CN110257519A - A kind of lung cancer early diagnosis kit causing the amplification of atom transition free radical polymerization reaction signal based on Nafion - Google Patents

A kind of lung cancer early diagnosis kit causing the amplification of atom transition free radical polymerization reaction signal based on Nafion Download PDF

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CN110257519A
CN110257519A CN201910587483.2A CN201910587483A CN110257519A CN 110257519 A CN110257519 A CN 110257519A CN 201910587483 A CN201910587483 A CN 201910587483A CN 110257519 A CN110257519 A CN 110257519A
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杨怀霞
张京玉
刘艳菊
巴妍妍
赵利营
郭壮壮
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Henan University of Traditional Chinese Medicine HUTCM
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Abstract

The invention discloses a kind of lung cancer early diagnosis kits for causing the amplification of atom transition free radical polymerization reaction signal based on Nafion, including amino Fe3O4Magnetic bead solution, Sulfo-SMCC solution, PNA solution, ZrClO2Solution, Nafion solution, fluorescein O- acrylate solution, AA solution, CuBr2/Me6TREN solution, PBS buffer solution, DMF.The present invention is applied in ATRP reaction using the polymerization property of Nafion, as initiator to improve monomer polymerization ability, a large amount of fluorescent monomers are polymerize by ATRP reaction forming, finally, it is positively correlated by the fluorescence signal intensity that Fluorescence Spectrometer measures with target DNA (tDNA) concentration, successfully constructs the detection kit for causing the lung cancer biomarker CYFRA21-1 of atom transition free radical polymerization reaction signal amplification based on Nafion.Kit of the present invention has the features such as high sensitivity, stability is high, strong interference immunity, and detection limit is low, opens new approach for the clinical detection of lung cancer DNA.

Description

A kind of lung causing the amplification of atom transition free radical polymerization reaction signal based on Nafion Cancer early diagnosis kit
Technical field
The present invention relates to a kind of lung cancer early stages for causing the amplification of atom transition free radical polymerization reaction signal based on Nafion Diagnostic kit belongs to bioassay technique field.
Background technique
Perfluorinated sulfonic acid (Nafion) is a kind of artificial synthesized polymer with ion characteristic, and referred to as ionomer is poly- Close object.It opens a kind of completely new type of polymer, is chiefly used in the modification etc. of membrane material, fuel cell and electrode.Due to The ion characteristic of Nafion is formed on sulfonic group to polymer nature by increasing, and sulfonic group is that have very strong ion Property chemical component, part C-F bond energy is lower in Nafion, can produce free radical, the easy free radical polymerization under polymerization reaction.Perfluor Sulfonic acid polymer has excellent ionic conductivity, excellent chemical stability and good thermal stability.
Atom transfer radical polymerization (ATRP) usually utilizes initiator (organohalogen compounds), carrier (Transition metal complexes Object), by redox reaction, make to establish a kind of dynamic equilibrium between reactive species and suspend mode kind, to reach to polymerization reaction Control.It has the characteristics that reaction process is controllable, reaction monomers are extensive, sensitivity is high etc..Therefore, gradually with nano material While in biomolecule detection by more and more extensive application, the biotinylated nucleic acid probe signals amplification strategy based on ATRP is in life Great popularization has also been obtained in terms of analyte detection.
It is most fast that lung cancer is that morbidity and mortality increase, to one of population health and the maximum malignant tumour of life threat, The trend constantly risen is presented in its disease incidence。It is most of due to the effective technology and means for lacking early detection with diagnosing the illness Middle and advanced stage is in when lung cancer is found, treatment can only be based on palliative treatment, poor prognosis, and diagnosis depends on cell It learns, other tests such as image, but these conventional methods are difficult to detect the micrometastasis in cancer cell, so that it diffuses to advanced stage. Therefore, a kind of highly desirable ultrasensitive biological detector of today's society is detected applied to lung cancer DNA.CYFRA21-1 cell angle egg White tiles section is considered as the most important biomarker of diagnosing non-small cell lung cancer, just when dissolution or necrosis occur for tumour cell Cytokeratin is released into body fluid, NSCLC is more common in raising in blood, is presently believed to be detection lung squamous cancer It is preferred.Mei Chen etc. has utilized the part DNA fragmentation (5'- of CYFRA21-1 Cytokeratin CGCCCCTGACACCATTCCTCCCTTC-3' detection (the Three-dimensional to CYFRA21-1) is realized electrochemical DNA biosensor based on 3D graphene-Ag nanoparticles for sensitive detection of CYFRA21-1in non-small cell lung cancer)。
In order to improve the sensitivity and selectivity of detection, it is applied to ATRP using Nafion polymerization property and reacts In, as initiator, polymerizing power can be improved.Compared with traditional antibody-antigene reaction method, this method greatly improves science of heredity The efficiency and sensitivity of detection and medical diagnosis, are won with high specific, common-path interference and good selective advantage, can be used It is detected in the CYFRA21-1tDNA of low concentration.
Summary of the invention
Aiming at the problem that presently, there are detection lung cancer DNA, the object of the present invention is to provide one kind to cause original based on Nafion The lung cancer early diagnosis kit of sub- transition free radical polymerization reaction signal amplification.
To achieve the goals above, the technical scheme is that
A kind of lung cancer early diagnosis kit causing the amplification of atom transition free radical polymerization reaction signal based on Nafion, Including amino Fe3O4Magnetic bead solution, Sulfo-SMCC solution, PNA solution, ZrClO2Solution, Nafion solution, fluorescein O- third Olefin(e) acid ester solution, AA solution, CuBr2/Me6TREN solution, PBS buffer solution, DMF.
Amino Fe3O4Magnetic bead solution concentration is 10mg/mL, and Sulfo-SMCC solution concentration is 100 μM, and PNA solution concentration is 0.5 μM, ZrClO2Solution concentration is 5mM, and Nafion solution is117 solution, fluorescein O- acrylate solution concentration For 10mM, AA solution concentration is 2mM, CuIIBr/Me6TREN solution concentration is 10mM.
The sequence of PNA is 5'-SH- (CH2)11(O Linker)3-GAAGGGAGGAATGGTGTCAGGGGCG-3'。
CuBr2/Me6TREN solution the preparation method comprises the following steps: 20mM CuBr2Solution and 20mM Me6TREN solution is with volume ratio It is mixed for the ratio of 1:1, the Cu that concentration is 10mM is madeIIBr/Me6TREN solution, 4 DEG C save backup.
A kind of application of mentioned reagent box in lung cancer early diagnosis.
A kind of method of mentioned reagent box diagnosing, comprising the following steps:
(1) processing and modification of amino magnetic bead:
1. measuring a certain amount of amino Fe3O4Magnetic bead solution, Magnetic Isolation remove supernatant, are washed with PBS buffer solution, magnetic Supernatant is removed in separation, and PBS buffer solution is added and is resuspended and restores to the magnetic bead liquor capacity of measurement；
2. measuring 20 μ L magnetic bead re-suspension liquids, add 20 μ L Sulfo-SMCC solution and 160 μ L PBS buffer solution, after mixing, 2h is protected from light under the conditions of 37 DEG C of constant-temperature table；
3. by step (1) -2. gained mixed liquor Magnetic Isolation, remove supernatant, washed with PBS buffer solution, Magnetic Isolation is gone Supernatant；
4. 195 μ L PBS buffer solution and 5 μ L PNA solution are added, after mixing, it is protected from light under the conditions of 37 DEG C of constant-temperature table anti- It should stay overnight；
(2) tDNA hybridizes:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (1)；
2. 20 μ L samples to be tested and 180 μ L PBS buffer solution are added, after mixing, it is protected from light under the conditions of 37 DEG C of constant-temperature table anti- Answer 2h；
(3) the magnetic bead modification based on DNA:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (2)；
2. the ZrClO of 20 μ L is added2Solution and 180 μ L PBS buffer solution after mixing, are kept away under the conditions of 37 DEG C of constant-temperature table Light reaction 0.5h；
3. by step (3) -2. gained reaction solution Magnetic Isolation, remove supernatant, washed with PBS buffer solution；
4. the Nafion solution and 190 μ L PBS buffer solution of 10 μ L is added, after mixing, kept away under the conditions of 37 DEG C of constant-temperature table Light reaction 40-60min；
(4) atom transfer radical polymerization (ATRP) is reacted:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (3)；10 μ L fluoresceins are added FA solution, 150 μ L PBS buffer solution, 20 μ L AA solution, 20 μ L CuIIBr/Me6TREN solution, in 37 DEG C of conditions of constant-temperature table Under be protected from light 100-120min；
2. by step (4) -1. gained reaction solution Magnetic Isolation, remove supernatant, successively washed with DMF and PBS buffer solution；So Afterwards by magnetic bead redistribution in 700 μ L PBS buffer solution, after mixing, mixed liquor carries out fluorescence detection as sample, determines knot Fruit.
The revolving speed reacted in step (1)~(4) is 200r/s.
The excitation wavelength of fluorescence detection is 489nm, slit width 2nm in step (4).
Kit of the present invention and the schematic diagram of detection method are as shown in Figure 1.
Beneficial effects of the present invention:
1, compared with traditional ATRP polymerization method, suitable copper bromide, Cu is added in the present invention in ATRP polymerization systemⅡIt is logical It crosses and Cu0Between electronics transfer be regenerated as CuⅠ, keep the direction of " suspend mode kind " Xiang Shengcheng " reactive species " mobile, so that polymerization efficiency It is significantly improved, to improve the stability and reproducibility of the biosensors.
2, Nafion is living as Raolical polymerizable using the feature of polymer as the initiator in ATRP system Property center, the C-F key section bond energy in Nafion is lower, is broken, generates free radicals under the effect of the catalyst, passes through free radical New free radical is generated to the shock of C in fluorescein (FA) C=C double bond, a series of polymerization makes the fluorescence signal finally detected Intensity is amplified.Since the presence of C-F key increases the rate of polymerization and stability of free radical, improved in amplification fluorescence signal While detection sensitivity, stability is also opposite to be improved.
3, it is applied in ATRP reaction using the polymerization property of Nafion, as initiator to improve monomer polymerization energy Power, a large amount of fluorescent monomers are polymerize by ATRP reaction forming, finally, the fluorescence signal intensity and target that are measured by Fluorescence Spectrometer DNA (tDNA) concentration is positively correlated, and successfully constructs and causes the amplification of atom transition free radical polymerization reaction signal based on Nafion Lung cancer biomarker CYFRA21-1 detection kit.The result shows that under optimal conditions, within the scope of 0.1fM~1nM TDNA concentration and fluorescence signal intensity be in good linear relationship, linear equation be F (au)=2178.67 × LogCtDNA+ 35838.62(R2=0.9975), detection limit is down to 35.5aM.The kit measures DNA in actual sample human serum simultaneously Show higher anti-interference (90.63%).In conclusion this kit is to lung cancer biomarker CYFRA21-1DNA's Detection has the features such as high sensitivity, stability is high, strong interference immunity, opens new approach for the clinical detection of lung cancer DNA.
Detailed description of the invention
Fig. 1 is the schematic diagram of kit and detection method.
Fig. 2 is the intensity of the fluorescence signal of different modifying amino magnetic bead.
Fig. 3 is the relationship of fluorescence signal intensity and ATRP time.
Fig. 4 is the relationship of fluorescence signal intensity and FA dosage.
Fig. 5 is the relationship of fluorescence signal intensity and Nafion time.
Fig. 6 is the relationship of fluorescence signal intensity and Nafion dosage.
Fig. 7 is influence of the different tDNA concentration to fluorescence signal intensity.The concentration of tDNA is followed successively by from top to bottom in figure 1nM、100pM、10pM、1pM、100fM、10fM、1fM、0.1fM。
Fig. 8 is the relationship of fluorescence signal intensity and tDNA concentration C；In figure the concentration of tDNA be followed successively by from right to left 1nM, 100pM、10pM、1pM、100fM、10fM、1fM、0.1fM。
Fig. 9 is the relationship of fluorescence signal intensity and different mispairing tDNA.
Figure 10 is the relationship of fluorescence signal intensity and 5%NHS and 0.1nM PBS.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Amino Fe3O4Magnetic bead is purchased from Pu Ruimai lattice Biotechnology Co., Ltd (Xiamen).
The sequence of PNA are as follows: 5'-SH- (CH2)11(O Linker)3-GAAGGGAGGAATGGTGTCAGGGGCG-3'(SEQ ID NO.1)。
The sequence (CYFRA21-1 partial sequence) of tDNA are as follows: 5'-CGCCCCTGACACCATTCCTCCCTTC-3'(SEQ ID NO.2)。
The sequence of SBM are as follows: 5 '-CGCCCCTAACACCATTCCTCCCTTC-3 ' (SEQ ID NO.3).
The sequence of TBM is 5 '-CGCCCATGACACTATTCCTCGCTTC-3 ' (SEQ ID NO.4).
The sequence of Control are as follows: 5 '-TATTATCCGTCAGTGGAAAGGACCG-3 ' (SEQ ID NO.5).
The building of embodiment 1, diagnostic kit
The kit includes amino Fe3O4Magnetic bead solution (10mg/mL), Sulfo-SMCC solution (100 μM), PNA solution (0.5μM)、ZrClO2Solution (5mM), Nafion solution (117 solution), fluorescein O- acrylate (FA) solution (10mM), AA solution (2mM), CuIIBr/Me6TREN solution (10mM), PBS buffer solution, DMF (N,N-dimethylformamide).
PBS buffer solution the preparation method comprises the following steps: weighing 5.8g Na2HPO4.12H2O、0.592g NaH2PO4.2H2O、2.34g NaCl and 200 μ L ultrapure waters mixing to get；Its concentration is 0.1M, pH 7.4.
Ascorbic acid (AA) solution the preparation method comprises the following steps: AA is mixed with the ethanol solution of volume fraction 70%, be made dense Degree is the AA solution of 2mM, and 4 DEG C save backup.
Fluorescein FA solution the preparation method comprises the following steps: FA is mixed with DMF, be made the fluorescein FA solution that concentration is 10mM, 4 It DEG C saves backup.
CuIIBr/Me6TREN solution the preparation method comprises the following steps: 20mM CuBr2Solution (being dissolved in DMF) and 20mMMe6TREN Solution (being dissolved in DMF) is mixed with volume ratio for the ratio of 1:1, and the Cu that concentration is 10mM is madeIIBr/Me6TREN solution, 4 It DEG C saves backup.
Embodiment 2, the detection method of kit
(1) processing and modification of amino magnetic bead:
1. measuring a certain amount of amino Fe3O4Magnetic bead solution (10mg/mL), Magnetic Isolation remove supernatant, use PBS buffer solution Washing, Magnetic Isolation remove supernatant, and PBS buffer solution is added and is resuspended and restores to the magnetic bead liquor capacity of measurement；
2. measuring 20 μ L magnetic bead re-suspension liquids, add 20 μ L Sulfo-SMCC solution and 160 μ L PBS buffer solution, after mixing, Be protected from light 2h under the conditions of 37 DEG C of constant-temperature table, 200r/s, make the one end crosslinking aid S MCC and amino magnetic bead with amido bond in conjunction with；
3. by step (1) -2. gained mixed liquor Magnetic Isolation, remove supernatant, washed with PBS buffer solution, Magnetic Isolation is gone Supernatant；
4. 195 μ L PBS buffer solution and 5 μ L PNA solution are added, after mixing, under the conditions of 37 DEG C of constant-temperature table, 200r/s It is protected from light overnight, is bonded PNA with the crosslinking agent other end；
(2) tDNA hybridizes:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (1)；
2. the tDNA solution (0.1nM) and 180 μ L PBS buffer solution of 20 μ L is added, after mixing, 37 DEG C of constant-temperature table, It is protected from light 2h under the conditions of 200r/s, makes the tDNA specific recognition in PNA and sample to be tested；
(3) the magnetic bead modification based on DNA:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (2)；
2. the ZrClO of 20 μ L is added2Solution and 180 μ L PBS buffer solution, after mixing, in 37 DEG C of constant-temperature table, 200r/s Under the conditions of be protected from light 0.5h, make Zr4+It is connected with phosphate radical by chemical reaction；
3. by step (3) -2. gained reaction solution Magnetic Isolation, remove supernatant, washed with PBS buffer solution；
4. the Nafion solution and 190 μ L PBS buffer solution of 10 μ L is added, after mixing, in 37 DEG C of constant-temperature table, 200r/s Under the conditions of be protected from light 40-60min, sulfonate radical and Zr in Nafion4+It chemically reacts to form basic zirconium phosphate sulfonate with phosphate radical；
(4) atom transfer radical polymerization (ATRP) is reacted:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (3)；10 μ L fluoresceins are added FA solution, 150 μ L PBS buffer solution, 20 μ L AA solution, 20 μ L CuIIBr/Me6TREN solution, 37 DEG C of constant-temperature table, 100-120min is protected from light under the conditions of 200r/s；
2. removing supernatant, DMF being added and cleans 2 times, PBS buffer solution is washed step (4) -1. gained reaction solution Magnetic Isolation It washs 3 times；Then by magnetic bead redistribution in 700 μ L PBS buffer solution, after mixing, mixed liquor is as sample in fluorescence spectrophotometer light It spends on instrument and carries out fluorescence detection (excitation wavelength 489nm, slit width 2nm).It is dense that tDNA is calculated according to fluorescence signal intensity Degree.
Embodiment 3, feasibility verifying
Firstly, carrying out fluorescence intensity detection by using amino magnetic bead of the Fluorescence Spectrometer to different modifying state.Such as Fig. 2 It is shown, PBS buffer solution (0.1M, pH7.4) is only added and does not detect any fluorescence intensity.On the contrary, SMCC/PNA/ is added tDNA/Zr4+/Nafion/CuIIBr/Me6Apparent fluorescence emission peak is still observed that by repeatedly washing after TREN, this It is to produce great fluorescence signal because a large amount of FA is introduced on magnetic bead by ATRP.Meanwhile it not being added respectively Zr4+、CuIIBr/Me6Under conditions of TREN, Nafion, PNA, tDNA, relatively weak fluorescence signal is only detected, this shows reality Test sensitivity with higher.By comparative experiments, sufficiently demonstrates and answered based on ATRP polymerization for fluorescence signal amplification strategy Detection for tDNA is feasible.
Embodiment 4, the optimization of ATRP testing conditions
(1) optimization in reaction time
By detecting the fluorescence intensity of the amino magnetic bead in different ATRP reaction time, reflected reaction time of ATRP with The relationship of fluorescence intensity.As a result as shown in figure 3, fluorescence signal intensity with the extension in ATRP reaction time be gradually increased and 100min has reached stabilization.This may be because the free radical for causing polymer chain growth loses its activity or its space bit It hinders and excessive hinders the growth of itself.Therefore, ATRP optimum reacting time is 100min.
(2) optimization of fluorescein dosage
Fluorescein number can influence the length of polymer chain and then influence detection performance.Obviously, when fluorescein in system When FA content deficiency, maximum is not achieved in fluorescence signal intensity.As a result as shown in figure 4, fluorescein is added in ATRP system respectively The dosage of FA is 2 μ L, the concentration of 4 μ L, 6 μ L, 8 μ L, 10 μ L, 15 μ L, 20 μ L, FA are 10mM, the magnetic when FA amount is added and is 10 μ L The fluorescence signal intensity of pearl reaches maximum, hereafter continues to increase the dosage of fluorescein, and fluorescence signal intensity does not significantly increase, Therefore, the optimum amount of fluorescein is 10 μ L.
In conclusion ATRP system optimum optimizing condition are as follows: the ATRP reaction time is 100min, and fluorescein dosage is 10 μ L。
Embodiment 5, initiator Nafion condition optimizing
(1) optimization in Nafion reaction time
Nafion can influence the length of polymer chain and then influence detection performance.When Nafion reaction is insufficient in system When, maximum is not achieved in fluorescence intensity.As a result as shown in figure 5, respectively measurement the Nafion reaction time be 0min, 20min, 40min, Magnetic bead fluorescence signal intensity when 60min, 80min, when the Nafion time is 40min, the fluorescence intensity of magnetic bead reaches maximum, Therefore selecting the Nafion reaction time is 40min in later experiment.
(2) optimization of Nafion dosage
The dosage of Nafion not only has an impact to polymer chain, but also influences detection performance.As a result as shown in fig. 6, respectively Measurement Nafion dosage is respectively 2 μ L, 4 μ L, 8 μ L, 10 μ L, 15 μ L, 20 μ L, the amino magnetic bead when Nafion is added and is 10 μ L Fluorescence signal intensity reaches maximum, hereafter continues the dosage for increasing Nafion, and fluorescence signal intensity does not significantly increase, therefore selects Selecting Nafion dosage is that 10 μ L are applied in following experiment.
In conclusion Nafion optimum optimizing condition are as follows: the reaction time of Nafion is 40min, and dosage is 10 μ L.
Embodiment 6 analyzes the detection performance of tDNA
According to the optimum experimental condition that embodiment 4,5 condition optimizings are obtained, detect various concentration tDNA (0.1fM, 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, 1nM) generate fluorescence signal intensity size, come study the present invention for tDNA Detection performance.As shown in Figure 7,8, the fluorescence signal intensity of magnetic bead increases as the concentration of tDNA increases, this is because TDNA concentration is bigger, causes and Zr4+The phosphate radical of reaction is more, thus keep the fluorescein for introducing magnetic bead more, the fluorescence of generation Intensity is stronger.Good linear pass is presented in 0.1fM to 1nM range in the logarithm of fluorescence emission maximum peak intensity and tDNA concentration System, linear equation are F (au)=2178.67 × LogCtDNA+35838.62(R2=0.9975), detection is limited to 35.5aM, as a result Show that experimental method detection sensitivity with higher, lower detection limit are had excellent performance.
Embodiment 7, selectivity experiment
The optimum experimental condition obtained by embodiment 4,5 condition optimizings, research tDNA analog, SBM, TBM, The fluorescence signal intensity that Control uses this detection method to generate.Particular by comparison under concentration tDNA, TBM, The fluorescence signal intensity of Control, SBM (0.1nM).As shown in figure 9, difference mispairing tDNA (Control, TBM, SBM) institute is right The fluorescence signal intensity answered is respectively 8.93%, 22.17%, the 30.66% of tDNA, the fluorescence signal that chaff interferent generates relative to Object is smaller, this is because PNA and tDNA specific recognition complementary pairing, as a result prove the method for the present invention choosing with higher Selecting property.
Embodiment 8, interference experiment
According to the optimum experimental condition that embodiment 4,5 condition optimizings are obtained, by the way that 5% (10/200) will be separately added into The fluorescence signal intensity of the amino magnetic bead of normal human serum (NHS) and 0.1nM PBS (pH 7.4) compares, such as Figure 10 institute Show, when the concentration of the two tDNA is respectively 0.1fM, 0.1pM, 0.1nM, the amino of measured 5% normal human serum (NHS) The fluorescence signal intensity of magnetic bead is respectively 89.67%, 88.48%, the 90.63% of PBS (pH 7.4).The result shows that the present invention It is had strong anti-interference ability in the practical blood serum sample of method, can be used for the detection of actual sample.
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Claims (8)

1. a kind of lung cancer early diagnosis kit for causing the amplification of atom transition free radical polymerization reaction signal based on Nafion, It is characterized in that, including amino Fe3O4Magnetic bead solution, Sulfo-SMCC solution, PNA solution, ZrClO2It is solution, Nafion solution, glimmering Light element O- acrylate solution, AA solution, CuBr2/Me6TREN solution, PBS buffer solution, DMF.
2. kit according to claim 1, which is characterized in that amino Fe3O4Magnetic bead solution concentration is 10mg/mL, Sulfo-SMCC solution concentration is 100 μM, and PNA solution concentration is 0.5 μM, ZrClO2Solution concentration is 5mM, and Nafion solution is117 solution, fluorescein O- acrylate solution concentration are 10mM, and AA solution concentration is 2mM, CuIIBr/Me6TREN is molten Liquid concentration is 10mM.
3. kit according to claim 1, which is characterized in that the sequence of PNA is 5'-SH- (CH2)11(O Linker)3- GAAGGGAGGAATGGTGTCAGGGGCG-3'。
4. kit according to claim 1, which is characterized in that CuBr2/Me6TREN solution the preparation method comprises the following steps: 20mM CuBr2Solution and 20mM Me6TREN solution is mixed with volume ratio for the ratio of 1:1, and the Cu that concentration is 10mM is madeIIBr/ Me6TREN solution, 4 DEG C save backup.
5. a kind of application of kit as described in claim any one of 1-4 in lung cancer early diagnosis.
6. a kind of method using any one of the claim 1-4 kit diagnosing, which is characterized in that including following step It is rapid:
(1) processing and modification of amino magnetic bead:
1. measuring a certain amount of amino Fe3O4Magnetic bead solution, Magnetic Isolation remove supernatant, are washed with PBS buffer solution, magnetism point From removing supernatant, PBS buffer solution be added and is resuspended and restores to the magnetic bead liquor capacity of measurement；
2. measuring 20 μ L magnetic bead re-suspension liquids, add 20 μ L Sulfo-SMCC solution and 160 μ L PBS buffer solution, after mixing, in constant temperature 2h is protected from light under the conditions of 37 DEG C of shaking table；
3. by step (1) -2. gained mixed liquor Magnetic Isolation, remove supernatant, washed with PBS buffer solution, Magnetic Isolation removes supernatant Liquid；
4. 195 μ L PBS buffer solution and 5 μ L PNA solution are added, after mixing, it was protected from light under the conditions of 37 DEG C of constant-temperature table Night；
(2) tDNA hybridizes:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (1)；
2. 20 μ L samples to be tested and 180 μ L PBS buffer solution are added, after mixing, it is protected from light under the conditions of 37 DEG C of constant-temperature table 2h；
(3) the magnetic bead modification based on DNA:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (2)；
2. the ZrClO of 20 μ L is added2Solution and 180 μ L PBS buffer solution after mixing, are protected from light anti-under the conditions of 37 DEG C of constant-temperature table Answer 0.5h；
3. by step (3) -2. gained reaction solution Magnetic Isolation, remove supernatant, washed with PBS buffer solution；
4. the Nafion solution and 190 μ L PBS buffer solution of 10 μ L is added, after mixing, it is protected from light under the conditions of 37 DEG C of constant-temperature table anti- Answer 40-60min；
(4) atom transfer radical polymerization (ATRP) is reacted:
1. removing supernatant, being washed with PBS buffer solution reaction solution Magnetic Isolation obtained by step (3)；It is molten that 10 μ L fluorescein FA are added Liquid, 150 μ L PBS buffer solution, 20 μ L AA solution, 20 μ L CuIIBr/Me6TREN solution is kept away under the conditions of 37 DEG C of constant-temperature table Light reaction 100-120min；
2. by step (4) -1. gained reaction solution Magnetic Isolation, remove supernatant, successively washed with DMF and PBS buffer solution；Then will Magnetic bead redistributes in 700 μ L PBS buffer solution, and after mixing, mixed liquor carries out fluorescence detection as sample, determines result.
7. according to the method described in claim 6, it is characterized in that, the revolving speed reacted in step (1)~(4) is 200r/s.
8. according to the method described in claim 6, it is characterized in that, in step (4) fluorescence detection excitation wavelength be 489nm, Slit width is 2nm.
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Publication number Priority date Publication date Assignee Title
CN111175507A (en) * 2020-03-13 2020-05-19 河南中医药大学 Lung cancer early diagnosis kit based on signal amplification of ring-opening polymerization reaction initiated by hydroxyl functionalized graphene oxide
CN114410735A (en) * 2022-01-25 2022-04-29 河南中医药大学 Electrochemical kit for detecting alkaline phosphatase by using amifostine as substrate and ATRP signal amplification strategy and use method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
H. QIAN等: "Surface-initiated activators generated by electron transfer for atom transfer radical polymerization in detection of DNA point mutation", 《ANAL CHEM》 *
J.ZHANG等: "F-containing initiatior for ultrasensitive fluorescent detection of lung cancer DNA via atom transfer radical polymerization", 《ANALYTICA CHIMICA ACTA》 *
K.-J. PENG等: "Atom Transfer Radical Addition/Polymerization of Perfluorosulfonic Acid Polymer with the C–F Bonds as Reactive Sites", 《ACS MACRO LETT.》 *
Q.HU等: "Electrochemically Mediated Surface-Initiated de Novo Growth of Polymers for Amplified Electrochemical Detection of DNA", 《ANAL. CHEM.》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111175507A (en) * 2020-03-13 2020-05-19 河南中医药大学 Lung cancer early diagnosis kit based on signal amplification of ring-opening polymerization reaction initiated by hydroxyl functionalized graphene oxide
CN114410735A (en) * 2022-01-25 2022-04-29 河南中医药大学 Electrochemical kit for detecting alkaline phosphatase by using amifostine as substrate and ATRP signal amplification strategy and use method
CN114410735B (en) * 2022-01-25 2023-10-27 河南中医药大学 Electrochemical kit for detecting alkaline phosphatase by using amifostine as substrate and utilizing ATRP signal amplification strategy and using method

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