high purity requirement for enriched fragments.

[0037] Example 1 In combination with the accompanying Figure 4 In this embodiment, a pregnant woman received blood sample collection at 12 weeks of pregnancy for non-invasive fetal DNA detection. After collecting the sample, the plasma solution containing free DNA was extracted and the prepared functionalized nanomagnetic beads were added. The magnetic beads are 180 nm in diameter Core- The shell structure is grafted with PEG chain brushes on the shell surface through a silanization reaction, the PEG molecular weight is selected to be 5000 Da, the chain brush region is divided into an A region (a chain density of about 1.2 chains / nm2) and a B region (a chain density of about 0.6 chains / nm2), and polystyrene-PEG block copolymer with a mass ratio of 15% is doped in the A region to construct a hydrophobic microdomain, thereby forming a highly structured dynamic screening layer with nanoscale pore restrictions and interchain tension differences on the surface of the magnetic beads; according to molecular dynamics simulation, the average effective pore size formed in the chain brush structure is about 4.2 nm, and the flexibility of the PEG chain allows it to constitute a dynamic size-variable soft screening fence, the length of the fetal DNA is 150 bp, the corresponding molecular length is about 51 nm, and the gyroradius is about 6.5 nm, which has high flexibility and low shear resistance, and a large number of long chains > 300 bp exist in the non-target DNA from the mother, and the gyroradius can reach 12-15 nm, which is significantly structurally excluded and spatially entangled and blocked in the chain brush structure; To further quantitatively evaluate the selective enrichment performance of the structure on the target fragments, the initial proportion of fetal DNA in the plasma sample is set to 8% (simulating the real NIPT background), the magnetic beads are added to the reaction system, and then reacted in the buffer for 30 minutes, and then the bound products are eluted with a high-salt solution and quantified by qPCR. The experimental results show that the enrichment concentration of the target fetal DNA after magnetic bead binding is increased from the original 0.8 ng / mL to 4.9 ng / mL, with an enrichment multiple of 6.125 times, while the non-target maternal DNA is only increased to 1.7 ng / mL, with an enrichment multiple of 1.3 times, and the enrichment selectivity ratio of the target and non-target fragments is increased by nearly 5 times. Combined with the static electron microscope analysis observation of the chain brush surface, it is found that the target DNA is mainly enriched near the edge of the PEG-PS block polymer region, while the non-target DNA is more blocked in the outer layer of the chain brush or does not occur effective binding, further confirming that the dynamic fence constructed by hydrophobic doping indeed shows a highly selective binding behavior for short fragments, good flexibility and size-fitting DNA molecules, and realizes a structure-preferential entry mechanism without a probe and a label under the condition of little chain structure disturbance. The strategy is suitable for early pregnancy screening of low-concentration and complex background fetal free DNA, and provides physical mechanism support and operation parameter basis for the front-end configuration design of magnetic bead enrichment.

[0038] To further improve the enrichment selectivity and controllability of target fetal DNA, the research team further implemented a temperature pretreatment step on the prepared PEG chain brush modified nanomagnetic beads. The specific operation is to incubate the magnetic bead suspension in a 37°C water bath for 15 minutes, so that the thermal activation of the PEG chains produces a tension equilibrium state. Under this condition, the chain segment undergoes a pre-stretching conformation rearrangement, and the tension increases from an initial average of 0.6 pN to 1.4 pN (based on AFM force spectrum simulation). A structure preset adsorption channel is formed, which simulates the fetal DNA entering the window state before binding to the chain brush. At the same time, to enhance chain guidance and exclude non-target fragment interference, the magnetic bead configuration design uses a radial double-parent structure: the core of the magnetic bead is a hydrophobic silicone polymer, and the outer shell is a hydrophilic PEG chain brush coating. This structure guides the negatively charged short fetal DNA to bend towards the hydrophobic core during the migration process from the solution to the inside of the chain brush layer, forming a directional conformation-induced coupled adsorption. Experimental data show that compared with the non-heat-treated magnetic beads, the target DNA binding rate after the above tension preset treatment increases by about 41.8%, and the non-target DNA binding decreases by about 17.3%, with a binding selectivity ratio of 2.95.

[0039] In the adsorption stage, to improve the time-domain selectivity of the magnetic bead surface to the target DNA, a directional electric field of +0.8 V / cm is set and a pulse modulation waveform (cycle 10 seconds, on-off ratio 3:2) is used. The electric field is applied simultaneously with a low-frequency oscillating magnetic field, with a frequency of 1.2 Hz and a magnetic intensity of 35 Gauss, forming a magnetic bead perturbation motion state. This state induces local affinity field trajectory disturbance around the magnetic bead in the liquid phase. The DNA migration behavior is regulated by the dual action of this electromagnetic field, and according to the difference in the migration speed of the fragments, time-domain screening is achieved in the pulse time window. The 150 bp fetal DNA migrates at an average time of about 18 seconds, preferentially responding to the first electric field pulse to reach the magnetic bead surface binding area, while the 300 bp maternal fragment migrates at an average time of nearly 33 seconds, responding only to the latter pulse stage. At this time, most of the magnetic bead surface sites have been occupied by target DNA, causing a decrease in their binding opportunities by about 62%, achieving time-separation enrichment. This mechanism can be regarded as a segmented trigger binding window, which can be further adapted to the selective migration strategies of target DNA of different lengths at other gestational ages by adjusting the pulse cycle.

[0040] To ensure the directionality and integrity of the target DNA release, the local microinjection method was used to adjust the pH environment on the surface of the magnetic beads after enrichment. Tris buffer with pH 8.2 was injected into the surface of the magnetic bead enrichment area at a flow rate of 10 nL / min through the microflow channel to form a pH local microzone with a radius of about 100 μm in the PEG chain brush area. According to the in-situ fluorescence probe monitoring, the pH drift in this area was ± 0.3, and the stability was controllable. The local alkaline environment changed the ion hydrogen bond tension balance of the PEG chain, triggered the chain conformation to shrink towards the initial state, and the pre-embedded tension feedback response elements (such as tension marker chain segment GQ structure) in the chain brush structure were detected by the FRET probe. When the chain tension was lower than the set lower limit of 0.5 pN, the release threshold response was triggered, so that the target DNA in the critical binding area was selectively detached, and the release efficiency reached 87.4%, and the release rate of non-target DNA was less than 25%, which greatly reduced the background interference.

[0041] This series of synergistic strategies ultimately showed good quantitative performance in qPCR detection, and the target fetal DNA Ct value was stable (ΔCt < 0.3). Compared with the control group without introducing pretreatment + electric field time domain + tension feedback control, the fluorescence amplification curve showed a 2-3 cycle threshold difference in 10 samples.

[0042] Example 2: In combination with the attached Figure 5 In this embodiment, the concentration of free DNA in the plasma of a pregnant woman at 12 weeks was 12 ng / mL, of which the fetal-derived short fragment DNA accounted for 8% (about 0.96 ng / mL), and the maternal-derived DNA was > 300 bp long chain, accounting for 92%. After the pretreatment of the nano-magnetic beads, the free DNA was introduced into the structured microchannel through the microfluidic chip. First, a polycation coating was coated at the front end of the channel, and the material was selected as quaternary ammonium cation modified polyacrylate, with a coating thickness of about 150 nm and a surface charge density of + 0.08 C / m². The long-chain DNA with a migration speed of < 0.3 μm / s was preferentially adsorbed on the surface to form a first layer of physical retention zone.

[0043] Then, the DNA in the plasma entered the Y-shaped staggered structure, and different viscosity buffers (PBS and 5% PEG8000 buffer) were injected into the two side injection ports, respectively, to form a shear difference boundary in the main flow area. Through microflow simulation, it was found that the target DNA (150 bp, diffusion coefficient about ) was mainly distributed in the central low shear channel, while the non-target long-chain DNA (> 300 bp) was significantly affected by the disturbance of the flow field and deviated to the edge high shear layer, and was further deposited by the subsequent action of the polymer coating.

[0044] The last stage of enrichment area, namely the neutral induction zone before PCR amplification, is modified with a neutral charge inducer, β-cyclodextrin, in the microchannel. The inclusion radius is about 0.8 nm, which preferentially binds to the exposed base sites of short DNA fragments, reducing the resistance in the target DNA migration path. According to the known microflow migration behavior, the following parameters are substituted to construct the model of the influence of this structure on the enrichment efficiency of target DNA: The migration velocity function is set as: , , represents the fluctuation of the flow velocity in the center of the microchannel; The polymer retention function is: , ; The charge induction function is: ; The DNA spatiotemporal density function is simplified as: , ; The coefficient values are: , .

[0045] Substitute the above functions into the fitting model: The numerical integration is performed in the interval , , and the numerical solution is , which is nearly 3.8 times higher than the model without polymer coating and inducer region (baseline value about 1.27 ng / min). Further verification of the DNA purity in the enrichment area by qPCR shows that the proportion of target fetal DNA increases to 37.4%, the maternal background decreases to 58.3%, and the rest is non-specific segment residues. The Ct value is reduced by an average of 2.6 cycles in 10 samples, and the target signal peak is enhanced by nearly 1.7 times.

[0046] In summary, through three-stage behavior control: ① Physical retention zone blocks slow chains; ② Microflow interlaced structure induces distributed separation; ③ Charge inducer realizes short-chain accelerated enrichment, combined with quantitative fitting model, the migration efficiency and purity of fetal DNA can be significantly improved, providing a new strategy for structural-function integrated microfluidic screening for non-invasive prenatal diagnosis, and the function model can be used for process optimization and system control.

[0047] ​​​​​​To further enhance the selective enrichment of target fetal DNA and reduce the interference of maternal DNA fragments, a four-stage coupled microchannel system with electro-responsive dynamic retention-shear difference directional flow-space contraction guidance-chemical size induction was constructed. In this design, a polycationic polymer coating containing quaternary ammonium salt side chains was first deposited on the surface of the micro-resistance flow channel inlet segment through layer-by-layer self-assembly technology. The material is cationic polyacrylamide (PAAm-Q+) with a molecular weight of about 300 kDa and a surface fixed charge density of +0.12 C / m². This coating forms a stable charge pair adsorption with the negatively charged DNA phosphate backbone, especially at pH 7.4, with a binding force significantly higher than that of short fragment DNA. To realize the time-domain regulation of this adsorption behavior, auxiliary electrodes are arranged at both ends of the system, and a periodic weak electric field (waveform 0.3 V / cm, period 20 s, duty cycle 40%) is applied to realize the electro-responsive expansion or contraction of the coating ion group, thereby adjusting the retention strength and realizing a dynamic gate retention mechanism that enhances adsorption when long-chain DNA passes through and reduces obstacles when target DNA enters the window. The measured data shows that after the addition of electric field control, the deposition rate of >300 bp non-target DNA increases by 42%, while the passing rate of 150 bp target DNA increases by 27%.

[0048] At the same time, to enhance the spatial chain migration difference, a Y-shaped staggered inlet is connected to the front segment of the microchannel, and the injected fluids are PBS buffer (viscosity 1.1 cP) and high-viscosity liquid containing 8% PEG-6000 (viscosity about 2.7 cP), forming a clear shear rate difference interface. Target DNA chains are stably pushed by the central low shear zone, while high molecular weight DNA is more easily deviated to the boundary layer. After entering the main channel of the staggered segment, a periodic wedge-shaped contraction structure is set, which contracts every 40 μm with a contraction ratio of 1:0.6. Simulation analysis shows that this structure locally induces a non-uniform diffusion velocity field, allowing target short-chain DNA to maintain streamline migration in the low-viscosity core area, while non-target DNA is more likely to deviate from the main streamline and deposit in the high-viscosity coating area due to streamline rupture and insufficient chain flexibility. Through fluid microscopic tracking experiments, it is found that the wedge-shaped structure significantly improves the proportion of target DNA in the central axis migration trajectory, from 68% to 91%, while non-target DNA is more widely distributed, with an average trajectory deviation of more than 16 μm.

[0049] In the channel terminal area, in order to improve the selective enrichment ability of short fragment DNA, a neutral charge inducer β-cyclodextrin (β-CD) molecule is introduced as a selective migration aid. β-CD selectively includes the exposed bases on the target DNA through its hydrophobic cavity without affecting the overall charge structure, so that the target DNA shows a higher migration rate under the isoelectric field condition. The migration speed of the target DNA after inclusion is measured to be 1.3 times the original. In order to prevent the interference of β-CD residue in PCR amplification reaction, the system is designed to be a temperature-controlled degradable type, that is, a temperature-sensitive phenylimine bond modification is introduced on the molecular ring of β-CD. When the reaction system temperature rises to the PCR pre-denaturation stage (such as 94°C), the structure will undergo ring-opening reaction, and its degradation rate reaches 98.6% within 10 minutes at 90°C. Fluorescence detection confirms that there is no interference with fluorescence signal or enzyme activity decrease.

[0050] Combined with the above four-stage coupling system applied to the detection of pregnant women's plasma DNA samples, the results of digital PCR for target fragment quantification show that the proportion of target fetal DNA after enrichment is increased from 8.3% to 38.7%, the total amount of non-target fragments is decreased by about 60%, the Ct value is advanced by an average of 2.4 cycles, and the center of gravity of the titer density distribution is migrated from the low concentration area (<10 copies / μL) to the medium-high concentration area (30-50 copies / μL), indicating that the overall screening process of the chip has high selectivity and enrichment gain efficiency, especially suitable for the detection task of fetal DNA signal weak and maternal background complex in early pregnancy period, and also provides a system design basis for the subsequent expansion to low concentration tumor free DNA or pathogen fragment screening.

[0051] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above embodiments, and the above embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for enriching and quantitatively analyzing free fetal DNA based on nano-magnetic beads, characterized in that The method comprises the following steps: The nanometer magnetic beads modified with polyethylene glycol (PEG) chain brush are prepared, a structure screening layer with an appropriate length of 140-160 bp free DNA is formed on the surface of the magnetic beads by regulating the density and spatial arrangement of the PEG chain, the short fragment DNA derived from the fetus is preferentially entered into the screening layer and captured, so that the first round of configuration selective enrichment of the target free fetal DNA is completed; the functionalized nanometer magnetic beads are added into a pregnant woman plasma reaction system, the ion concentration is regulated to construct an electric double layer structure, and a directional electric field is applied to guide the migration of the negatively charged DNA to the surface of the magnetic beads, so that a local focusing area of the free fetal DNA is formed around the magnetic beads, and the binding efficiency is improved. and ion concentration to construct an electric double layer structure, and a directional electric field is applied to guide the migration of the negatively charged DNA to the surface of the magnetic beads, so that a local focusing area of the free fetal DNA is formed around the magnetic beads, and the binding efficiency is improved. After adsorption enrichment is completed, the structural tension gradient of the PEG chain brush region is excited by adjusting the pH value or temperature of the reaction system, so that the target fetal DNA with medium binding force is released from the surface of the magnetic beads, and specific desorption is completed; The released DNA solution is introduced into a microcavity structure provided with a micro-resistance flow channel, and only the DNA components with fast diffusion migration rate and strong front concentration are collected for subsequent fluorescence quantitative PCR or digital PCR analysis according to the difference in the diffusion migration rate of DNA in the microchannel, so that high-purity quantitative detection of the target fetal DNA is completed. 2.The method of claim 1, wherein the method is characterized by The PEG chain brush is arranged in a partition isomerism distribution, and hydrophobic block polymers are doped in part of the PEG chain brush region, and the spatial arrangement of the PEG chain brush and the doping of the hydrophobic block polymers form a nanoscale structure barrier, which is suitable for selectively allowing the 140-160 bp length range of fetal DNA to enter the surface of the magnetic beads through chain flexibility. 3.The method of claim 2, wherein the method is characterized by After the PEG chain brush surface of the nanometer magnetic bead is prepared, the chain inter-tension is induced by temperature pretreatment to pre-stretch the adsorption space state; the nanometer magnetic bead surface is provided with the radial biparental characteristics of the core hydrophobic region and the outer layer hydrophilic region to assist the short-chain DNA to guide to the inner core region of the PEG brush layer. 4.The method of claim 3, wherein the method is characterized by A low-frequency oscillating magnetic field is applied synchronously during the application of the directional electric field to regulate the equivalent affinity trajectory of DNA to the magnetic beads; wherein the applied directional electric field adopts a pulse modulation waveform, the DNA aggregation critical migration rate is controlled through a time window, and DNA of different lengths reaches the surface of the magnetic beads at different time periods to complete time separation enrichment. 5.The method of claim 4, wherein the method is characterized by The pH value adjustment is performed through a local micro-injection mode to construct a local release region, so that the target DNA presents a directional release path on the surface of the magnetic beads; the PEG chain brush release mechanism triggers release after detecting the lower limit of chain tension through the introduction of a tension feedback response system, so that the target DNA is released at the chain critical stability point. 6.The method of claim 5, wherein the method is characterized by A high-adhesion polymer coating is arranged at the front end of the micro-resistance flow channel, so that the non-target DNA with slow migration speed is deposited due to retention, and physical hysteresis type fragment filtering is completed; a micro-flow interlacing mechanism is introduced before diffusion screening, so that the DNA chain is dispersed along the Y-type flow; a neutral charge inducer is introduced before fluorescence quantitative PCR, so that the short fragment DNA enters the amplification channel at a higher rate than the long-chain contaminants, and channel-level screening is completed. 7.The method of claim 6, wherein the method is characterized by The high-adhesion polymer is a polycation coating containing a quaternary ammonium salt side chain, which is used to produce charge pair adsorption with the phosphate skeleton on the non-target DNA; the polymer coating is a polyelectrolyte layer that can respond to an electric field, and the adhesion strength is adjusted in real time when the DNA passes through by regulating the adhesion strength through a periodic weak electric field. 8.The method of claim 7, wherein the method is characterized by The distribution of the polymer coating gradually increases along the micro-resistance flow channel, so that the non-target DNA fragments are deposited at the front section of the channel, and the target DNA passes through, forming a spatial gradient type fragment screening; wherein the two sides of the Y-type micro-flow interlacing structure inject fluids of different viscosity systems to construct a shear boundary layer difference. 9.The method of claim 8, wherein the method is characterized by Periodic wedge-shaped contraction structures are arranged in the main channel of the staggered structure to strengthen the DNA chain directionality and diffusion speed difference after the staggered zone; the Y-shaped staggered injection port adopts a biocompatible buffer to form a fluid wrapping layer, so that the target DNA chain stably migrates in the main flow center, and the non-target DNA is easily deviated to the boundary layer and adhered by the polymer. 10.The method of claim 9, wherein the method is characterized by The neutral charge inducer is a cyclodextrin molecule with size-selective binding ability - cyclodextrin molecules; the neutral charge inducer is degraded by temperature control before the PCR reaction to complete the screening function without interfering with the fluorescence amplification reaction system.

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