CN115725743A - Probe set, kit and detection system for detecting tumor exosomes and application of probe set and kit - Google Patents
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Abstract
The invention provides a probe set, a kit, a detection system and application for detecting tumor exosomes, and relates to the technical field of exosome detection. The probe set comprises three groups of probes with different purposes, and a new biosensing technology for the exosome detection with high sensitivity and high specificity is constructed by combining a double-Aptamer sandwich detection method, a loop-mediated isothermal amplification technology and a G-quadruplex-heme DNase enzymatic reaction. The detection system/detection kit (or called detection platform) developed based on the probe set accords with the development trend of miniaturization, integration and portability, and provides a specific, noninvasive or minimally invasive clinical diagnosis and analysis technology for the fields of early diagnosis, preoperative evaluation, prognosis and the like of malignant tumors.
Description
Technical Field
The invention belongs to the technical field of exosome detection, and particularly relates to a probe set, a kit, a detection system and application for detecting tumor exosomes.
Background
Malignant tumors are currently one of the leading causes of death worldwide, and therefore, early diagnosis and intervention are critical to reduce the morbidity and mortality of tumors. With the rapid development of tumor molecular biology, liquid specimen biopsy becomes a hot spot of current research. Compared with tissue biopsy, the liquid biopsy specimen is easy to collect, has small wound and can dynamically feed back the progress of the tumor, so the liquid biopsy specimen has important values in the aspects of clinical diagnosis, disease condition evaluation, accurate treatment and prognosis evaluation of the tumor. Recent studies have shown that exosomes as potential biomarkers in liquid biopsy strategies are the hot spots of current research. The exosome can carry various bioactive molecules to circulate in blood/body fluid and mediate long-distance intercellular communication, the molecules such as protein, nucleotide and lipid rich in the tumor-derived exosome can reflect the physiological and pathological states of cells from the exosome, and the lipid bilayer membrane structure of the exosome ensures that the exosome has certain stability both inside and outside the body, so that the detection of the tumor exosome becomes a remarkable advantage of liquid biopsy.
In recent years, various analytical methods have been established for the detection of exosomes, for example, colorimetric methods (Anal chem.2017, 89, 12327-12333, biosens bioelectrron.2018, 102, 204-210), electrochemical methods (talanta.2021, 221, 121379-121387, chem Commun.2020, 56, 269-272), surface enhanced Raman scattering (Theranostics.2018, 8, 2722-2738 science advances.2020,6, eaax3223), microfluidic methods (Nano letters.2018, 18, 4226-4232. Although these methods are very effective, most of them use antibody as recognition unit, and have the defects of high cost, complex production, poor stability, complex modification and the like, which results in difficult coupling of signal molecules.
As an important functional nucleic acid, the aptamer is one of the important routes of exosomes in extraction and application. Compared with an antibody, the aptamer has the advantages of easiness in synthesis, easiness in chemical modification, good stability, low price and the like. Meanwhile, due to the recognition mechanism of the double proteins, high sensitivity and high specificity, the sandwich-type biosensor is widely applied to the detection of the surface protein of the exosome. In such an assay platform, two probes are used for both signal capture and signal generation, in order to identify two spatially remote distances of a macromolecular target. The advantages enable the aptamer to show wide application prospects in detection of exosomes. In recent years, a number of aptamer sensing platforms have been constructed for detecting exosomes (Anal chem.2018, 90, 4507-4513, acs nano chem.2017, 11, 3943-3949, anal chem.2020, 92, 5546-5553. Although these techniques have unique and irreplaceable advantages and characteristics and are well applied in the related fields, some limitations (such as higher cost, low sensitivity, unsuitability for developing high-throughput technology, etc.) restrict the wider and deeper application of these techniques in the field of tumor clinical diagnosis.
Disclosure of Invention
The invention aims to provide a probe set, a kit, a detection system and application for detecting tumor exosomes, and the new biosensing technology for detecting the exosomes with high sensitivity and high specificity is constructed by combining a double Aptamer sandwich detection method, a loop-mediated isothermal amplification technology and a G-quadruplex-heme DNA enzyme enzymatic reaction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a probe group for detecting tumor exosomes, which comprises a probe P1, a probe P2, a probe P3-1, a probe P3-2, a probe P3-COM and a loop-mediated isothermal amplification primer;
the nucleotide sequence of the probe P1 is shown as SEQ ID NO. 1; the nucleotide sequence of the probe P2 is shown as SEQ ID NO.2, the nucleotide sequence of the probe P3-1 is shown as SEQ ID NO.3, the nucleotide sequence of the probe P3-2 is shown as SEQ ID NO.4, and the nucleotide sequence of the probe P3-COM is shown as SEQ ID NO. 5;
the loop-mediated isothermal amplification primers comprise FIP, BIP, FLP and BLP;
the nucleotide sequence of the FIP is shown as SEQ ID NO.6, the nucleotide sequence of the BIP is shown as SEQ ID NO.7, the nucleotide sequence of the FLP is shown as SEQ ID NO.8, and the nucleotide sequence of the BLP is shown as SEQ ID NO. 9.
Preferably, the 3' end of the P1 also comprises a biotin label;
the 5' end of the P3-2 is also modified with a phosphate group.
The invention also provides a kit for detecting tumor exosomes, which comprises the probe set and microspheres modified with streptavidin.
Preferably, the kit also comprises ligase, nicking endonuclease, heme, ABTS and H 2 O 2 。
The invention also provides a system for detecting tumor exosomes, which comprises the steps of fixing P1 in the probe set on microspheres modified with streptavidin, mixing the microspheres with exosomes to be detected, and mixing the microspheres with P2 in the probe set to construct a sandwich structure;
mixing P3-1 and P3-2 in the probe group, and constructing to obtain an LAMP primary basic structure with a free end at the 3' end in the presence of a P3-COM probe and ligase;
mixing the sandwich structure with the LAMP primary basic structure, and constructing to obtain an LAMP basic structure P4 under the action of incision endonuclease;
performing loop-mediated isothermal amplification on the LAMP base structure P4 and a loop-mediated isothermal amplification primer in the probe set to construct a G-quadruplex sequence;
forming a G-quadruplex-heme DNase from the G-quadruplex sequence under the action of heme, and reacting the G-quadruplex-heme DNase with ABTS and H 2 O 2 And (4) mixing.
The invention also provides application of the probe set or the kit or the system in preparing a medicament for diagnosing and/or preventing and/or treating tumors.
Preferably, the tumor comprises leukemia.
Preferably, the sample for diagnosis comprises a serum sample of a leukemia patient or exosomes extracted from CCRF-CEM cells.
Has the beneficial effects that: the invention provides a probe group for detecting tumor exosomes, which comprises three groups of probes with different purposes, and combines a double Aptamer sandwich detection method, a loop-mediated isothermal amplification technology and a G-quadruplex-heme DNA enzyme enzymatic reaction to construct a new biosensing technology for detecting the tumor exosomes with high sensitivity and high specificity. The invention is based on a detection system/detection kit (or called a detection platform) developed by the probe set, accords with the development trend of miniaturization, integration and portability, and provides a specific, noninvasive or minimally invasive clinical diagnosis and analysis technology for the fields of early diagnosis, preoperative evaluation, prognosis and the like of malignant tumors.
The invention designs an Aptamer nucleic acid sequence to be combined with a specific site of a tumor cell exosome to form a double Aptamer sandwich structure (or sandwich structure), then the double Aptamer sandwich structure is mixed with an LAMP primary basic structure to form the LAMP basic structure under the action of Nb.BsrDI nicking endonuclease, loop-mediated isothermal amplification is carried out under the action of primers, heme is added into an amplification product, and ABTS-H is utilized 2 O 2 The color reaction is detected, and the detection limit can reach 2 multiplied by 10 after statistics 4 Exosome Particles (Particles)/mL.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a schematic diagram of detection of tumor exosomes;
FIG. 2 is a CEM extracellular exosome concentration detection signal saturation curve;
FIG. 3 is a calibration curve for the extracellular fluid response of CEM at different concentrations;
FIG. 4 is a graph of the ultraviolet absorption spectrum of exosomes of different concentrations; wherein 1 is blank control, and 2 is 1.05 × 10 4 Absorption curve of the exosome sample of Particles/mLCEM cell, 3 is 3.5X 10 4 Absorption curve of extracellular fluid sample of Particles/mLCEM cell, 4 is 1.05X 10 5 Absorption curves for the samples of CEM extracellular secretion from Particles/mL, 5 is 1.05X 10 6 Absorption curve of the exosome sample of Particles/mLCEM cell, 6 is 1.05X 10 7 Absorption curve of the extracellular fluid sample of Particles/mLCEM, 7 is 3.5X 10 7 The absorption curve of the exosome sample of Particles/mLCEM cell, 8 is 1.05X 10 8 Absorption profile of exosome samples of Particles/mLCEM cells;
FIG. 5 is a feasibility analysis of tumor exosome detection system;
FIG. 6 is a graph of the particle size and concentration distribution of exosomes;
fig. 7 is an exosome image captured for NTA detection.
Detailed Description
The invention provides a probe group for detecting tumor exosomes, which comprises a probe P1, a probe P2, a probe P3-1, a probe P3-2, a probe P3-COM and a loop-mediated isothermal amplification primer;
the nucleotide sequence of the probe P1 is shown as SEQ ID NO. 1; the nucleotide sequence of the probe P2 is shown as SEQ ID NO.2, the nucleotide sequence of the probe P3-1 is shown as SEQ ID NO.3, the nucleotide sequence of the probe P3-2 is shown as SEQ ID NO.4, and the nucleotide sequence of the probe P3-COM is shown as SEQ ID NO. 5;
the loop-mediated isothermal amplification primer comprises FIP, BIP, FLP and BLP;
the nucleotide sequence of FIP is shown as SEQ ID NO.6, the nucleotide sequence of BIP is shown as SEQ ID NO.7, the nucleotide sequence of FLP is shown as SEQ ID NO.8, and the nucleotide sequence of BLP is shown as SEQ ID NO. 9.
The invention preferably further comprises a biotin label at the 3' end of the P1; the 5' end of the P3-2 is preferably modified with a phosphate group.
In the present invention, the complete structure of each probe is as follows:
(1) P1 has the following structure (5 '-3', SEQ ID NO. 1-biotin):
in the structure, the elongation chain is underlined, and the exosome-recognition sequence is in bold.
(2) The structure of P2 is as follows (5 '-3'):
wherein the bold is an exosome recognition sequence, the scribed region is completely complementary with the 3' free end of the LAMP primary basic structure, a hybrid dimer is formed after combination, and the italic is the free end.
(3) The P3-1 structure is as follows (5 '-3'):
wherein the underlined part is an intramolecular hybridization complementary region, and the black-bold part at the 3' end is a connection complementary region complementary to the P3-COM probe;
(4) The P3-2 structure is (5 '-3', (P) -SEQ ID NO. 4):
the 5 'end of the probe is modified with a phosphate group for connecting with the hydroxyl at the 3' end of the P3-1 probeCrosslinking under the action of enzyme, wherein a black thickened part at the 5' end is a connecting complementary region and is complementary with the P3-COM probe;
(5) The P3-COM structure is as follows (5 '-3'):
the left thickened area can be complementary with the 5' end of the P3-2 probe, the right underlining area can be complementary with the 3' end of the P3-1 probe, the P3-1 probe and the P3-2 probe are simultaneously combined with the P3-COM probe, and the P3 probe, namely a loop-mediated isothermal amplification (LAMP) primary basic structure with a free end at the 3' end, is formed under the action of ligase.
(6) Designing a loop-mediated isothermal amplification primer:
FIP：GGCAGAGGCATCTTCAACGAGGAAGGTGGCTCCTACAA；
BIP：CACGAGGAGCATCGTGGAAACGTCAGTGGAGATATCACATC；
FLP：
BLP：
in the loop-mediated isothermal amplification primers, FLP and BLP carry complementary sequences (bold regions) of the G-quadruplex sequence at the 5' end, and after LAMP, heme is added to form a large amount of G-quadruplex-heme DNase for enzymatic signal amplification.
The invention also provides a kit for detecting tumor exosomes, which comprises the probe set and microspheres modified with streptavidin.
The kit preferably also comprises ligase, nb 2 O 2 . The ligase of the present invention preferably comprises a high temperature ligase, and the Ampligase ligase is exemplified in the examples, but it is not considered to be the full scope of the present invention. The nicking endonuclease of the present invention preferably comprises a nb.
The invention also provides a system for detecting tumor exosomes, which comprises the steps of fixing P1 in the probe set on microspheres modified with streptavidin, mixing the microspheres with exosomes to be detected, and mixing the microspheres with P2 in the probe set to construct a sandwich structure;
mixing P3-1 and P3-2 in the probe group, and constructing to obtain an LAMP primary basic structure with a free end at the 3' end in the presence of a P3-COM probe and ligase;
mixing the sandwich structure and the LAMP primary basic structure, and constructing under the action of incision endonuclease to obtain an LAMP basic structure P4;
performing loop-mediated isothermal amplification on the LAMP base structure P4 and loop-mediated isothermal amplification primers in the probe set to construct a G-quadruplex sequence;
forming a G-quadruplex-heme DNase from the G-quadruplex sequence under the action of heme, and reacting the G-quadruplex-heme DNase with ABTS and H 2 O 2 And (4) mixing.
In the invention, a probe P1, a probe P2, a probe P3-1, a probe P3-2 and a probe P3-COM exist as nucleic acid aptamers, when a sandwich structure (double Aptamer sandwich structure) is constructed, the nucleic acid aptamers P1 are biotin-modified nucleic acid aptamers and are fixed on a microsphere carrier, exosomes are captured through specific recognition with exosome membrane target proteins to be detected and are enriched, finally, another nucleic acid Aptamer P2 is added to form the sandwich structure, and at the moment, the 3' end of the nucleic acid aptamers P2 carries a free single-stranded sequence. The invention fixes P1 on the microsphere carrier modified with streptavidin, thereby functionally modifying the microsphere, leading the microsphere to be further subjected to affinity action with CCRF-CEM extracellular secretion membrane target protein PTK7, and realizing the enrichment of CCRF-CEM extracellular secretion. The kind of the microsphere carrier is not particularly limited in the present invention, and polystyrene microspheres with a diameter of 15.8 μm are preferably used in the examples.
According to the invention, when the LAMP primary foundation structure is constructed, the probe P3-1 and the probe P3-2 are mixed, and the probe P3-1 and the probe P3-2 are connected to form the LAMP primary foundation structure with a 3 'free end in the presence of the P3-COM probe and the Ampligase ligase, namely, the P3-COM probe and the Ampligase ligase can be reused after P3 is denatured at 94 ℃, so that more 3' protruding loop-mediated isothermal amplification (LAMP) primary foundations are constructed.
According to the invention, when an LAMP basic structure P4 is constructed, under the action of Nb.BsrDI nicking endonuclease, a double-Aptamer sandwich structure and an LAMP primary basic structure are mixed, wherein a free single-chain sequence at the 3 'end of P2 in the double-Aptamer sandwich structure is completely complementary with the free end at the 3' end of the LAMP primary basic structure, and a hybrid dimer, namely the LAMP basic structure P4, can be formed through mixing; and the Nb.BsrDI nicking endonuclease cuts the cutting site at the 3' end of the LAMP basic structure in the hybrid dimer to generate a notch, a shorter chain is formed and is unstable, and a double-Aptamer sandwich structure and the LAMP basic structure are released. The dual Aptamer sandwich may reassociate with the 3' overhanging ends of another LAMP primary infrastructure to form another cycle.
The LAMP basic structure P4 is subjected to loop-mediated isothermal amplification under the action of 4 primers (FIP, BIP, FLP-G4 and BLP-G4) to amplify a large amount of G-quadruplex sequences. Adding heme into the formed amplification product containing the G-quadruplex sequence, unwinding the double-helix structure at 90-95 ℃, and rapidly cooling to 4 ℃ to form G-quadruplex-heme DNase. The resulting G-quadruplex-heme DNase solution is combined with ABTS and H 2 O 2 Mixed, capable of catalyzing ABTS-H 2 O 2 Reacting to generate light green ABTS ·+ And has ultraviolet absorption at 420nm, and can be measured by visual observation or spectrophotometry.
The invention also provides application of the probe set or the kit or the system in preparing a medicament for diagnosing and/or preventing and/or treating tumors.
The tumor according to the invention preferably comprises leukemia, which is exemplified by CCRF-CEM cells in the examples, but which should not be considered as the full scope of the invention.
The sample for diagnosis according to the present invention preferably comprises a serum sample of a leukemia patient (for detecting the exosomes contained therein) or exosomes extracted from CCRF-CEM cells (human T-type acute lymphoblastic leukemia cells), and is used for diagnosis of leukemia.
The invention also provides a tumor exosome detection method based on aptamer functionalized microbead enrichment and LAMP signal amplification, preferably as shown in figure 1, and the method comprises the following steps: constructing a double Aptamer sandwich structure, constructing an LAMP primary base structure, constructing an LAMP base structure, constructing a loop-mediated isothermal amplification (LAMP) primary base structure and carrying out loop-mediated isothermal amplification.
In the construction of the double Aptamer sandwich structure, the method is preferably the same as that described above, and the description thereof is omitted.
In the invention, the probe P3-1 and the probe P3-2 are preferably mixed, and then react for 10 to 50s at the temperature of 90 to 98 ℃ in the presence of a P3-COM probe and Ampligase ligase, and then react for 5 to 10min at the temperature of 30 to 40 ℃ to perform 20 to 40 cyclic reactions, so that the probe P3-1 and the probe P3-2 are connected into a 3' LAMP primary basic structure with a free end.
In the present invention, since the 3 'free single-stranded sequence of P2 in the double Aptamer sandwich structure is completely complementary to the 3' free end of the LAMP primary infrastructure, a hybrid dimer, i.e., LAMP infrastructure P4, can be formed upon mixing. The formed LAMP base structure has 2 Nb.BsrDI nicking endonuclease cutting sites at the 3 'protruding end, and the Nb.BsrDI nicking endonuclease can cut the cutting sites at the 3' end of the LAMP base structure in the hybrid dimer, so that a gap is generated, a shorter chain is formed and is unstable, and a double Aptamer sandwich structure and the LAMP base structure are released. The double Aptamer sandwich can reassociate to the 3' overhanging ends of another LAMP primary infrastructure to form another cycle.
According to the invention, the LAMP basic structure is preferably subjected to loop-mediated isothermal amplification under the action of 4 primers (FIP, BIP, FLP-G4 and BLP-G4) to amplify a large amount of G-quadruplex sequences; adding heme into the formed amplification product containing the G-quadruplex sequence, unwinding the double-helix structure at 90-95 ℃, and rapidly cooling to 4 ℃ to form the G-quadruplex-heme DNase. The resulting G-quadruplex-heme DNase solution is combined with ABTS and H 2 O 2 Mixed, capable of catalyzing ABTS-H 2 O 2 Reacting to generate light green ABTS ·+ Has ultraviolet absorption at 420nm, and can be used for treating diabetesMeasured by visual observation or spectrophotometry.
The probe set, the kit and the detection system for detecting tumor exosomes and the application thereof provided by the present invention are described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Microsphere functionalization
By modifying with biotin
And microspheres modified with streptavidin: 100 μ L of the microspheres were placed in a 1.5mL EP tube and centrifuged (3500rpm, 5min, in all cases for subsequent centrifugation steps), and the supernatant was removed. Washing with Binding/wash buffer solution, centrifuging and removing supernatant. Add 4.5. Mu.L of 10. Mu.M P1 and incubate for 30min at room temperature. Washed three times with Binding/wash buffer. Add 100. Mu.L of 10% BSATBbuffer, block at 37 ℃ for 1h, store at 4 ℃.
(2) Purification of CCRF-CEM extracellular exosomes
CCRF-CEM cells are obtained through subculture, supernatant is taken and added with an ECS reagent, the mixture is uniformly mixed through a vortex oscillator and then placed at 4 ℃ for standing for 2 hours; centrifuging at 10000g, discarding supernatant, and precipitating to obtain exosome particles; redissolving it in PBS, centrifuging at 12000g for 2min at 4 deg.C, and retaining the supernatant, which is rich in exosome particles; transferring the obtained crude product of the exosome particles into an EPF column upper chamber, centrifuging at 3000g at 4 ℃, collecting liquid at the bottom of the EPF column after centrifuging for 10min, wherein the liquid is the purified exosome particles, subpackaging by 50-100 mu L, and storing in a low-temperature refrigerator at-80 ℃ for later experiments. The exosomes were characterized for particle size and particle concentration by the ZetaviewPMX 110.
As a result, as shown in FIG. 6, zetaview analysis showed that the measured exosome particle size was mainly distributed between 20-230 nm, with an average particle size of 127nm. Fig. 7 is an exosome image captured at the time of measurement. The exosome is quantitatively detected by an NTA technology, and the particle concentration of the exosome diluted by 100 times is 1.05E + 7. Can be used for subsequent experimental analysis.
(3) Specific recognition of CCRF-CEM exosomes (construction of double Aptamer sandwich structure)
5 μ L of CEM extracellular secretion (1.05X 10) at different concentrations 4 Particles/mL～1.05×10 8 Particles/mL) was added 4. Mu.L of the above P1 probe-immobilized microspheres, 91. Mu.L of 2% BSATBbuffer, 100. Mu.L in total volume per tube, and subjected to constant-temperature shaking at 37 ℃ for 60min, after the reaction, centrifugation was performed, the microsphere complex was washed 3 times with 100. Mu.L of an eluent (20 mM Tris-HCl,0.1% Tween20, pH 7.4) containing 2% BSA, centrifuged, the supernatant was removed, and 92. Mu.L of 2% BSATBbuffer and 8. Mu.L of 1. Mu.M P2 probe were added
Incubated at 37 ℃ for 60min with gentle shaking. After the reaction, the microsphere complex was washed 3 times with 100. Mu.L of an eluent containing 2% BSA, centrifuged, and the supernatant was removed.
(4) Formation of LAMP infrastructure
(1) Construction of the primary infrastructure: a clean centrifuge tube was taken, 64.5. Mu.L of sterilized deionized water was added by using a pipette, 10. Mu.L of 10 × reactionbuffer solution and 5. Mu.L of 5. Mu.M P3-COM probe were sequentially added
10μL10μMP3-1
And 10. Mu.L of 10. Mu.MP 3-2
And 0.5 mu LAmpligase enzyme to make the total volume reach 100 mu L, setting the temperature of 94 ℃ for 30s by a PCR instrument, and then setting the temperature of 37 ℃ for 8min to carry out 25 cycles in total, so that the two added DNA probes are fully connected to form a 3' prominent LAMP basic structure. The PCR instrument was set at 95 ℃ for 5min and rapidly cooled to room temperature.
(2) Construction of the basic structure: the double Aptamer sandwich structure was mixed with the 3' overhanging LAMP primary infrastructure formed above, forming the LAMP infrastructure under the action of nb. Taking 2 mu L of the solution, adding a sandwich structure, then adding 0.4 mu L of 2UNb.BsrDI incision enzyme and 2.5 mu L of NEBuffer2.1 buffer solution, adding sterile water to 25 mu L, reacting for 1h at 65 ℃ of a PCR instrument, and then reacting for 20min at 80 ℃.
(5) LAMP amplification
(1) To a 0.2ml LEP tube were added 3. Mu.L of 10 Xbuffer, 4. Mu.L of 10. Mu.MFIP (GGCAGAGGCATCTTCACGAGGAAGGGTGGCTCCTACAA), 4. Mu.L of 10. Mu.MBIP (CACGAGGAGCATCGTGGAACGTCAGTGGAGATATCACATC), and 10. Mu.MFLP in this order
2μL，10μMBLP
2 mu L, 1.6 mu L of 10mdNTPs1.6 mu L, 18.6 mu L of LAMP basic structure, setting the temperature of a PCR instrument to be 95 ℃ for 5min, and cooling to 60 ℃;
(2) then, 1. Mu.L of 8U/. Mu.LBstDNA polymerase was added, and sterile water (10.4. Mu.L) was added to 30. Mu.L. (or adding 7.5 μ L of 30% dimethyl sulfoxide, adding sterile water to 30 μ L.) and mixing, and setting PCR at 63 deg.C 60min and 85 deg.C for 2min.
(6) G-quadruplex peroxidase formation and signal amplification
10mM heme (hemin) stock was diluted to 10. Mu.M with 0.05% aqueous TritonX-100. Adding 10 mu L of 10 mu Mhemin into the LAMP system after amplification, cooling the temperature from 95 ℃ to 4 ℃ after 2min at 95 ℃ (PCR setting: 95 ℃ for 5min,60 ℃ for 1min,30 ℃ for 4min,20 ℃ for 5min, and 4 ℃ for 60 min), and completing G-quadruplex folding.
Adding 10 μ L of the above solution into 0.2mL of LEP tube, adding 45 μ L of 20 mL MH 2 O 2 And 45. Mu.L of 20mM ABTS, reacted in PCR at 37 ℃ for 8min.
The absorbance at 420nm was chosen as the measurement value, defining Δ A 420nm ＝A 420nm －A 0 Wherein A is 420nm For sample measurements, A 0 Is the background value when the CCRF-CEM extracellular body concentration is 0.
When CCRF-CEM extracellular secretion concentration is 3.5X 10 4 Particles/mL～3.5×10 7 When the concentration of the particle is within the range of particle/mL, the ultraviolet absorbance value is gradually increased along with the increase of the concentration, namely the signal is gradually increased, the absorbance value and the exosome concentration have a better linear relation (FIG. 3), the linear regression equation is y =5.19136E-9x +0.04857 (y is the difference value of the exosome absorbance value and the blank absorbance value, and x is the concentration of the CEM extracellular exosome), and the linear correlation coefficient R is 2 =0.9929, and the detection limit is 2 × 10 by dividing the standard deviation of the blank group by the slope of the standard curve 4 Particles/mL。
EXAMPLE 2 feasibility analysis
Three sets of comparative experiments were set up. The same volume of PBS buffer containing 3.5X 10 7 The procedures of example 1 were followed with Particles/mL of PBS buffer of CEM exosomes, and the results were detected by UV spectrophotometer; another 50. Mu.L of 10mM ATBTS was used as a control for UV detection.
The results of three experiments are shown in FIG. 5, which contains 3.5X 10 of UV absorption spectrum 7 The PBS absorbance value of CEM cell exosome of Particles/mL is obviously higher than that of the other two groups, and the method is feasible.
EXAMPLE 3 actual sample detection analysis
And detecting the CEM cell exosome in a 20% human serum system by adopting a labeling method, and calculating the detection recovery rate according to the ultraviolet absorption result. Specifically, exosomes (exosomes) are bioactive regenerative nanoparticles naturally secreted by adult stem cells, exosome particles are added into 20% of human serum, 4 serum samples with different exosome particle concentrations are constructed, and actual samples are simulated. And (4) calculating the concentration of the exosome according to a linear equation after detection, comparing the actual concentration with the detected concentration, and calculating the recovery rate and the Relative Standard Deviation (RSD).
The detection result is shown in table 1, the detection recovery rate of the CEM extracellular exosome is between 88.9% and 114.7%, and the recovery rate is good, which indicates that the detection technology still has feasibility in complex samples and can be used for better detecting the CEM extracellular exosome.
TABLE 1 detection of CEM extracellular exosomes in human serum samples
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.
Claims (8)
1. A group of probe groups for detecting tumor exosomes, which is characterized by comprising a probe P1, a probe P2, a probe P3-1, a probe P3-2, a probe P3-COM and a loop-mediated isothermal amplification primer;
the nucleotide sequence of the probe P1 is shown as SEQ ID NO. 1; the nucleotide sequence of the probe P2 is shown as SEQ ID NO.2, the nucleotide sequence of the probe P3-1 is shown as SEQ ID NO.3, the nucleotide sequence of the probe P3-2 is shown as SEQ ID NO.4, and the nucleotide sequence of the probe P3-COM is shown as SEQ ID NO. 5;
the loop-mediated isothermal amplification primer comprises FIP, BIP, FLP and BLP;
the nucleotide sequence of the FIP is shown as SEQ ID NO.6, the nucleotide sequence of the BIP is shown as SEQ ID NO.7, the nucleotide sequence of the FLP is shown as SEQ ID NO.8, and the nucleotide sequence of the BLP is shown as SEQ ID NO. 9.
2. The probe set of claim 1, further comprising a biotin label at the 3' end of P1;
the 5' end of the P3-2 is also modified with a phosphate group.
3. A kit for detecting tumor exosomes, comprising a probe set according to claim 1 or 2, and streptavidin-modified microspheres.
4. The kit of claim 3, wherein the kit further comprises ligase, nicking endonuclease, heme, ABTS and H 2 O 2 。
5. A system for detecting tumor exosomes is characterized by comprising the steps of fixing P1 in a probe set of claim 1 or 2 on microspheres modified with streptavidin, mixing the microspheres with exosomes to be detected, and mixing the microspheres with P2 in the probe set of claim 1 or 2 to construct a sandwich structure;
constructing a LAMP primary basic structure with a free end at the 3' end in the presence of a P3-COM probe and ligase after mixing P3-1 and P3-2 in the probe set of claim 1 or 2;
mixing the sandwich structure and the LAMP primary basic structure, and constructing under the action of incision endonuclease to obtain an LAMP basic structure P4;
performing loop-mediated isothermal amplification on the LAMP base structure P4 and a loop-mediated isothermal amplification primer in the probe set of claim 1 or 2 to construct a G-quadruplex sequence;
forming a G-quadruplex-heme DNase from the G-quadruplex sequence under the action of heme, and reacting the G-quadruplex-heme DNase with ABTS and H 2 O 2 And (4) mixing.
6. Use of a set according to claim 1 or 2 or a kit according to claim 3 or 4 or a system according to claim 5 for the preparation of a medicament for the diagnosis and/or prevention and/or treatment of a tumour.
7. The use of claim 6, wherein the tumor comprises leukemia.
8. The use of claim 6, wherein the sample for diagnosis comprises a serum sample from a leukemia patient or exosomes extracted from CCRF-CEM cells.
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