CN105044184A - Meta-zinc tetraphenylporphyrin-based electrogenerated chemiluminescence body as well as preparation method and application thereof - Google Patents
Meta-zinc tetraphenylporphyrin-based electrogenerated chemiluminescence body as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a meta-zinc tetraphenylporphyrin-based electrogenerated chemiluminescence body. The chemiluminescence body is prepared from aniline imidazole, footballene pyrrolidine tricarboxylic acid and meta-zinc tetraphenylporphyrin. The invention also provides a preparation method of the chemiluminescence body, comprising the step that on the commercial footballene pyrrolidine tricarboxylic acid, reacting with the meta-zinc tetraphenylporphyrin in an axial coordination way by taking the aniline imidazole as a bridging object, so as to generate the efficient and stable novel electrogenerated chemiluminescence body. On this basis, according to the envelope characteristics of beta-cyclodextrin molecules, a stable electrogenerated chemiluminescence biological sensing system with beta-cyclodextrin enveloping fixed chemiluminescence body molecules is designed and built, realizes high sensitivity and high specificity detection on DNA (deoxyribonucleic acid), and is stable in electrogenerated chemiluminescence output signal and lower in detection lower limit. The detection system can be applied to biological medicine, clinical diagnosis, environmental monitoring and the like.
Description
Technical field
The invention belongs to electrochemical analysis detection technique field, be specifically related to a kind of electrogenerated chemiluminescence body based on a Tetraploid rice, preparation method and the application in DNA detects thereof.
Background technology
Electrogenerated chemiluminescence (electrochemiluminescence, ECL) with high sensitivity and low background famous, as a kind of powerful analysis means, be applied to food hygiene medical industry at present.The luminescence efficiency of luminophor is depended in the sensitivity of ECL in essence.Wherein, cathodic electroluminescence body is occupied an leading position with semiconductor nanocrystal, but such material is generally containing toxic ingredient, and ECL intensity is not high and rely on exogenous strong oxidizer as coreagent enhanced sensitivity, greatly more the widely using of restriction ECL technology.Therefore efficient negative electrode ECL luminophor is found and the ECL detection method utilizing its development simple and easy, stable, very necessary.
Porphyrin Molecule, due to its special electrochemical activity, has been widely used in photoelectric sensor and electrochemical analysis.Between Tetraploid rice (ZnTPP) using dissolved oxygen DO as under the condition of coreagent, there is efficiently excellent electrogenerated chemiluminescence response, and there is better stability, the reaction of any photobleaching does not occur.
On Fe in Heme porphyrin central metal atom and histidine imidazole ring can there is axial coordination in free atom N.Carbon nano-tube is connected with containing conjugation group compound such as pyrene butyric acid based on π-pi-conjugated mode by the people such as Chitta, and designed and synthesized a kind of nano-complex by pyrene butanoic acid derivative terminal imidazole ring atom N lone pair electrons and metalloporphyrin axial coordination, find that this compound has certain quantity of photogenerated charge and is separated and transmission effect (ChittaR, SandanayakaASD, SchumacherAL, etal.Donor-acceptornanohybridsofzincnaphthalocyanineorzi ncporphyrinnoncovalentlylinkedtosingle-wallcarbonnanotub esforphotoinducedelectrontransfer [J] .TheJournalofPhysicalChemistryC, 2007, 111 (19): 6947-6955).But embedded photoluminescent material might not realize electroluminescence.
Current studied maximum electrogenerated chemiluminescence body comprises ruthenium dipyridine (Ru (bpy)
3 2+), luminol and quantum dot etc.For ruthenium dipyridine, it is expensive, also exist constantly consumed cause that analysis cost is high, environmental pollution and the series of problems such as experimental provision is complicated, it needs to add using tripropyl amine (TPA) as coreagent in the application simultaneously, and the easy intoxicating of the latter and to bad environmental, the application of ruthenium dipyridine is subject to certain restrictions.Porphyrin comes from haemoglobin and chlorophyll, itself has excellent security and biocompatibility, does not need other coreagent in addition, only need can realize stronger electroluminescence signal under aerobic environment.Usually utilize the photoelectric properties of Porphyrin and its derivative in prior art, carry out photoelectricity bionical aspect research, the light absorbent etc. of such as solar panel.
Summary of the invention
The object of the present invention is to provide a kind of novel electroluminescent chemical luminophor simple for production and preparation method thereof, with aniline imidazoles (4-(1H-Imidazol-1-yl) Aniline, ImAn) bridge joint football alkene pyrrolidine tricarboxylic acids (C60pyrrolidinetris-acid, C
60-(COOH)
3) and between axial coordination the nanocluster of Tetraploid rice as novel electroluminescent chemical luminophor, and utilize beta-schardinger dextrin-bag by this nanocluster, devise a kind of high selectivity, easy ECL sensing system, and be applied to electrochemiluminescdetection detection DNA.
To achieve these goals, technical scheme of the present invention is:
Based on an electrogenerated chemiluminescence body for a Tetraploid rice, this luminophor is C
60-ImAn-ZnTPP, is made up of aniline imidazoles, football alkene pyrrolidine tricarboxylic acids and a Tetraploid rice, described aniline imidazoles bridge joint football alkene pyrrolidine tricarboxylic acids Tetraploid rice between axial coordination.
Prepare a method for above-mentioned luminophor, concrete steps are as follows:
(1) successively by football alkene pyrrolidine tricarboxylic acids, N-ethyl-N '-(3-Dimethylaminopropyl) carbodiimide (N-Ethyl-N '-(3-Dimethylaminopropyl) Carbodiimidehydrochloride, EDC), N-hydroxysuccinimide (N-HydroxySuccinimide, NHS) and aniline imidazoles join methylene chloride (DiChloroMethane, DCM) in, isolated air stirring reacts completely, reaction terminates rear dialysis treatment, removing unreacted reactant completely;
(2) Tetraploid rice between aniline imidazoles equimolar amounts is joined in the solution in step 1 after dialysis, sealing lucifuge, stirring reaction is complete, reaction terminates rear dialysis treatment, removing unreacted reactant completely, is drying to obtain with aniline imidazoles bridge joint football alkene pyrrolidine tricarboxylic acids and the electrogenerated chemiluminescence body (C of Tetraploid rice between axial coordination
60-ImAn-ZnTPP).
In step 1, the mol ratio of described EDC and NHS is 4 ~ 1:1, and described football alkene pyrrolidine tricarboxylic acids and the mol ratio of aniline imidazoles are 1:3 ~ 6.
Described dialysis treatment is proceed in bag filter by reactant liquor after reaction terminates, take DCM as dislysate, change every 2 ~ 3h, room temperature dialysis 3 ~ 4 days, wherein, the molecular cut off of the bag filter of the dialysis treatment in step 1 is 1000, and the molecular cut off of the bag filter of the dialysis treatment in step 2 is 2000.
The application of above-mentioned luminophor in DNA detects, comprises the steps:
(1) successively by 2wt.% diallyl dimethyl ammoniumchloride (PolyDiallylDimethylAmmonium, PDDA) aqueous solution and nano-Au solution drip and are coated in glassy carbon electrode surface, then three (2-carboxy ethyl) phosphine (Tris (2-CarboxyEthyl) Phosphine be coated with containing capture dna (cDNA) is dripped at electrode surface, TCEP) solution, leave standstill in 37 DEG C of incubation casees, with being placed on 4 DEG C, spending the night under saturated humidity condition;
(2) PBS drip washing electrode, removes loose cDNA, drips the bovine serum albumin(BSA) (BovineSerumAlbumin being coated with 2wt.% subsequently, BSA) aqueous solution, close avtive spot in case non-specific adsorption, and be placed in 37 DEG C of incubations, drip washing after taking out;
(3) the TCEP solution containing target dna to be measured (tDNA) is dripped the electrode surface being coated in step 2 and obtaining, react at being placed in 37 DEG C, reacted rear drip washing;
(4) prepare the mixed solution of EDC and NHS, then with 2-hexane diamine group-beta-cyclodextrin (2-HexyldiAmino-β-CycloDextrin, β-CDNH
2) the mixing of saturated methanol solution equal-volume, obtain EDC/NHS/ β-CDNH
2mixed solution, then by EDC/NHS/ β-CDNH
2mixed solution drips the electrode surface being coated in above-mentioned steps 3 and obtaining, amidation process under room temperature;
(5) C is prepared
60the ethanolic solution of-ImAn-ZnTPP, subsequently by C
60the ethanolic solution of-ImAn-ZnTPP drips the electrode surface being coated in step 4 and obtaining, and reacts under room temperature;
(6) electrode obtained with step 5 is for working electrode, with tetrabutylammonium perchlorate (TetrabutylammoniumPerchlorate, TBAP) as adopting cyclic voltammetry to detect ECL signal in dielectric DCM solution, finally calculate the concentration of tDNA according to the linear equation of tDNA log concentration value and ECL signal intensity value, and then draw the content of tDNA in liquid to be measured.
In step 1, the concentration range of described capture dna is 1 μM ~ 10 μMs.
In step 5, described C
60the concentration range of the ethanolic solution of-ImAn-ZnTPP is 0.5mM ~ 1mM.
In step 6, the method for described detection ECL signal is: the electrode obtained with step 5 is working electrode, and Ag/AgCl is contrast electrode, and platinum electrode is to electrode, and adjustment current potential is-2.2 ~ 0V, and sweep velocity is 100mVS
-1, the concentration of the DCM solution of tetrabutylammonium perchlorate is 0.1M.
By adjusting the base sequence of capture dna, method provided by the present invention can carry out accommodation according to the base of target dna, thus meets various different DNA testing requirement.
The present invention compared with prior art, its remarkable advantage is: (1) adopts football alkene pyrrolidine tricarboxylic acids as raw material, football alkene pyrrolidine tricarboxylic acids is as a kind of carbon nanomaterial, there is excellent electric conductivity, with porphyrin compound bridge joint, can improve charge separation and the transmission efficiency of compound, three carboxyls had in addition can realize the enrichment of three porphyrins, realize the amplification of signal; (2) luminophor preparation process is simple and quick, nontoxic, and obtained electrogenerated chemiluminescence body, directly using air as coreagent, has preferably electrogenerated chemiluminescence response, can be used as general easy signal mark; (3) utilize beta-schardinger dextrin-bag by this luminophor, be applied in electrogenerated chemiluminescence DNA detection, detection method is easy, controllability, selectivity and favorable reproducibility, highly sensitive, detectability is low, signal stabilization, parallel experiment and be only 8% and 10% with the ECL signal relative standard deviation exported under condition different batches.
Accompanying drawing explanation
Fig. 1 is that the present invention prepares novel electroluminescent chemical luminophor and produces the schematic diagram of ECL signal for specific detection DNA.
Fig. 2 is the C that embodiment 4 obtains
60-ImAn-ZnTPP electrogenerated chemiluminescence body (a), ZnTPP (b), C
60(COOH)
3the ultraviolet spectrum phenogram of (c).
Fig. 3 is the C that embodiment 5 obtains
60-ImAn-ZnTPP electrogenerated chemiluminescence body (a), ZnTPP (b), C
60(COOH)
3c the electrogenerated chemiluminescence collection of illustrative plates of (), interior illustration is ECL light intensity time stability collection of illustrative plates.
Fig. 4 is that the beta-schardinger dextrin-bag that obtains of embodiment 6 is by C
60-ImAn-ZnTPP electrogenerated chemiluminescence body PDDA+AuNPs+ β-CDSH+C
60-ImAn-ZnTPP (a) with do not wrap by the electrogenerated chemiluminescence collection of illustrative plates of PDDA+AuNPs+ β-CDSH (b) of luminophor.
Fig. 5 be embodiment 7 obtain based on C
60the electrochemiluminescence signal that H7N9 viral DNA produces is surveyed in the health check-up of-ImAn-ZnTPP electrogenerated chemiluminescence, and interior illustration is the linear relationship chart of H7N9 viral DNA concentration logarithm value and ECL luminous intensity.
Fig. 6 is the base mispairing interference test result figure that embodiment 9 obtains, and in upper right, illustration is the electrogenerated chemiluminescence collection of illustrative plates of ECL light intensity potential change, and in bottom right, illustration is ECL light intensity time stability collection of illustrative plates.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Embodiment 1
(1) in 5mL dichloromethane solvent, add 4.5mg football alkene pyrrolidine tricarboxylic acids, 38.3mgN-ethyl-N '-(3-Dimethylaminopropyl) carbodiimide, 5.8mgN-hydroxysuccinimide and 4mg aniline imidazoles successively, isolated ambient magnetic stirs 24h;
(2) above-mentioned reaction product solution is proceeded to molecular cut off after terminating by reaction is in the bag filter of 1000, take DCM as dislysate, changes every 2 ~ 3h, and room temperature dialysis 3 ~ 4 days, to remove unreacted reactant completely;
(3) get reaction mixture after dialysis, add Tetraploid rice between 17.0mg, sealing lucifuge magnetic agitation reaction 2h, use molecular cut off be 2000 bag filter above-mentioned reaction product is purified, dialysis condition is identical with back;
(4) obtain reaction product solution after having dialysed, the also vacuum drying that naturally volatilizees obtains with ImAn bridge joint C
60and the nanocluster (C of axial coordination ZnTPP
60-ImAn-ZnTPP), products therefrom is placed in 4 DEG C of refrigerators and preserves.
Embodiment 2
(1) in 5mL dichloromethane solvent, add 4.5mg football alkene pyrrolidine tricarboxylic acids, 19.2mgN-ethyl-N '-(3-Dimethylaminopropyl) carbodiimide, 5.8mgN-hydroxysuccinimide and 2.4mg aniline imidazoles successively, isolated ambient magnetic stirs 24h;
(2) above-mentioned reaction product solution is proceeded to molecular cut off after terminating by reaction is in the bag filter of 1000, take DCM as dislysate, changes every 2 ~ 3h, and room temperature dialysis 3 ~ 4 days, to remove unreacted reactant completely;
(3) get reaction mixture after dialysis, add Tetraploid rice between 10.2mg, sealing lucifuge magnetic agitation reaction 2h, use molecular cut off be 2000 bag filter above-mentioned reaction product is purified, dialysis condition back is identical;
(4) obtain reaction product solution after having dialysed, the also vacuum drying that naturally volatilizees obtains with ImAn bridge joint C
60and the nanocluster (C of axial coordination ZnTPP
60-ImAn-ZnTPP), products therefrom is placed in 4 DEG C of refrigerators and preserves.
Embodiment 3
(1) in 5mL dichloromethane solvent, add 4.5mg football alkene pyrrolidine tricarboxylic acids, 9.6mgN-ethyl-N '-(3-Dimethylaminopropyl) carbodiimide, 5.8mgN-hydroxysuccinimide and 4.7mg aniline imidazoles successively, isolated ambient magnetic stirs 24h;
(2) above-mentioned reaction product solution is proceeded to molecular cut off after terminating by reaction is in the bag filter of 1000, take DCM as dislysate, changes every 2 ~ 3h, and room temperature dialysis 3 ~ 4 days, to remove unreacted reactant completely;
(3) get reaction mixture after dialysis, add Tetraploid rice between 20.3mg, sealing lucifuge magnetic agitation reaction 2h, use molecular cut off be 2000 bag filter above-mentioned reaction product is purified, dialysis condition back is identical;
(4) obtain reaction product solution after having dialysed, the also vacuum drying that naturally volatilizees obtains with ImAn bridge joint C
60and the nanocluster (C of axial coordination ZnTPP
60-ImAn-ZnTPP), products therefrom is placed in 4 DEG C of refrigerators and preserves.
Embodiment 4
Ultraviolet characterizes:
Using absolute ethyl alcohol as solvent, prepare the C of 1mM respectively
60-ImAn-ZnTPP, ZnTPP and C
60(COOH)
3solution, measures the ultra-violet absorption spectrum of each solution, and result as shown in Figure 2.Wherein, a is C
60-ImAn-ZnTPP, b are ZnTPP, c is C
60(COOH)
3.As seen from the figure, C
60the maximal ultraviolet absorption peak of-ImAn-ZnTPP and ZnTPP appears at 422nm place, and C at that wavelength
60(COOH)
3without uv absorption.C simultaneously
60the ultraviolet absorption value of-ImAn-ZnTPP approximates three times of the ultraviolet absorption value of ZnTPP, and coincidence theory is expected, can think C thus
60the synthesis success of-ImAn-ZnTPP.
Embodiment 5
ECL input:
Using absolute ethyl alcohol as solvent, prepare the C of 1mM respectively
60-ImAn-ZnTPP, ZnTPP and C
60(COOH)
3solution, drips respectively afterwards and is coated in polished glassy carbon electrode surface, and then scribble the electrode of different solutions for working electrode to drip, take Ag/AgCl as contrast electrode, platinum electrode is to electrode, and potential window is-2.2V ~ 0V, and sweep velocity is 100mVS
-1, in dielectric DCM solution, carrying out ECL input using the tetrabutylammonium perchlorate of 0.1M, result as shown in Figure 3.Wherein, a is C
60-ImAn-ZnTPP electrogenerated chemiluminescence body, b is ZnTPP, c is C
60(COOH)
3, interior illustration is the ECL light intensity time stability collection of illustrative plates of corresponding detection sample.As seen from the figure, C
60-ImAn-ZnTPP and ZnTPP has obvious ECL to respond between-1.80V ~-2.0V, and C
60(COOH)
3respond without ECL within the scope of same potential.During interior list of illustrations bright corresponding pattern detection, ECL light intensity time variations has good stability.Can think that this electrogenerated chemiluminescence body has good electrogenerated chemiluminescence effect thus, can be used as excellent signal mark.
Embodiment 6
The orderly self assembly in ECL interface is implemented with beta-schardinger dextrin-bag
(1) dry up after glass-carbon electrode being carried out polishing, cleaning;
(2) the water-soluble drop 20 μ L being contained 2wt.% diallyl dimethyl ammoniumchloride is coated in pretreated glassy carbon electrode surface, dries in 60 DEG C of blowing-type drying boxes;
(3) electrode surface obtained in step 2 drips 20 μ L nm of gold (AuNPs) solution, dries in 60 DEG C of blowing-type drying boxes;
(4) the saturated methanol solution 20 μ L being contained 2-hexyl mercapto group-beta-cyclodextrin (main body) drips the electrode surface being coated in step 3 and obtaining, and 1h is reacted under room temperature, after reaction terminates, be phosphate buffered solution and the abundant drip washing electrode surface of ethanol solution 3 ~ 4 times of 7.4 by 10mM, pH value;
(5) electrode surface obtained in step 4 drips painting 10 μ L containing 1mMC
60the ethanolic solution of-ImAn-ZnTPP (object), room temperature reaction is to realize Subjective and Objective envelope 1h, after reaction terminates, be phosphate buffered solution and the abundant drip washing electrode surface of ethanol solution 3 ~ 4 times of 7.4 by 10mMpH value, in blank group, do not add C
60the ethanolic solution of-ImAn-ZnTPP;
(6) electrode obtained with step 5 is for working electrode, and Ag/AgCl is contrast electrode, and platinum electrode is to electrode, and adjustment current potential is-2 ~ 0V, and sweep velocity is 100mVS
-1, scan in dielectric DCM solution using 0.1MTBAP at 4mL, detect ECL signal.
Testing result as shown in Figure 4.PDDA+AuNPs+ β-CDSH+C
60-ImAn-ZnTPP (Fig. 4, a) with C
60(Fig. 3, the identical-1.90V ~-2.0V of luminous current potential a) has obvious ECL Intensity response to-ImAn-ZnTPP, and PDDA+AuNPs+ β-CDSH is without ECL Intensity response (Fig. 4, b), contrasts with this, can prove that β-CDSH can wrap by C
60the latter is also fixed on electrode surface by-ImAn-ZnTPP, successfully builds ECL system.
Embodiment 7
C
60the application of-ImAn-ZnTPP luminophor in DNA detects, capture dna can design according to DNA to be measured, and embodiment is detected as example with H7N9 viral DNA.
(1) dry up after glass-carbon electrode being carried out polishing, cleaning;
(2) the water-soluble drop that 20 μ L gather (diallyldimethylammonium chloride) containing 2wt.% is coated in pretreated glassy carbon electrode surface, dries in 60 DEG C of blowing-type drying boxes;
(3) drip 20 μ LAuNPs to electrode surface, dry in 60 DEG C of blowing-type drying boxes;
(4) by three (2-carboxy ethyl) phosphine (Tris (2-CarboxyEthyl) Phosphine of 10 μ L containing 1 μM of capture dna (HA1RStem-LoopCaptureProbe (cDNA)), TCEP) solution modifies above-mentioned electrode, and 3h is left standstill in 37 DEG C of incubation casees, spend the night under saturated damp condition with being placed in 4 DEG C of moistening refrigerators, wherein, the sequence of cDNA is 5'-HOOC-GGAAGAGGCCTATTTTAAGTCGACGCCTCTTCCAAAAA-SH-3', the electrodes selective obtained is for the target dna (HA1RTarget (tDNA)) in Acquisition Detection system, its sequence is 5'-GTCGACTTAAAATAGGCCTCTTCC-3',
(5) be the PBS drip washing electrode of 7.4 by appropriate 10mM, pH value after electrode step 4 obtained takes out, remove loose cDNA, then 20 μ L are dripped containing 2wt.% bovine serum albumin(BSA) (BovineSerumAlbumin, BSA) aqueous solution with closed avtive spot in case non-specific adsorption, and be placed in 37 DEG C of incubation case 1h, with method drip washing after taking-up;
(6) with the electrode that the TCEP solution modification step 5 that 10 μ L contain variable concentrations tDNA (100nM, 10nM, 1nM, 0.1nM, 0.01nM, 0.001nM) obtains, be placed in 37 DEG C of incubators and react 1h; Reaction terminates rear same method drip washing.
(7) 2mL deionized water dissolving 15.3mgEDC and 2.3mgNHS is used respectively, mixed solution is mixed to get again with the saturated methanol solution equal-volume of 2-hexane diamine group-beta-cyclodextrin, the electrode surface that step 6 obtains is coated in the above-mentioned mixing drop of 20 μ L, in room temperature generation amidation process 1h, and rinse electrode surface 3 ~ 4 times to remove the reagent of non-specific binding with the PBS of 10mM, pH7.4;
(8) 0.5mMC is contained with 10 μ L
60β-the CDNH that what the ethanolic solution of-ImAn-ZnTPP (object) and step 7 obtained be fixed on electrode interface
2(main body) carries out Subjective and Objective envelope, after room temperature reaction 1h, uses ethanolic solution to rinse electrode surface 3 ~ 4 times to remove the C of non-specific binding
60-ImAn-ZnTPP, obtains novel electroluminescent chemical luminophor based on ZnTPP for detecting the bio-sensing system of H7N9 avian influenza genes;
(9) electrode obtained with step 8 is for working electrode, and Ag/AgCl is contrast electrode, and platinum electrode is to electrode, and adjustment current potential is-2.2 ~ 0V, and sweep velocity is 100mVS
-1, in dielectric DCM solution, carrying out ECL detection using 0.1MTBAP, result as shown in Figure 5.
As can be seen from the figure, along with the reduction of target DNA concentration, the corresponding reduction of electrogenerated chemiluminescence intensity, interior cut line concentration and luminous intensity linear.And only using dissolved oxygen DO as under the condition of coreagent, this detection system Monitoring lower-cut reaches 10
-12mol/L, describes the practicality that this sensor is good.
Embodiment 8
(1) dry up after glass-carbon electrode being carried out polishing, cleaning;
(2) the water-soluble drop that 20 μ L gather (diallyldimethylammonium chloride) containing 2wt.% is coated in pretreated glassy carbon electrode surface, dries in 60 DEG C of blowing-type drying boxes;
(3) drip 20 μ LAuNPs to electrode surface, dry in 60 DEG C of blowing-type drying boxes;
(4) by three (2-carboxy ethyl) phosphine (Tris (2-CarboxyEthyl) Phosphine of 10 μ L containing 10 μMs of capture dnas (HA1RStem-LoopCaptureProbe (cDNA)), TCEP) solution modifies above-mentioned electrode, and 3h is left standstill in 37 DEG C of incubation casees, spend the night under saturated damp condition with being placed in 4 DEG C of moistening refrigerators, wherein, the sequence of cDNA is 5'-HOOC-GGAAGAGGCCTATTTTAAGTCGACGCCTCTTCCAAAAA-SH-3', the electrodes selective obtained is for the target dna (HA1RTarget (tDNA)) in Acquisition Detection system, its sequence is 5'-GTCGACTTAAAATAGGCCTCTTCC-3',
(5) be the PBS drip washing electrode of 7.4 by appropriate 10mM, pH value after electrode step 4 obtained takes out, remove loose cDNA, then 20 μ L are dripped containing 2wt.% bovine serum albumin(BSA) (BovineSerumAlbumin, BSA) aqueous solution with closed avtive spot in case non-specific adsorption, and be placed in 37 DEG C of incubation case 1h, with method drip washing after taking-up;
(6) with the electrode that the TCEP solution modification step 5 that 10 μ L contain variable concentrations tDNA (100nM, 10nM, 1nM, 0.1nM, 0.01nM, 0.001nM) obtains, be placed in 37 DEG C of incubators and react 1h; Reaction terminates rear same method drip washing.
(7) 2mL deionized water dissolving 15.3mgEDC and 2.3mgNHS is used respectively, mixed solution is mixed to get again with the saturated methanol solution equal-volume of 2-hexane diamine group-beta-cyclodextrin, the electrode surface that step 6 obtains is coated in the above-mentioned mixing drop of 20 μ L, in room temperature generation amidation process 1h, and rinse electrode surface 3 ~ 4 times to remove the reagent of non-specific binding with the PBS of 10mM, pH7.4;
(8) 1mMC is contained with 10 μ L
60β-the CDNH that what the ethanolic solution of-ImAn-ZnTPP (object) and step 7 obtained be fixed on electrode interface
2(main body) carries out Subjective and Objective envelope, after room temperature reaction 1h, uses ethanolic solution to rinse electrode surface 3 ~ 4 times to remove the C of non-specific binding
60-ImAn-ZnTPP, obtains novel electroluminescent chemical luminophor based on ZnTPP for detecting the bio-sensing system of H7N9 avian influenza genes;
(9) electrode obtained with step 8 is for working electrode, and Ag/AgCl is contrast electrode, and platinum electrode is to electrode, and adjustment current potential is-2.2 ~ 0V, and sweep velocity is 100mVS
-1, in dielectric DCM solution, carrying out ECL detection using 0.1MTBAP.
Embodiment 9
Interference is tested
The present embodiment selects concentration to be that the tDNA of 100nM is as test sample to be checked in step 6 as different from Example 5, as a control group respectively with the tDNA sequence of the single base mismatch of 100nM (1-Base), double alkali yl mispairing (2-Base) and three base mispairings (3-Base) simultaneously, other steps are identical with embodiment 5, carry out the contrast of electrogenerated chemiluminescence collection of illustrative plates, result as shown in Figure 6.As can be seen from the figure, based on C
60the DNA detection system of-ImAn-ZnTPP electrogenerated chemiluminescence body has higher detection sensitivity to target dna, and has good antijamming capability to interference DNA, also illustrate that this sensor has good practicality simultaneously.
Claims (8)
1. based on an electrogenerated chemiluminescence body for a Tetraploid rice, it is characterized in that, described luminophor is C
60-ImAn-ZnTPP, is made up of aniline imidazoles, football alkene pyrrolidine tricarboxylic acids and a Tetraploid rice, described aniline imidazoles bridge joint football alkene pyrrolidine tricarboxylic acids Tetraploid rice between axial coordination.
2. prepare a method for luminophor as claimed in claim 1, it is characterized in that, concrete steps are as follows:
(1) join in methylene chloride by football alkene pyrrolidine tricarboxylic acids, EDC, NHS and aniline imidazoles successively, isolated air stirring reacts completely, and reaction terminates rear dialysis treatment;
(2) Tetraploid rice between aniline imidazoles equimolar amounts is joined step 1 dialyse after solution in, sealing lucifuge, stirring reaction is complete, reaction terminates rear dialysis treatment, is drying to obtain with aniline imidazoles bridge joint football alkene pyrrolidine tricarboxylic acids and the electrogenerated chemiluminescence body of Tetraploid rice between axial coordination;
Described dialysis treatment is proceed in bag filter by reactant liquor after reaction terminates, and take methylene chloride as dislysate, changes every 2 ~ 3h, room temperature dialysis 3 ~ 4 days;
Wherein, in step 1, the molecular cut off of the bag filter of dialysis treatment is 1000, in step 2, and the molecular cut off of the bag filter of dialysis treatment is 2000.
3. prepare the method for luminophor as claimed in claim 2, it is characterized in that, in step 1, the mol ratio of described EDC and NHS is 4 ~ 1:1, and described football alkene pyrrolidine tricarboxylic acids and the mol ratio of aniline imidazoles are 1:3 ~ 6.
4. one kind as arbitrary in claim 1-3 as described in luminophor DNA detect in application.
5. apply as claimed in claim 4, it is characterized in that, described application comprises the steps:
(1) three (2-carboxy ethyl) the phosphine solution containing capture dna is dripped the electrode surface being coated in and being modified with diallyl dimethyl ammoniumchloride and nm of gold, be placed in 37 DEG C of incubations, with being placed on 4 DEG C, spending the night under saturated humidity condition;
(2) PBS drip washing electrode, removes loose capture dna, drips painting Bovine Serum Albumin in Aqueous Solution subsequently, closes avtive spot, and is placed in 37 DEG C of incubations, drip washing after taking out;
(3) three (2-carboxy ethyl) the phosphine solution containing target dna to be measured is dripped the electrode surface being coated in step 2 and obtaining, react at being placed in 37 DEG C, reacted rear drip washing electrode;
(4) prepare the mixed solution of EDC and NHS, then with 2-hexane diamine group-beta-cyclodextrin (β-CDNH
2) the mixing of saturated methanol solution equal-volume, obtain EDC/NHS/ β-CDNH
2mixed solution, then by EDC/NHS/ β-CDNH
2mixed solution drips the electrode surface being coated in above-mentioned steps 3 and obtaining, and reacts under room temperature;
(5) C is prepared
60the ethanolic solution of-ImAn-ZnTPP, subsequently by C
60the ethanolic solution of-ImAn-ZnTPP drips the electrode surface being coated in step 4 and obtaining, and reacts under room temperature;
(6) electrode obtained with step 5 is for working electrode, ECL signal is detected adopting cyclic voltammetry in dielectric DCM solution using tetrabutylammonium perchlorate, finally calculate the concentration of target dna according to the linear equation of target DNA concentration logarithm value and ECL signal intensity value, and then draw the content of target dna in liquid to be measured.
6. apply as claimed in claim 5, it is characterized in that, in step 1, the concentration range of described capture dna is 1 μM ~ 10 μMs.
7. apply as claimed in claim 5, it is characterized in that, in step 5, described C
60the concentration range of the ethanolic solution of-ImAn-ZnTPP is 0.5mM ~ 1mM.
8. apply as claimed in claim 5, it is characterized in that, in step 6, the method of described detection ECL signal is: the electrode obtained with step 5 is working electrode, and Ag/AgCl is contrast electrode, and platinum electrode is to electrode, adjustment current potential is-2.2 ~ 0V, and sweep velocity is 100mVS
-1, the concentration of the DCM solution of tetrabutylammonium perchlorate is 0.1M.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106248758A (en) * | 2016-09-30 | 2016-12-21 | 南京理工大学 | A kind of analysis method that DNA probe interacts with electrode surface |
| CN106442690A (en) * | 2016-09-30 | 2017-02-22 | 南京理工大学 | ECL detection method of unlabeled DNA based on porphyrin and DNA double helix groove mosaicism action |
| CN106977522A (en) * | 2017-04-14 | 2017-07-25 | 南京理工大学 | A kind of preparation method of the electrogenerated chemiluminescence material based on zinc protoporphyrin |
| CN107286193A (en) * | 2017-01-20 | 2017-10-24 | 南京理工大学 | (3,4,5 3 (dodecyloxy) benzyl) triphenyl Si Fu Peng Suan Phosphonium and its application in electrogenerated chemiluminescence |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062330A (en) * | 2014-06-11 | 2014-09-24 | 汕头大学 | Electrochemical luminous biosensor for detecting thrombin and manufacture method of biosensor |
| CN104698056A (en) * | 2015-03-11 | 2015-06-10 | 南京理工大学 | Ion selectivity electrode based on ZnPPIX electrogenerated chemiluminescence and application of electrode |
-
2015
- 2015-08-14 CN CN201510501355.3A patent/CN105044184B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062330A (en) * | 2014-06-11 | 2014-09-24 | 汕头大学 | Electrochemical luminous biosensor for detecting thrombin and manufacture method of biosensor |
| CN104698056A (en) * | 2015-03-11 | 2015-06-10 | 南京理工大学 | Ion selectivity electrode based on ZnPPIX electrogenerated chemiluminescence and application of electrode |
Non-Patent Citations (3)
| Title |
|---|
| SUNISH K. SUGUNAN等: "Photophysics of Untethered ZnTPP-Fullerene Complexes in Solution", 《THE JOURNAL OF PHYSICAL CHEMISTRY A》 * |
| TAKANE IMAOKA等: "Enhancing the Photoelectric Effect with a Potential-Programmed Molecular Rectifier", 《J. AM. CHEM. SOC.》 * |
| WENWEN TU等: "Noncovalent nanoassembly of porphyrin on single-walled carbon nanotubes for electrocatalytic reduction of nitric oxide and oxygen", 《ELECTROCHEMISTRY COMMUNICATIONS》 * |
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