Circulating biomarker detection system based on magnetic particle response

Abstract

The invention belongs to the field of magnetic particle imaging, particularly relates to a circulating biomarker detection system based on magnetic particle response, and aims to provide a novel circulating biomarker detection tool. The invention provides a circulating biomarker detection system based on magnetic nanoparticle response, which is characterized in that magnetic nanoparticles are embedded by using a responsive hydrogel technology, and specific biomolecule recognition and magnetic nanoparticle release are realized through a specific enzyme; in combination with a high-sensitivity magnetoelectric induction effect, an alternating magnetic field is used for driving magnetic nanoparticles to perform nonlinear response, quantitative analysis is realized through a magnetic particle spectrum signal reconstruction and machine learning method, and monitoring is performed in combination with a confocal fluorescence imaging method.

Description

Technical Field

[0001] The present invention relates to the field of magnetic particle imaging, and particularly to a circulating biomarker detection system based on magnetic particle response. Background Art

[0002] In recent years, studies have found that magnetic nanoparticle imaging has many advantages such as high spatio-temporal resolution, high sensitivity, and high-precision quantification. Currently, it is also widely used in fields such as cell tracing, inflammation imaging, and angiography. However, it also has limitations in losing tiny signal fluctuation information and being difficult to achieve analysis and quantification for specific biomolecules. In addition, the current established biomolecular electromagnetic induction detection relies on specific modification of magnetic nanoparticles, making it difficult to achieve detection and analysis of multiple biomolecules.

[0003] In view of this, the present invention is specifically provided. Summary of the Invention

[0004] The purpose of the present invention is to provide a new circulating biomarker detection tool.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is as follows:

[0006] A circulating biomarker detection system based on magnetic particle response, the system comprising: a magnetic particle-responsive hydrogel detection chip and a restriction enzyme;

[0007] The chip comprises: a sample loading hole, an identification groove communicating with the sample loading hole, a reaction groove communicating with the identification groove, and a detection hole communicating with the reaction groove;

[0008] The identification groove includes a DNA recognition strand and a DNA translation strand that forms a partial reverse complementary pairing with it. After the DNA recognition strand binds to the circulating biomarker, the DNA translation strand is released;

[0009] The reaction groove includes a hydrogel crosslinked by a DNA crosslinking agent, and the hydrogel entraps magnetic nanoparticles; the released DNA translation strand and the DNA crosslinking agent have at least a partial reverse complementary pairing to form a DNA double strand containing a restriction site, and the double strand is recognized and cleaved by the restriction enzyme, thereby destroying the crosslinked structure of the hydrogel and releasing the magnetic nanoparticles;

[0010] The detection hole is used to receive and detect the released magnetic nanoparticles;

[0011] Before sample loading, the restriction enzyme and the chip exist independently of each other; after sample loading, the restriction enzyme is loaded onto the chip through the sample loading hole and is delivered to the reaction groove for cutting the hydrogel.

[0012] In some specific embodiments, the system further includes a control module, a magnetic field driving module, and a signal acquisition module. The control module generates an alternating magnetic field by controlling the magnetic field driving module, and the acquisition module is used to collect the magnetoelectric induction signals of the magnetic nanoparticles.

[0013] In some specific embodiments, the DNA crosslinker includes DNA crosslinker 1 and DNA crosslinker 2, and there is at least partial reverse complementary pairing between DNA crosslinker 1 and DNA crosslinker 2.

[0014] In some specific embodiments, DNA crosslinker 1 and DNA crosslinker 2 respectively include the sequences shown in SEQ ID NO: 1 and 2.

[0015] In some specific embodiments, the circulating biomarker recombines with the DNA recognition strand or unwinds the DNA recognition strand, thereby releasing the DNA translation strand.

[0016] In some specific embodiments, the circulating biomarker is a protein, and the DNA recognition strand is an aptamer that specifically binds to the circulating biomarker.

[0017] In some specific embodiments, the circulating biomarker is RNA, for example, mRNA, and the DNA recognition strand is reversely complementary paired with the circulating biomarker, preferably completely reversely complementary paired.

[0018] In some specific embodiments, the circulating biomarker is CD63 protein, the DNA recognition strand includes the sequence shown in SEQ ID NO.3, and the DNA translation strand includes the sequence shown in SEQ ID NO.4.

[0019] In some specific embodiments, the circulating biomarker is IL-6 mRNA, the DNA recognition strand includes the sequence shown in SEQ ID NO.5, and the DNA translation strand includes the sequence shown in SEQ ID NO.6.

[0020] In some specific embodiments, the monomer of the hydrogel is sodium acrylate, and the DNA crosslinker is DNA crosslinking modified with acrylate.

[0021] In some specific embodiments, the magnetic nanoparticles are magnetic nanoparticles modified with fluorescent protein. Preferably, the fluorescent protein is AF647.

[0022] In some specific embodiments, the system further includes a fluorescence imaging module.

[0023] In some specific embodiments, the control module includes a PC and an FPGA board, the magnetic drive module includes Helmholtz coils and permanent magnets to apply an alternating drive magnetic field and a gradient selection magnetic field, and the signal acquisition module includes a Litz coil with forward and reverse windings, a signal amplifier, and a high-pass filter.

[0024] In some specific embodiments, the magnetic particle response chip is made of polydimethylsiloxane. Preferably, the polydimethylsiloxane is bonded to glass.

[0025] In some specific embodiments, the acquired signals are processed by machine learning.

[0026] On the other hand, the present invention also provides a method for detecting circulating biomarkers, and the method uses the aforementioned system for detection.

[0027] Advantageous effects:

[0028] In summary, the present invention provides a circulating biomarker detection system based on magnetic particle response. The advantageous effects of the present invention are mainly reflected in:

[0029] First, the present invention realizes the recognition of circulating biomarkers (specific nucleic acids or proteins) and the monitoring of the release of magnetic nanoparticles through a magnetic particle-responsive hydrogel, providing a new tool for detecting circulating biomarkers. Second, the present invention combines the highly sensitive magnetoelectric induction effect, uses an alternating magnetic field to drive electromagnetic signal acquisition to achieve quantification of specific molecules, and in some cases, realizes quantitative analysis through magnetic particle spectrum signal reconstruction and machine learning methods. Finally, in some cases, the magnetic particles are labeled with fluorescent proteins and can be monitored by combining fluorescence imaging methods (such as confocal fluorescence imaging methods). Description of the drawings

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 For constructing a magnetic particle response hydrogel chip provided by an embodiment of the present invention;

[0032] Figure 2 For the magnetic particle response hydrogel detection chip provided by an embodiment of the present invention (a hydrogel column constructed by the chip is placed in the reaction tank) Figure 1 ;

[0033] Figure 3Schematic diagram of the detection principle of the magnetic particle-responsive hydrogel detection chip provided by the embodiment of the present invention;

[0034] Figure 4 Schematic diagram of the detection process of the magnetic particle-responsive hydrogel detection chip provided by the embodiment of the present invention;

[0035] Figure 5 Signal diagram of the magnetic particle-responsive hydrogel detection chip provided by the embodiment of the present invention.

[0036] Icons: 1 - hydrogel tank; 2 - polydimethylsiloxane; 3 - glass; 4 - sample injection hole; 5 - recognition tank; 6 - reaction tank; 7 - hydrogel column; 8 - detection hole. Detailed implementation manners

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Usually, the components of the embodiments of the present invention described and illustrated herein can be arranged and designed in various different configurations.

[0038] Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0039] Embodiment

[0040] Step Sa: Construct a magnetic particle-responsive hydrogel detection chip

[0041] (1) Use polydimethylsiloxane to construct a microfluidic chip structure and construct a hydrogel column embedded with magnetic nanoparticles:

[0042] Use polydimethylsiloxane to construct a microfluidic chip in a mold and perform plasma bonding with glass to form a chip structure as shown in Figure 1 and Figure 2 shown. Figure 1 The shown chip structure is used to construct a hydrogel column responsive to magnetic particles. The constructed hydrogel column responsive to magnetic particles is placed in the reaction tank of the chip structure shown in Figure 2 shown to construct a magnetic particle-responsive hydrogel detection chip. As shown in Figure 2As shown, the detection chip includes: a sample loading hole (4); an identification groove (5) communicating with the sample loading hole (4); a reaction groove (6) communicating with the identification groove (5), in which a hydrogel column (7) embedding magnetic nanoparticles is placed; and a detection hole (8) communicating with the reaction groove (6).

[0043] Among them, the formation of the hydrogel column is as follows: Sodium acrylate, photoinitiator 2959, acrylate-modified DNA crosslinker and magnetic nanoparticles are mixed in proportion and injected into Figure 1 the hydrogel groove (1) made of polydimethylsiloxane as shown. After ultraviolet irradiation for 2 min, a column with a diameter of 1 mm and a length of 1 mm is formed. Three of the hydrogel columns are in a group and placed in the reaction groove of the magnetic particle-responsive hydrogel detection chip. The concentration of sodium acrylate is 20%, the molar ratio to the DNA crosslinker is 80:1, the concentration of the initiator is 1 mg / ml, and the magnetic nanoparticles are modified with AF647 fluorescent protein and the concentration is 2 mg / ml. The acrylate-modified DNA crosslinker in this embodiment includes DNA crosslinker 1 and DNA crosslinker 2, and there is at least partial reverse complementary pairing in the nucleic acid parts of DNA crosslinker 1 and DNA crosslinker 2. The nucleic acid sequences of DNA crosslinker 1 and DNA crosslinker 2 are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0044] SEQ ID NO.1: TCGCTCGTGCCACTTTGCTAG.

[0045] SEQ ID NO.2: GCTCTAGCAAAGTGG.

[0046] (2) Specific recognition of the molecule to be detected is achieved on the magnetic particle-responsive chip by using the recognition DNA strand, the translation DNA strand and restriction endonucleases:

[0047] The glass slide bonded with polydimethylsiloxane by plasma is pretreated to fix the biotinylated specific DNA recognition strand and DNA translation strand. The identification groove part of the glass slide is dropped with a mixed solution of biotin PEG thiol and methyl PEG thiol for 12 h. The mixing ratio of biotin PEG thiol and methyl PEG thiol is 1:1 and the concentration is 10 nmol. After dropping 5% concentration of bovine serum albumin to block for 1 h, streptavidin and the mixed solution of DNA recognition strand and DNA translation strand are dropped in sequence and incubated for 1 h each. The concentration of streptavidin is 1 mg / ml, the mixing ratio is 1:1, and the concentration is 20 μmol.

[0048] The DNA recognition strand and the DNA translation strand have partial reverse complementary pairing. The DNA recognition strand can bind to the molecule to be detected, and after binding, the DNA translation strand is released. In some cases, the molecule to be detected is a protein, and the DNA recognition strand is an aptamer that can bind to it. In some cases, the molecule to be detected is mRNA, and the ability of the DNA recognition strand to form a double-stranded structure with the molecule to be detected is stronger than its ability to form a double-stranded structure with the DNA translation strand.

[0049] Exemplarily, the molecule to be detected is CD63 protein, and the sequences of the corresponding DNA recognition strand and DNA translation strand are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. Exemplarily, taking the molecule to be detected as IL-6 mRNA as an example, the sequences of the corresponding DNA recognition strand and DNA translation strand are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0050] SEQ ID NO.3: TCAACATCAGTCTGATAAGCTA.

[0051] SEQ ID NO.4: CACGAGCGACTGGCACTGGTA.

[0052] SEQ ID NO.5:

[0053] CACCCCACCTCGCTCCCGTGACACTAATGCTA.

[0054] SEQ ID NO.6: AAAGTGGCACGAGCGATAGCATTAGTG.

[0055] Restriction endonuclease: Before adding the sample to be detected, the restriction endonuclease is stored independently from the magnetic particle-responsive hydrogel detection chip. After adding the sample to be detected, the restriction endonuclease is loaded into the sample addition hole, and is delivered to the reaction tank through the sample addition hole and the recognition groove for reaction.

[0056] The principle of specific recognition and detection of the molecule to be detected is as Figure 3 shown: If the sample to be detected contains the molecule to be detected, the molecule to be detected binds to the DNA recognition strand in the reaction tank and releases the DNA translation strand. The released DNA translation strand forms a double-stranded structure containing a restriction enzyme cleavage site with the DNA cross-linker of the hydrogel column in the reaction tank. This double-stranded structure is recognized and cleaved by the restriction endonuclease, thereby destroying the hydrogel structure and releasing the magnetic particles embedded in the hydrogel, and then the released magnetic particles are detected in the detection hole. If the sample to be detected does not contain the molecule to be detected, the restriction endonuclease does not cleave the DNA cross-linker, and thus does not release magnetic particles, and the detection hole cannot detect the magnetic signal.

[0057] Step Sb: Specific biomolecular recognition

[0058] Using mouse serum as the experimental detection object, the serum is injected into the recognition groove through the sample loading hole of the magnetic particle response chip. The molecule to be detected (such as specific nucleic acid or protein) recombines with the DNA recognition strand or the DNA recognition strand unwinds to release the DNA translation strand. The serum sample loading volume is 100 μL, and the reaction time is 20 min.

[0059] Step Sc: Responsive release of magnetic particles

[0060] Using 5 μl of restriction endonuclease (100 units of Nb.BssSI) and the released DNA translation strand, complementary pairing is formed through the DNA translation strand and the DNA crosslinker of the hydrogel network to cut the responsive hydrogel DNA crosslinker network, and the magnetic nanoparticles bound with AF647 fluorescent protein are released by the restriction endonuclease. The reaction time with the magnetic particle responsive release hydrogel column is 20 min.

[0061] Step Se: Move the magnetic particle response chip into the center of the magnetic field driving system

[0062] Move the detection hole of the magnetic particle response chip to the center position of the receiving coil composed of litz wire.

[0063] Step Sf: The control module starts the system

[0064] Use the PC side to control the magnetic field driving module. The start signal of the magnetic field driving module passes through the ADDA chip and power amplifier of the FPGA board to generate a stable alternating driving magnetic field with a frequency of 20 kHz and a peak value of 15 mT.

[0065] Step Sg: Electromagnetic induction signal acquisition

[0066] Use the start signal of the received signal acquisition module to start the signal amplifier, high-pass filter, and the ADDA chip of the FPGA board, so as to realize the real-time acquisition of the magnetic induction electrical signal.

[0067] Step Sh: Confocal fluorescence image acquisition

[0068] Use a confocal fluorescence microscope to perform fluorescence imaging on the responsive hydrogel column and magnetic particle release groove of the magnetic particle response chip.

[0069] Step Si: Acquisition signal processing

[0070] After using the machine learning method to construct a training model with the gradient concentration magnetic particle received signal, import the collected magnetic induction electrical signal spectrum for processing and analysis. Machine learning plots the gradient signal as Figure 5 shown.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A circulating biomarker detection system based on magnetic particle response, characterized in that: The system comprises: a magnetic particle response hydrogel detection chip and a restriction endonuclease; The chip comprises: a sample adding hole, an identification slot connected to the sample adding hole, a reaction slot connected to the identification slot, and a detection hole connected to the reaction slot; The recognition groove includes a DNA recognition chain and a DNA translation chain that forms a local reverse complementary pairing therewith, and after the DNA recognition chain binds to the circulating biomarker, the DNA translation chain is released; The reaction tank comprises a hydrogel cross-linked by a DNA cross-linking agent, wherein the hydrogel is embedded with magnetic nanoparticles; the released DNA translation chain is at least partially reversely complementary to the DNA cross-linking agent to form a DNA double-strand containing a restriction site, and the double-strand is recognized and cut by the restriction endonuclease, thereby destroying the cross-linking structure of the hydrogel and releasing the magnetic nanoparticles; The detection hole is used to receive and detect the released magnetic nanoparticles; Before loading the sample, the restriction endonuclease and the chip exist independently of each other; after loading the sample, the restriction endonuclease is loaded into the chip through the loading hole and delivered to the reaction tank for cutting the hydrogel.

2. The system according to claim 1, characterized in that The system also includes a control module, a magnetic field driving module and a signal acquisition module. The control module generates an alternating magnetic field by controlling the magnetic field driving module. The acquisition module is used to collect the magnetoelectric induction signals of the magnetic nanoparticles.

3. The system according to claim 1, characterized in that The DNA crosslinker comprises a DNA crosslinker 1 and a DNA crosslinker 2, wherein the DNA crosslinker 1 and the DNA crosslinker 2 are at least partially reverse complementary to each other; preferably, the DNA crosslinker 1 and the DNA crosslinker 2 comprise the sequences shown in SEQ ID NOs: 1 and 2, respectively.

4. The system according to claim 1, characterized in that The circulating biomarker is CD63 protein, the DNA recognition strand includes the sequence shown in SEQ ID NO.3, and the DNA translation strand includes the sequence shown in SEQ ID NO.

4.

5. The system according to claim 1, characterized in that The circulating biomarker is IL-6 mRNA, the DNA recognition strand comprises the sequence shown in SEQ ID NO.5, and the DNA translation strand comprises the sequence shown in SEQ ID NO.

6.

6. The system according to claim 1, characterized in that The monomer of the hydrogel is sodium acrylate, and the DNA cross-linking agent is a DNA cross-linking agent modified by acrylate.

7. The system according to claim 1, characterized in that The system further comprises a fluorescence imaging module, preferably a confocal fluorescence imaging module, and the magnetic nanoparticles are magnetic nanoparticles modified with fluorescent proteins, and preferably, the fluorescent protein is AF647.

8. The system according to claim 1, characterized in that The control module includes a PC terminal and an FPGA board, the magnetic drive module includes a Helmholtz coil, a permanent magnet applying an alternating drive magnetic field and a gradient selection magnetic field, and the signal acquisition module includes a Litz coil with forward and reverse windings, a signal amplifier and a high-pass filter.

9. The system according to claim 1, characterized in that The chip is made of polydimethylsiloxane, and preferably, the polydimethylsiloxane is bonded to glass.

10. A method for detecting circulating biomarkers, characterized in that: The detection is performed using the system described in any one of claims 1 to 9.

Images