CN113684246B - High-sensitivity nucleic acid detection method without amplification and application thereof - Google Patents
High-sensitivity nucleic acid detection method without amplification and application thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The application discloses a nucleic acid detection method, which comprises the following steps: providing a sample to be tested, magnetic beads, a primary antibody and a secondary antibody; mixing and reacting a sample to be detected, magnetic beads, a primary antibody and a secondary antibody to form a magnetic bead complex; and detecting according to the detection mark of the magnetic bead complex to obtain a detection result. The nucleic acid detection method according to the embodiment of the application has at least the following beneficial effects: the magnetic beads are used as a reaction substrate and a minimum reaction unit of the whole reaction, so that the concentration and amplification effects can be achieved in the follow-up immune reaction. The nucleotide chain marked by the magnetic beads and the nucleic acid fragment to be detected in the sample to be detected are mutually paired to form a hybridization duplex, the hybridization duplex is captured by the primary antibody, and the hybridization duplex is detected by the secondary antibody, so that the specificity and the sensitivity of detection are improved, and the nucleic acid fragment to be detected is not required to be amplified additionally.
Description
Technical Field
The application relates to the technical field of nucleic acid detection, in particular to a high-sensitivity nucleic acid detection method without amplification and application thereof.
Background
The hybridization technology based on the base complementary pairing principle is the earliest developed nucleic acid diagnosis technology, and the basic idea is to utilize the complementary pairing of oligonucleotide chains, and generate a unique reading signal after hybridization and complementation of a probe and single-stranded nucleic acid to be detected in a sample, so as to realize the detection of a known sequence. The nucleic acid hybridization technology is mature, the cost is low, but the sensitivity is relatively low, and the high-sensitivity nucleic acid detection cannot be satisfied. Thus, conventional methods still require PCR amplification of the target fragment. The complex temperature cycle required for the amplification process has the possibility of aerosol pollution and is extremely easy to cause false positive results. Therefore, there is a need to provide a nucleic acid detection method with high sensitivity that does not require amplification.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides a high-sensitivity nucleic acid detection method without amplification and application thereof.
The application also aims to provide a nucleic acid detection kit.
In a first aspect of the present application, there is provided a nucleic acid detection method comprising the steps of:
Providing a sample to be detected, magnetic beads, a primary antibody and a secondary antibody, wherein the magnetic beads are marked with nucleotide chains, the nucleotide chains are used for complementarily pairing with a nucleic acid fragment to be detected in the sample to be detected to form a hybridization duplex, the primary antibody is used for specifically combining with the hybridization duplex, the secondary antibody is used for specifically combining with the primary antibody, and the secondary antibody is provided with a detection mark;
mixing and reacting a sample to be detected, magnetic beads, a primary antibody and a secondary antibody to form a magnetic bead complex;
and detecting according to the detection mark of the magnetic bead complex to obtain a detection result.
The nucleic acid detection method according to the embodiment of the application has at least the following beneficial effects:
(1) The magnetic beads are used as a reaction substrate and a minimum reaction unit of the whole reaction, so that the concentration and amplification effects can be achieved in the follow-up immune reaction; in addition, after various reaction reagents are mixed to participate in the reaction, the magnetic beads can also accelerate the speed of immune reaction and reduce the reaction time. In addition, the separation, the cleaning and the concentration can be further carried out by utilizing the magnetic beads.
(2) The nucleotide chain marked by the magnetic beads and the nucleic acid fragment to be detected in the sample to be detected are mutually paired to form a hybridization duplex, the hybridization duplex is captured by the primary antibody, and the hybridization duplex is detected by the secondary antibody, so that the specificity and the sensitivity of detection are improved, and the nucleic acid fragment to be detected is not required to be amplified additionally.
The magnetic beads refer to magnetic particles which can show stronger magnetism under the action of an external magnetic field, generally comprise internal magnetic particles and shell layers (non-limiting examples of which include polymer shell layers, silicon dioxide shell layers and the like) wrapped on the outer sides of the magnetic particles, and also comprise sandwich-type magnetic beads formed by inner and outer polymer layers and middle magnetic particles. Hybridization duplex refers to a double strand formed by the combination of a single strand of a nucleic acid fragment to be detected and a single strand of a nucleotide strand labeled on a magnetic bead through complementary bases on both strands. The primary antibody is a capture antibody for specifically binding to the hybridization duplex, and after the nucleic acid fragment to be detected forms the hybridization duplex with the nucleotide chain on the magnetic bead, the primary antibody is specifically bound with the antigen-antibody, so that a corresponding binding site is provided for the secondary antibody with the detection label. The secondary antibody is a detection antibody for specifically binding to the primary antibody, and after the primary antibody is bound to the hybridization duplex, a part of other binding sites are also arranged on the primary antibody, so that the primary antibody can be specifically bound with the secondary antibody, and a magnetic bead complex such as a magnetic bead- (nucleotide chain-nucleic acid fragment to be detected) -primary antibody-secondary antibody-detection label is formed. Detection label refers to an optional label in an immunoassay, non-limiting examples of which are radioisotopes, enzymes, fluorescent substances, chemiluminescent substances, bioluminescent substances, pigment molecules, and the like.
In some embodiments of the application, the means of detection are: and encapsulating the magnetic bead complex to form liquid drops, and detecting according to detection marks in the liquid drops to obtain detection results. The method of forming the liquid drops through encapsulation enables the magnetic bead complex in the liquid drops to be used as a minimum reaction unit in the reaction process, so that the reaction speed is further increased, and the reaction time is reduced. The packaging mode can be specifically a mode of processing a microfluidic chip as is well known in the art, and detection is performed by using a digital droplet microfluidic technology.
In some embodiments of the application, the volume of the droplet is (1-10) x 10 -15 L. The detection limit is further reduced by confining the magnetic bead complex to a droplet of a size of about a femto liter, thereby further concentrating the signal of the detection label or the signal generated by the reaction of the detection label into a signal that can be directly detected.
In some embodiments of the application, a method of encapsulating a magnetic bead complex comprises the steps of: providing an aqueous solution of an oil phase and a magnetic bead complex; the oil phase is used as a mobile phase, the aqueous solution of the magnetic bead complex is used as a disperse phase, and the water-in-oil droplets are formed by encapsulation under the action of pressure and/or shearing force. The encapsulation method uses the capillary action of the oil phase to shear the aqueous solution of the magnetic bead complex, so that the dispersed phase is intercepted by the mobile phase to form liquid drops which encapsulate the magnetic bead complex.
In some embodiments of the application, the oil phase further comprises a surfactant. The formed droplets are more stable by adding surfactant to the oil phase.
In some embodiments of the application, S3 is: and analyzing to obtain the concentration of the nucleic acid fragments to be detected in the sample to be detected according to the number of the liquid drops packed with the magnetic bead complex. Under the protection of the surfactant, the liquid drops can not be fused and exchanged in the reaction process, and finally, the complete and independent liquid drop form is maintained, so that the liquid drops can be directly detected without designing a micropore structure corresponding to the liquid drop in size. In addition, as the microporous structure is not required to be designed for segmentation and isolation, the size of the liquid drops in the detection area can be more uniform and have larger density so as to facilitate detection. In addition, since the liquid drop with the volume can ensure that only one magnetic bead exists in one liquid drop, the proportion of the liquid drop of the encapsulated magnetic bead complex to the total number of liquid drops can be directly compared to judge the content or the concentration of the nucleic acid fragments to be detected in the sample to be detected.
In some embodiments of the application, the detection label is an enzyme. Enzyme-linked immunosorbent assay is utilized to enable the enzyme on the magnetic bead complex to react with the provided substrate for color development, and a light signal for detection is generated. Compared with other immune detection methods, the method has the advantages of good specificity, high sensitivity and lower environmental pollution.
In some embodiments of the application, the enzyme is selected from at least one of beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease, glucose oxidase.
In some embodiments of the application, the secondary antibody is a porous nanomaterial immobilized with an antibody, the porous nanomaterial carrying a plurality of detection labels, the antibody being for specific binding to the primary antibody. The porous nano material (such as metal organic framework material, covalent organic framework material and the like) has the characteristics of large specific surface area, high porosity and the like, and can further amplify detection signals by loading a plurality of detection marks on the porous nano material, so that the detection sensitivity is improved.
In some embodiments of the application, the magnetic beads have a diameter of 0.5 to 2 μm.
In a second aspect of the present application, there is provided a nucleic acid detection kit comprising:
the magnetic beads are marked with nucleotide chains, and the nucleotide chains are used for complementarily pairing with the nucleic acid fragments to be detected to form hybridization duplex;
a primary antibody for specific binding to the hybridization duplex;
the secondary antibody is used for specifically combining with the primary antibody, and the secondary antibody is also provided with a detection mark;
the microfluidic chip is provided with a reaction chamber and a detection chamber which are mutually communicated, the reaction chamber is used for supplying magnetic beads, primary antibodies and secondary antibodies to react to form a magnetic bead complex, and the detection chamber is used for detecting signals of detection marks.
The nucleic acid detection kit provided by the embodiment of the application has at least the following beneficial effects:
The traditional biochemical reaction has higher requirements on conditions such as time, temperature, mixing efficiency and the like, and when the microfluidic chip is used for reaction, because the conditions required by different functional areas are different, a plurality of biochemical reaction functional areas cannot be directly connected in sequence. The reaction in the scheme does not need complicated temperature circulation, and all parts can be connected in an integrated sequence, so that manual operation is reduced, and the possibility of pollution is further reduced. On the other hand, the magnetic beads are adopted as a reaction substrate and a minimum reaction unit of the whole reaction, so that the concentration and amplification effects can be realized in the follow-up immune reaction, the speed of the immune reaction is accelerated, and the reaction time is shortened. The nucleotide chain marked by the magnetic beads and the nucleic acid fragment to be detected in the sample to be detected are mutually paired to form a hybridization duplex, the hybridization duplex is captured by the primary antibody, and the hybridization duplex is detected by the secondary antibody, so that the specificity and the sensitivity of detection are improved, and the nucleic acid fragment to be detected is not required to be amplified additionally.
In some embodiments of the application, the microfluidic chip further comprises a droplet formation region provided with an oil phase inlet for injecting an oil phase, a water phase inlet for injecting an aqueous solution of magnetic beads, and an outlet for forming droplets.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
FIG. 1 is a schematic illustration of a magnetic bead complex according to one embodiment of the present application.
Fig. 2 is a schematic diagram of a microfluidic chip according to one embodiment of the present application.
Fig. 3 is an enlarged partial view of the position a of fig. 2 in accordance with the present application.
Fig. 4 is a photograph taken by a CCD camera of the present application of the drop generation zone drop generation at different diameters for the outlet of fig. 3.
Fig. 5 is a schematic diagram of detection of a microfluidic chip according to an embodiment of the present application using an enzyme-linked immunosorbent assay.
FIG. 6 is a fluorescence image of samples of different concentrations in example 1 of the present application.
FIG. 7 is a standard curve plotted for different concentration standards in example 1 of the present application.
Reference numerals: magnetic bead 100, streptavidin 110, biotin 120, nucleotide chain 130, nucleic acid fragment to be tested 140, primary antibody 150, secondary antibody 160, detection label 170, droplet generation region 210, first liquid inlet 211, second liquid inlet 212, third liquid inlet 213, mixing reaction region 220, reaction flow channel 221, detection region 230, detection chamber 231, liquid outlet 232, first oil phase inlet 310, second oil phase inlet 320, aqueous phase inlet 330, outlet 340, and droplet 350.
Detailed Description
The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.
The following detailed description of embodiments of the application is exemplary and is provided merely to illustrate the application and is not to be construed as limiting the application.
In the description of the present application, the meaning of a number is one or more, the meaning of a number is two or more, and greater than, less than, exceeding, etc. are understood to exclude the present number, and the meaning of a number is understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, unless explicitly defined otherwise, terms such as arrangement, installation, connection, etc. should be construed broadly and the specific meaning of the terms in the present application can be reasonably determined by a person skilled in the art in combination with the specific contents of the technical scheme.
In the description of the present application, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Referring to FIG. 1, there is shown the structure of a magnetic bead complex comprising a magnetic bead 100, a nucleotide chain 130, a nucleic acid fragment to be detected 140, a primary antibody 150, and a secondary antibody 160, which is produced in the nucleic acid detection method according to one embodiment of the present application. The nucleotide chain 130 is connected with the magnetic bead 100 and is connected with the nucleic acid fragment to be detected according to the base complementary pairing principle to form a hybridization duplex, the primary antibody 150 can be specifically combined with the hybridization duplex through antigen-antibody interaction, and the secondary antibody 160 can be specifically combined with the primary antibody 150 through antigen-antibody interaction.
In the above examples, the magnetic bead 100 is a magnetic particle, and in some embodiments, the magnetic bead 100 includes an inner magnetic particle and a polymer shell layer surrounding the outer side of the magnetic particle.
In some embodiments, the nucleotide strand 130 is an RNA probe capable of base complementary pairing with the nucleic acid fragment 140 to be tested to form a hybridization duplex, and in some embodiments, the hybridization duplex is DNA: RNA hybridizes to double strands.
In some embodiments, the nucleotide chain 130 is modified with a linking element by which it can be linked to the magnetic bead 100. For example, the linking element may be biotin 120, and the magnetic bead 100 is modified with streptavidin 110, and the nucleotide chain 130 is immobilized to the magnetic bead 110 by the attachment of streptavidin 110 to biotin 120.
In the above embodiment, the secondary antibody 160 has a detection label 170 thereon, and the specific binding between the secondary antibody 160 and the primary antibody 150 allows the detection label 170 to be attached to the magnetic bead complex for detection. In some embodiments, the detection label 170 may be a radioisotope, an enzyme, a fluorescent substance, a chemiluminescent substance, a bioluminescent substance, a pigment molecule, or the like. In some preferred embodiments, the detection label 170 is an enzyme. Compared with other immune detection methods, the enzyme-linked immune detection has good specificity, high sensitivity and lower environmental pollution, and the enzyme can be at least one of beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease, glucose oxidase and the like.
Referring to fig. 2, a schematic diagram of the structure of a microfluidic chip used in packaging and detection in one embodiment of the present application is shown. The microfluidic chip has a flow channel layer that, in some embodiments, includes a droplet generation region 210, a mixing reaction region 220, and a detection region 230. The droplet generation region 210 is provided with a first liquid inlet 211 and a second liquid inlet 212, and the first liquid inlet 211 and the second liquid inlet 212 are respectively intersected at the position A through connected runners and are communicated with the mixing reaction region 220. The first liquid inlet 211 is used for introducing the aqueous solution of the magnetic bead complex into the droplet generation region 210, and the second liquid inlet 212 is used for introducing the oil phase into the droplet generation region 210. The reaction chamber formed by the reaction flow channel 221 is provided in the mixing reaction zone 220, and the droplets generated in the droplet generation zone 210 enter the reaction flow channel 221, wherein the reaction raw materials react with each other. After the reaction, the droplets and the magnetic bead complexes wrapped in the droplets are sent into the detection chamber 231 of the detection region 230 to be dispersed, and the detection is carried out according to whether the magnetic beads in the droplets are provided with detection marks or not so as to judge the content of the nucleic acid fragments to be detected. In some embodiments, the liquid drop generating device further comprises a third liquid inlet 213, and the third liquid inlet 213 is close to the first liquid inlet 211 and is used for introducing other raw materials participating in the reaction into the liquid drop generating region 210. In some of these embodiments, the third liquid inlet 213 and the first liquid inlet 211 have completed merging before point a, and the mixed liquid forming the aqueous phase participates in the formation of liquid droplets. For example, when the detection label is an enzyme, the substrate for the enzyme reaction can be introduced into the microfluidic chip through the third liquid inlet 213, so that the substrate can be mixed with the enzyme to react. After the detection, the waste is discharged outwards through the waste liquid port 232 connected with the detection chamber 231, and the magnetic beads therein can be separated and cleaned by an external magnetic field, so that the recovery is completed.
Referring to fig. 3, an enlarged view of a portion of the present application at position a of fig. 2 is shown. Referring to fig. 2, the flow path communicating with the second inlet 212 forms a first oil phase inlet 310 and a second oil phase inlet 320 therein, while the communication communicating with the first inlet 211 forms an aqueous phase inlet 330 therein, the first oil phase inlet 310 and the second oil phase inlet 320 are disposed opposite to each other and communicate with the aqueous phase inlet 330 to form an intersection region and form an outlet 340, the oil phase fed from the second inlet 212 is injected into the intersection region by the first oil phase inlet 310 and the second oil phase inlet 320, and the aqueous solution of the magnetic bead complex is injected by the aqueous phase inlet 330, in the intersection region, the oil phase intercepts the aqueous solution of the magnetic bead complex flowing under the action of pressure and shearing force, and encapsulates these intercepted aqueous solution portions to form water-in-oil droplets 350, and then is fed into the reaction chamber 220 by the outlet 340 under the action of pressure.
In some embodiments, the volume of the droplets is controlled to be (1-10) x 10 -15 L. After the magnetic bead complex is limited in the liquid drop with the size of about flying liter, the signal of the detection mark or the signal generated by the reaction of the detection mark is concentrated into a signal which can be directly detected, the detection limit can be further reduced, and the sensitivity and the accuracy of detection are improved. In the microfluidic chip, the droplet size may be controlled by adjusting the flow rate of the liquid at each inlet and the difference in the components and proportions of the aqueous phase and the oil phase, or by adjusting the size of the outlet. Referring to FIG. 4, photographs of droplet sizes were generated from a to c when the diameters of the outlets in FIG. 3 were 10 μm, 20 μm and 30 μm, respectively. As can be seen, the larger the size of the outlet 340, the larger the diameter of the droplet 350 produced. Thus, the droplet size may be adjusted by any of the at least one means described above or other means known in the art.
Referring to fig. 5, a schematic diagram of detection of a microfluidic chip using an enzyme-linked immunosorbent assay in an embodiment of the application is shown. Where a represents uniformly arranged droplets transferred into the detection chamber, and since the volume of the droplets is small, only one magnetic bead can be packed in a single droplet, the droplets are divided into droplets packed with a magnetic bead complex and droplets not containing a magnetic bead complex. In a droplet comprising a magnetic bead complex, the nucleotide chains on the magnetic bead complex form a hybridization duplex, such that the primary and secondary antibodies bind to the magnetic beads and carry the corresponding detection label (olive shaped label in part of the droplet in a). While other droplets do not have a detectable label. b represents the color development result of the droplet in a. After the magnetic beads in the liquid drops wrapped with the magnetic bead complex are mixed with the substrate, a color reaction occurs, so that the liquid drops can detect corresponding optical signals; in contrast, other droplets are not developed due to the absence of a detectable label, and remain as they are. c represents a quantitative count of the corresponding result in b. Referring to fig. 5c, since the droplet volume is smaller, only one magnetic bead is wrapped in one droplet, so that the number of the droplets emitted in the detection chamber can be counted directly, or can be further compared with the total number of the droplets, thereby obtaining the content of the nucleic acid fragments to be detected in the sample.
In some specific embodiments, as the enzyme-linked immunosorbent assay method is adopted, the requirements of all parts of the microfluidic chip on the reaction temperature are not too severe, so that a continuous flow design mode can be adopted to connect the generation, mixing and reaction of liquid drops and the imaging of the liquid drops through a micro-channel, the advantages of small reagent consumption and high detection efficiency of the microfluidic chip are exerted, and the analysis and detection cost and the artificial interference are greatly reduced. In the mixing reaction zone of the liquid drop, a plurality of mixing units are designed in the flow channel and matched with the spiral flow channel so as to fully mix the generated liquid drop, the temperature of the area can be further regulated by an instrument, the combination of the substrate and the enzyme is promoted, the enzymatic fluorogenic substrate is generated, the whole liquid drop generates corresponding fluorescence, and finally the liquid drop is imaged in the detection zone.
The present embodiment provides a nucleic acid detecting method including the steps of: s1: providing a sample to be tested, magnetic beads, a primary antibody and a secondary antibody; s2: mixing and reacting a sample to be detected, magnetic beads, a primary antibody and a secondary antibody to obtain the magnetic bead complex; s3: and detecting according to the detection mark of the magnetic bead complex to obtain a detection result. The nucleic acid detection method adopts magnetic beads as a reaction substrate and a minimum reaction unit of the whole reaction, and can play roles in concentration and amplification in the subsequent immune reaction of primary antibodies and secondary antibodies; in addition, after various reaction reagents are mixed to participate in the reaction, the magnetic beads can also accelerate the speed of immune reaction and reduce the reaction time. The hybridization duplex is captured through the primary antibody, and the hybridization duplex is detected through the secondary antibody, so that the specificity and the sensitivity of detection are improved, and the nucleic acid fragment to be detected does not need to be amplified additionally.
In some embodiments, the magnetic beads participate in the reaction in the form of encapsulated droplets during detection. The method of forming liquid drops by encapsulation makes the magnetic beads in the liquid drops as the smallest reaction units in the reaction process, so that the speed of immune reaction is further increased, and the reaction time is reduced. In some preferred embodiments, the encapsulation to form droplets is by way of microfluidic chip processing.
In some embodiments, the volume of the droplet encapsulating the magnetic bead is (1-10) x 10 -15 L. By generating and confining the magnetic bead complex within droplets of a size around the femto, the signal of the detection label or the signal generated by the reaction of the detection label can be further concentrated, so that a signal which is not easily detected is more easily detected, to further reduce the detection limit thereof.
In some embodiments, the magnetic beads are encapsulated by a microfluidic chip, an oil phase is taken as a mobile phase, an aqueous solution containing the magnetic beads is taken as a disperse phase, and the magnetic beads are encapsulated by the magnetic beads under the action of pressure and/or shearing force to form water-in-oil droplets. Wherein the oil phase can be liquid hydrocarbon, ester, etc., such as fluorine oil, silicone oil, mineral oil, vegetable oil, petroleum ether, etc. In some of the preferred embodiments, the oil phase further comprises a surfactant, non-limiting examples of which may be span, triton, EM 90, fluorocarbon surfactant, and the like. The formed liquid drops are more stable by adding the surfactant into the oil phase, so that the liquid drops can not be fused and exchanged in the subsequent reaction process, and the complete and independent liquid drop forms are always kept. Therefore, in detection, it is not necessary to design a microporous structure corresponding to the size of the droplet and sink the droplet therein by means of magnetism or the like, but detection can be performed directly. Meanwhile, the size of the liquid drop in the detection area can be more uniform and the liquid drop has larger density because the micro-pore structure is not needed for segmentation and isolation, so that the detection can be conveniently carried out and higher flux can be provided.
In some embodiments, the detection label on the secondary antibody is an enzyme. Enzyme-linked immunosorbent assay is utilized to enable the enzyme on the magnetic bead complex to react with the provided substrate for color development, and a light signal for detection is generated. Compared with other immune detection methods, the method has the advantages of good specificity, high sensitivity and lower environmental pollution. Non-limiting examples of enzymes used as detection labels include beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease, glucose oxidase. In addition, in some preferred embodiments, the secondary antibody may take the form of a porous nanomaterial loaded with a plurality of detection labels and labeled therein for specific binding of the primary antibody. In this case, the plurality of detection markers loaded can further amplify the detection signal, improving the detection sensitivity.
In some embodiments, the magnetic beads used in the assay have a diameter of 0.5 to 2 μm and the droplets have a diameter of 1 to 50 μm.
The application is illustrated below with reference to specific examples.
Example 1
The present embodiment provides a nucleic acid detection kit, wherein the microfluidic chip is as described above. The kit also comprises magnetic beads, a primary antibody, a secondary antibody and a substrate. Wherein the magnetic beads are streptavidin magnetic beads with the concentration of 20 mu L and the particle size of 1 mu m and the concentration of 10mg/mL, and HPV mRNA probes corresponding to the nucleic acid fragments to be detected are coupled on the magnetic beads. Anti-DNA at a concentration of 1. Mu.g/mL at 10. Mu.L: RNA hybrid antibody (S9.6 antibody). The secondary antibody is 10 mu L of beta-galactosidase marked goat anti-mouse antibody with the concentration of 1 mu g/mL. The substrate is FDG (fluorescein 2-beta-D-galactopyranoside). In addition, the volume of the sample to be measured containing the nucleic acid fragment to be measured was 1mL.
The present embodiment also provides a nucleic acid detection method comprising the steps of:
(1) Mixing and incubating the solution of the magnetic beads coupled with the probes, a sample to be detected and a hybridization buffer solution (50 mM sodium citrate, 750mM NaCl,pH 7.2) for 5min to obtain a magnetic bead-DNA: solution of RNA complex.
(2) Incubating the reaction product obtained in the step (1) with a primary antibody and a secondary antibody for 5min to form magnetic bead-DNA: solution of magnetic bead complex of RNA-primary-secondary antibody.
(3) And injecting the mixed solution into the microfluidic chip from the first liquid inlet, simultaneously injecting the substrate solution into the microfluidic chip from the third liquid inlet, injecting fluorine oil HFE7500 (fluorocarbon surfactant FSA,1 wt%) into the microfluidic chip from the second liquid inlet, wherein the flow rates of the first liquid inlet, the second liquid inlet and the third liquid inlet are respectively 0.1mL/h, 0.4mL/h and 0.1mL/h, and carrying out mixed reaction to form liquid drops.
(4) After 20 minutes from the start of the droplet entering the detection chamber, the detection chamber was imaged by a fluorescence microscope.
In the above detection method, the volume of the droplet finally formed by controlling the flow rate is about 1×10 -14 L.
In the above detection method, a standard curve is formed using purified HPV DNA as a standard. HPV DNA concentration was started at 1X 10 6 copies/mL and 10-fold gradient diluted. Microscopic images were recorded and analyzed to quantify the concentration of HPV DNA. Since the droplets are filled in the imaging region, the fraction of the total amount of droplets that is a bright droplet with fluorescence is used as an output signal for calculating HPV DNA concentration.
The results are shown in FIG. 6 and FIG. 7, wherein FIG. 6 is a fluorescence image of samples of different concentrations in example 1, a-f respectively represent concentration gradients of 10 3、104、105、106、107、108 copies/mL, and FIG. 7 is a standard curve drawn for different concentration standards. Referring to fig. 7, the detection range is 10 6 copies/mL to 10 3 copies/mL, with a linear relationship of y= 5.829 ×log (X) -18.59, and r 2 =0.964. Thus, the detection method provided in this example can detect nucleic acid molecules on the order of 10 3 at the lowest.
Compared with the commercial product Hybrid Capture 2, the method for detecting HPV provided by the embodiment has lower LOD, and has lower time and reagent consumption. Compared with the PCR-based method, the detection method avoids the problems of high cost, complex processing procedures and aerosol pollution. The method is simple, versatile and has excellent quantification capability.
From the above embodiments, it can be seen that the solution of the present application can concentrate micro biochemical signals by compressing conventional biochemical reactions in a micro-droplet reactor of a fly-lift stage, so that signals which are not easily detected can be more easily detected. Modification of the probe with magnetic beads, DNA after capture of the target nucleic acid molecule: hybridization double strand of RNA is replaced by Anti-DNA: RNA hybrid antibody capture. The mouse source Anti-DNA-RNA hybrid antibody is marked by enzyme-labeled goat Anti-mouse antibody to form the final immune complex. The immune complex and the fluorogenic substrate thereof generate hundreds of thousands of liquid drops with the size of about ten micrometers through a liquid drop microfluidic chip. When immunocomplexes are present in the droplets, enzymes therein are capable of catalyzing the fluorescent substrate to generate a fluorescent signal and performing a photographic analysis. According to the proportion of fluorescent liquid drops to all liquid drops, absolute quantitative results of nucleic acid molecules can be calculated.
In summary, the droplet-based nucleic acid hybridization method can realize amplification-free high-sensitivity nucleic acid detection, on one hand, high-sensitivity detection in complex samples is realized by utilizing the specificity of antigen-antibody combination, and on the other hand, the method has the advantage of short detection time, and the whole detection process can be controlled within 30 minutes, so that rapid diagnosis is facilitated. In addition, detection and analysis of multiple targets can be realized by designing multiple microfluidic channels.
Example 2
The present example provides a nucleic acid detection kit, which is different from example 1 in that the secondary antibody is horseradish peroxidase-labeled goat anti-mouse antibody, and the substrate is TMB (3, 3', 5' -Tetramethylbenzidine). The detection was performed by the method of example 1, and the analysis of the detection results revealed that the detection range was similar to that of example 1 and the LOD was low.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.
Claims (2)
1. A method for detecting a nucleic acid for the purpose of non-disease diagnosis, comprising the steps of:
S1, providing a sample to be tested, a magnetic bead, a primary antibody and a secondary antibody, wherein the magnetic bead is marked with a nucleotide chain, the nucleotide chain is used for complementarily pairing with a nucleic acid fragment to be tested in the sample to be tested to form a hybridization duplex, the primary antibody is used for specifically combining with the hybridization duplex, the secondary antibody is used for specifically combining with the primary antibody, and the secondary antibody is provided with a detection mark;
S2, mixing and reacting the sample to be detected, the magnetic beads, the primary antibody and the secondary antibody to form a magnetic bead-containing DNA: a mixed aqueous solution of the magnetic bead complex of the RNA-primary antibody-secondary antibody;
S3, providing a mixed aqueous solution of an oil phase and the magnetic bead complex; taking the oil phase as a mobile phase, taking the mixed aqueous solution of the magnetic bead complex as a disperse phase, and encapsulating with a fluorogenic substrate solution under the action of pressure and/or shearing force to form water-in-oil droplets, wherein the method comprises the following steps: injecting the mixed aqueous solution of the magnetic bead complex into a microfluidic chip from a first liquid inlet, simultaneously injecting the fluorogenic substrate solution into the microfluidic chip from a third liquid inlet, and injecting the fluorocarbon oil HFE7500 containing 1wt% of fluorocarbon surfactant into the microfluidic chip from a second liquid inlet, wherein the flow rates of the first liquid inlet, the second liquid inlet and the third liquid inlet are respectively 0.1mL/h, 0.4mL/h and 0.1mL/h, and mixing and reacting to form water-in-oil liquid drops;
s4, detecting according to the detection marks in the liquid drops to obtain detection results;
Wherein the diameter of the magnetic beads is 0.5-2 mu m, and the primary antibody is Anti-DNA: the secondary antibody is a goat anti-mouse antibody containing a detection label, and the detection label is enzyme; the enzyme is at least one of beta-galactosidase, horseradish peroxidase, alkaline phosphatase, urease and glucose oxidase; the enzyme is capable of catalyzing the fluorescent substrate to generate a fluorescent signal; the volume of the liquid drop is (1-10) multiplied by 10 -15 L.
2. The method for detecting nucleic acid according to claim 1, wherein the detection is performed by: and analyzing and obtaining the content of the nucleic acid fragments to be detected in the sample to be detected according to the number of the liquid drops packed with the magnetic bead complex.
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