CN103399005A - Method for determining lysozyme based on interaction between carboxylation carbon nanoparticles and DNA (Deoxyribose Nucleic Acid) - Google Patents
Method for determining lysozyme based on interaction between carboxylation carbon nanoparticles and DNA (Deoxyribose Nucleic Acid) Download PDFInfo
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- 235000010335 lysozyme Nutrition 0.000 title claims abstract description 76
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- 239000011852 carbon nanoparticle Substances 0.000 title claims abstract description 48
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a lysozyme content detection method which is used for detecting lysozyme content in cells and biological samples and belongs to the technical field of a chemiluminiscence aptasensor. The lysozyme content detection method comprises the following steps of: modifying amination magnetic beads by using carboxylation carbon nanoparticles so as to obtain carbon nanoparticle modified magnetic beads; and modifying lysozyme aptamer by using a chemiluminiscence agent ABEI (3-aminophthalic acid cyclic hydrazide) so as to prepare a marked aptamer; combining the marked aptamer and the lysozyme in the presence of lysozyme, mixing a solution, which is subjected to the action of the lysozyme-marked aptamer, with the carbon nanoparticle modified magnetic beads, wherein liquid supernatant generates chemiluminiscence in the presence of H2O2 and Co<2+> after magnetic separation, thus realizing the detection of the lysozyme. The lysozyme content detection method is simple and low in cost.
Description
Technical field
The invention belongs to the fit sensor field of chemiluminescence, be specially a kind of method based on carboxylated carbon nano-particles and DNA interaction mensuration lysozyme.
Background technology
Lysozyme (Lysozyme, Lyz) is called again kytoplasm enzyme (Muramidase) or N-acetyl murein polysaccharide hydrolase (N-axetylmuramide glycanhydrolase).Because lysozyme can be hydrolyzed the cell membrane blocking-up murein polysaccharide of gram-positive bacteria, reach the effect of destroying cell membrane, therefore have the effect of sterilization.Lysozyme is requisite in the animals and humans immune system, and its content is different in different tissues, and content is different when disease occurs Yu under normal circumstances.In tissue, lysozyme content reduces and easily causes numerous disease.Therefore lysozyme content detects and is significant.
The classic method sensitivity of measuring at present lysozyme is not high, and complicated operation, be not easy to meet actual testing requirement.Technology screening out for the phyletic evolution that fit (aptamers, aptamer) is utilization index enrichment aglucon (Systematic Evolution of Ligands by Exponential Enrichment, SELEX).It can carry out the combination of high affinity, high specific with target protein etc., its specificity can compare favourably with antigen-antibody.Simultaneously fit stable, cheap, easily synthetic, renewable, thereby become in recent years one of powerful of protein analysis.Utilizing at present fit identification to carry out the lysozyme detection method mainly contains enzyme and cuts [the Xu Hun such as signal amplification technique, the fit technology of electrochemiluminescence, DNA zymotechnic; Huaicheng Chen; Wei Wang. Design of ultrasensitive chemiluminescence detection of lysozyme in cancer cells based on nicking endonuclease signal amplification technology. Biosensors and Bioelectronics; (2010), 26:248; Haiyan Wang; Wu Gong; Zhian Tan; Xunxun Yin; Lun Wang. Label-free bifunctional electrochemiluminescence aptasensor for detection of adenosine and lysozyme. Electrochimica Acta; (2012), 76:416; Aptamer DNAzyme hairpins for amplified biosensing. Carsten Teller, Simcha Shimron, Itamar Willner. Analytical Chemistry. (2009), 81 (21): 9114].These methods respectively have its advantage, can meet in various degree the testing requirement to lysozyme, but the sensitivity of method are not high, complicated operation.So necessary development is a kind of highly sensitive, simple novel detection method.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide a kind of preparation method who detects the fit sensor of chemiluminescence of lysozyme, and a kind of method that adopts described chemiluminescence sensor to detect lysozyme is provided.
The present invention realizes by following measures: based on carboxylated carbon nano-particles and the fit sensor of DNA interaction chemiluminescence, it is characterized in that comprising the following steps:
(1) utilize glutaraldehyde method that the fit apt of amidized bacteriolyze enzyme dna and chemical illuminating reagent N-(4-ammonia butyl)-different luminol of N-ethyl (ABEI) are carried out crosslinked, prepare mark fit (ABEI-apt);
(2) with microwave process for synthesizing, prepare carboxylated carbon nano-particles (cCNP);
(3) utilize amino effect with carboxyl that carboxylated carbon nano-particles and amination magnetic bead are carried out to the crosslinked carbon nano-particles modification magnetic bead (cCNP-MB) for preparing;
(4) utilize the fit and carbon nano-particles of mark to modify magnetic bead and form the fit sensor of chemiluminescence.
The fit sensor of the chemiluminescence that utilization of the present invention is set up detects the lysozyme method, it is characterized in that:
In ABEI-apt solution, add Mg
2+Ion, then add the lysozyme of variable concentrations, isothermal reaction.Get this solution of certain volume, add carbon nano-particles to modify in magnetic bead solution after incubation reaction, after magnetic resolution, supernatant utilizes the portable injection chemiluminescence method to carry out chemiluminescence detection, take lysozyme concentration as horizontal ordinate, chemiluminescence signal intensity is ordinate, the drawing standard curve; Measure the chemiluminescence signal intensity of unknown sample, according to typical curve, obtain the content of lysozyme in sample, thereby realize lysozyme is carried out to quantitative measurement.
Described portable injection chemiluminescence stream as shown in Figure 1.A: sample/blank solution (supernatant after magnetic resolution); B: current-carrying-buffer solution; C:CoCl
2Solution; D:H
2O
2V: introduction valve; F: chemiluminescence flow cell; PMT: photomultiplier; BPCL: chemiluminescent analyzer; PC: computing machine.
The fit sensor of chemiluminescence and the assay method of the present invention's development have following features:
By the Competition between electrostatic repulsion and pi-pi accumulation effect, utilize the fit and carboxylated carbon nano-particles non-covalent bond self assembly principle of mark to set up the fit sensor of a kind of chemiluminescence, utilize lysozyme to cause the variation of the fit concentration of mark in solution, thereby utilize chemiluminescence to realize lysozyme assay.
Only need the fit sensor of chemiluminescence that the DNA chain has just been set up lysozyme, this design has simply, advantage fast.In addition, according to experimental chemistry luminous signal (Fig. 5), can find out, when sensor of the present invention during for detection of fibrin ferment, ATP, BSA, codeine, cocaine and immunoglobulin G, its luminous intensity is well below the chemiluminescence signal that detects lysozyme, and this illustrates that this sensor has very high selectivity and detects lysozyme.Therefore, the chemiluminescence sensor that the present invention relates to embodies good development prospect in building the research method that detects protein.
The accompanying drawing explanation
Fig. 1 is portable injection chemiluminescence flow circuit diagram of the present invention.
Fig. 2 is concrete experimental principle figure of the present invention.
Fig. 3 is that lysozyme and the fit binding time of mark (Time) are to chemiluminescence signal (Δ I
CL) affect situation map.
Fig. 4 is chemiluminescence signal and lysozyme concentration (C
lyz) typical curve.
Fig. 5 is to sample (Sample) lysozyme (a), fibrin ferment (b), ATP(c), BSA(d), the testing result of codeine (e), cocaine (f) and immunoglobulin G (g).
Embodiment
Be below the specific embodiment that the present invention relates to, technical scheme of the present invention is done to further the description, but protection scope of the present invention is not limited to these embodiment.All do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
A kind of chemiluminescence sensor that detects lysozyme that the present invention relates to, modify amination magnetic bead (carbon nano-particles modification magnetic bead) by carboxylated carbon nano-particles, take it as capturing carrier; With chemical illuminating reagent ABEI, to lysozyme is fit, modify, prepare mark fit.Under the existence of lysozyme, mark is fit is combined with lysozyme, obtains the fit solution of lysozyme-mark; Again the fit solution of lysozyme-mark and carbon nano-particles are modified to the magnetic bead effect.Not fit with the mark of lysozyme reaction, by the Competition between electrostatic repulsion and pi-pi accumulation effect, be adsorbed on carbon nano-particles and modify magnetic bead surfaces, the recycling magnetic separation technique, isolate solution, and in solution, only lysozyme-the mark of residue reaction is fit; This lysozyme-mark is fit at hydrogen peroxide, Co
2+There is lower generation chemiluminescence (h υ), with this, realize the detection of lysozyme.
The present invention realizes by following measures: a kind of preparation method who detects the chemiluminescence sensor of lysozyme comprises the following steps:
(1) preparation of ABEI-apt:
The preparation of described ABEI-apt, preferred, utilize glutaraldehyde as cross linker that amidized DNA fit (apt) and ABEI are reacted and prepared ABEI-apt.Detailed process is as follows:
(a) 20 μ L 10
-6~ 10
-4The amidized DNA of M is fit to add in the phosphate buffer solution of 5% glutaraldehyde, continues to stir 2 h;
(b) add again 1 ~ 10 mg ABEI solution, at room temperature stir and spend the night, obtain.
(2) preparation of carboxylated carbon nano-particle:
The preparation of described carboxylated carbon nano-particles, preferred, detailed process is as follows:
(a) carbon nano-particles preparation:
Preferably, 0.1 ~ 1.0 g shitosan is dissolved in 10 mL water, puts into micro-wave oven (900 W) heating 5 ~ 15 minutes (min) to solution colour and change.Above-mentioned solution is removed to impurity with centrifugal 30 min of 13000 rpm, then use bag filter (molecular cut off 3000 ~ 10000) in pure water to its dialysis, continue to remove remaining shitosan in 2 ~ 6 days, obtain carbon nano-particles.
(b) utilize carbon nano-particles to prepare carboxylated carbon nano-particles:
Preferably, the nitric acid of 0.5 ~ 2 gram carbon nano-particles and 1.0 mL 63% and 1.0 mL dimethyl formamides are mixed after reaction 8 hours under 100 degrees centigrade.Then, product utilization 0.22 μ M ultra filtration membrane is filtered, by under the centrifugal 10 min(14000 rmp conditions of filtrate), and with second distillation water washing three times, then being dispersed in redistilled water and being prepared into concentration is 2.0 mg/mL solution, obtains.
(3) carbon nano-particles is modified the preparation of magnetic bead:
Described carbon nano-particles is modified the preparation of magnetic bead, and detailed process is as follows:
Preferably, the imidazole buffer (pH is 6.8) that adds the carboxylated carbon nano-particles of 10 ~ 150 μ L and 2000 μ L 0.1 M in little centrifuge tube, reaction 30 min under 37 ℃, then EDC, the NHS and the 50 μ L amination magnetic beads that add 1000 μ L to contain 0.1 M continue reaction 12 h.Then with phosphate buffer solution, wash three times, be settled to 500 μ L.Make carbon nano-particles and modify magnetic bead.
Utilize the Competition between DNA recognition reaction, electrostatic repulsion and pi-pi accumulation effect, build the fit sensor of chemiluminescence, principle as shown in Figure 2.
The detection of lysozyme:
With the fit sensor of described chemiluminescence, measure lysozyme, detailed process is as follows:
Preferably, the Mg that adds 10.0 mM in ABEI-apt solution
2+Ion, then add the lysozyme of variable concentrations, 37 ℃ of isothermal reaction 15 min.Get 1 ~ 5 this solution of μ L, after adding carbon nano-particles to modify in magnetic bead solution 58 ℃ of reaction 15 min, get supernatant and carry out chemiluminescence detection.
The described supernatant of getting carries out chemiluminescence flow circuit diagram that chemiluminescence detection adopts as shown in Figure 1.
Described chemiluminescence detection chemiluminescence experiment condition, specific features is as follows:
(1) pH of detection system.Preferably, when pH is 11.5, the chemiluminescence intensity maximum.So it is 11.5 for top condition that pH is selected in experiment.
(2) H of detection system
2O
2Concentration.Preferably, work as H
2O
2When concentration is 1.5 mM, the chemiluminescence intensity maximum.So H is selected in experiment
2O
2Concentration is that 1.5 mM are top condition.
(3) Co of detection system
2+ concentration.Preferably, work as Co
2+When concentration is 0.1 mM, the chemiluminescence intensity maximum.So Co is selected in experiment
2+Concentration is that 0.1 mM is top condition.
Lysozyme and mark fit action time.Specific features is as follows:
Preferably, in 1～15 min scope, the increase of chemiluminescence signal intensity lysozyme action time and strengthening, until reach maximum value during 15 min, little greater than after 15 min, changing.Therefore experiment determines that the Best Times of combination is 15 min.
Embodiment: based on the method for carboxylated carbon nano-particles and DNA interaction mensuration lysozyme.
1. experimental section
1.1 instrument and reagent
IFFM-E type Flow Injection Analysis/Chemiluminescence instrument (Xi'an Rui Mai Analytical Instrument Co., Ltd); Lmax II/II384 chemiluminescent analyzer (U.S. MD company); THZ-82A gas bath constant temperature oscillator (Quan Tan city Medical Instruments factory).
Chemical illuminating reagent (ABEI) is purchased from Aladdin reagent company; Amination magnetic bead (preferred, diameter 2～3 μ m) is doubly thought happy chromatographic technique development centre purchased from Tianjin; Cocaine, codeine are purchased from Nat'l Pharmaceutical & Biological Products Control Institute; Lysozyme (Lysozyme, Lyz), fibrin ferment, atriphos (ATP), bovine serum albumin(BSA) (BSA) and immunoglobulin G are all purchased from Sigma company; N-hydroxy-succinamide (NHS), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimides (EDC) are purchased from Acros(New Jersey, USA).
DNA artificial sequence synthetic used (purchased from match Parkson, Beijing bioengineering company limited) is as follows.
The partial sequence of described DNA is:
5’-NH
2-（CH
2）
6-ATCTACGAATTCATCAGGGCTAAAGAGTGCAGAGTTCTTAG-3’（apt）（SEQ?ID?NO：1）。
1.2 experimental procedure
1.2.1ABEI-apt preparation:
Utilize glutaraldehyde as cross linker that amidized DNA fit (apt) and ABEI are reacted and prepared
ABEI-apt.Detailed process is as follows: (1) 20 μ L 10
-4The amidized DNA of M is fit to add in the phosphate buffer solution of 5% glutaraldehyde, continues to stir 2 h; (2) add again 10 mg ABEI solution, at room temperature stir and spend the night.
1.2.2 the preparation of carboxylated carbon nano-particles:
(1) carbon nano-particles preparation:
0.5 g shitosan is dissolved in 10 mL water, puts into micro-wave oven (900 W) heating 9.5 min and change to solution colour.Above-mentioned solution is removed to impurity with the centrifugal 30min of 13000 rpm, then use bag filter (molecular cut off 8000) in pure water to its dialysis, continue to remove remaining shitosan in 4 days, obtain carbon nano-particles.
(2) utilize carbon nano-particles to prepare carboxylated carbon nano-particles:
Reaction 8 hours under 100 degrees centigrade after the nitric acid of 2 gram carbon nano-particles and 1.0 mL 63% and 1.0 mL dimethyl formamides are mixed.Then, product utilization 0.22 μ M ultra filtration membrane is filtered, by under the centrifugal 10 min(14000 rmp conditions of filtrate), and with second distillation water washing three times, then being dispersed in redistilled water and being prepared into concentration is 2.0 mg/mL solution.
1.2.3 carbon nano-particles is modified the preparation of magnetic bead:
The imidazole buffer (pH is 6.8) that adds the carboxylated carbon nano-particles of 150 μ L and 2000 μ L 0.1 M in little centrifuge tube, reaction 30 min under 37 ℃, then EDC, the NHS and the 50 μ L amination magnetic beads that add 1000 μ L to contain 0.1 M continue reaction 12 h.Then with phosphate buffer solution, wash three times, be settled to 500 μ L.Make carbon nano-particles and modify magnetic bead.
The detection of lysozyme:
In ABEI-apt solution, add the Mg of 10.0 mM
2+Ion, then add the lysozyme of variable concentrations, 37 ℃ of isothermal reaction 15 min.Get 5 these solution of μ L, after adding carbon nano-particles to modify in magnetic bead solution 58 ℃ of reaction 15 min, get supernatant and carry out chemiluminescence detection.
Chemiluminescence detection is tested portable injection chemiluminescence stream used as shown in Figure 1.In four transfer pipelines: two are used for carrying sample and current-carrying-buffer solution, carry CoCl for another two
2(Co
2+) and H
2O
2Solution.With the six-way valve sample thief, inject, each sampling volume is 100 μ L, and the sample of injecting is along with current-carrying mixes in first intersection, and then this potpourri is again at the 3rd intersection and CoCl
2+ H
2O
2Mixed solution mixes at the flow cell leading portion, chemical reaction occurs and produce chemiluminescence, utilizes photomultiplier to detect luminous signal, thereby according to the intensity of chemiluminescence signal, determines the concentration of the solution of surveying, and with this, carrys out the content of lysozyme in working sample.
1.3 result and discussion
(1) pH of detection system.When pH is 11.5, the chemiluminescence intensity maximum.So it is 11.5 for top condition that pH is selected in experiment.
(2) H of detection system
2O
2Concentration.Work as H
2O
2When concentration is 1.5 mM, the chemiluminescence intensity maximum.So H is selected in experiment
2O
2Concentration is that 1.5 mM are top condition.
(3) Co of detection system
2+Concentration.Work as Co
2+When concentration is 0.1 mM, the chemiluminescence intensity maximum.So Co is selected in experiment
2+Concentration is that 0.1 mM is top condition.
(4) lysozyme and mark fit action time.As shown in Figure 3, in 1～15 min scope, the increase of chemiluminescence signal intensity lysozyme action time and strengthening, until reach maximum value during 15 min, little greater than after 15 min, changing.Therefore experiment determines that the Best Times of combination is 15 min.
Under preferred condition, between variable concentrations lysozyme and chemiluminescence signal, become some linear, obtained detecting typical curve, the range of linearity and the linear equation of lysozyme.When the concentration of lysozyme 1.0 * 10
-10～1.0 * 10
-8In the time of between M, the chemiluminescence signal of system increases (Fig. 4) along with the increase of lysozyme concentration.Equation of linear regression is Δ I
CL =691.45 C+ 78.2(Δ I
CLChemiluminescence signal for system; C is the concentration of lysozyme, 10
-9M; N=7, R=0.9989).The method detects and is limited to 3.0 * 10
-11M (3 σ).To concentration 2.0 * 10
-9The RSD that the lysozyme of M carries out 7 parallel replications is 3.7%, shows that this law has reappearance preferably.
Select fibrin ferment, ATP, BSA, codeine, cocaine and immunoglobulin G as detecting the comparison of the fit sensor of chemiluminescence to the lysozyme detection.As shown in Figure 5: by the method detectable concentration, be 2.0 * 10
-9M lysozyme, lysozyme, codeine, immunoglobulin G and BSA, and 1.0 * 10
-5M ATP, cocaine, and chemiluminescence signal is compared.Can find out, detect the chemiluminescence signal of fibrin ferment, ATP, BSA, codeine, cocaine and immunoglobulin G well below the chemiluminescence signal (Fig. 5) that detects lysozyme, illustrate that the fit sensor of this chemiluminescence has sufficiently high selectivity and surveys lysozyme.
From above-described embodiment, can find out, by the Competition between electrostatic repulsion and pi-pi accumulation effect, utilize the fit and carboxylated carbon nano-particles non-covalent bond self assembly principle of mark to build the fit sensor of chemiluminescence, set up detection lysozyme method.Utilize a DNA chain to realize lysozyme assay, method is simple.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any deviates from change, modification, replacement, combination, the simplification of making under Spirit Essence of the present invention and principle; comparatively the equivalence substitute mode, within being included in protection scope of the present invention.
Claims (3)
1. one kind interacts and measures the method for lysozyme based on carboxylated carbon nano-particles and DNA, it is characterized in that comprising the following steps:
1.1 the ABEI-apt preparation, at 20 μ L 10
-6~ 10
-4In the fit solution of amidized DNA of M, add in the phosphate buffer solution of 5% glutaraldehyde, continue to stir 2 h(hour), then add 1 ~ 10 mg chemical illuminating reagent (ABEI) solution, at room temperature stir and spend the night, transfer in brown bottle and preserve in the place, cool place;
1.2 carboxylated carbon fabrication, 0.1 ~ 1.0 g shitosan is dissolved in 10 mL water, putting into micro-wave oven (900 W) heating 5 ~ 15 minutes (min) to solution colour changes, solution is removed to impurity with centrifugal 30 min of 13000 rpm, use again bag filter (molecular cut off 3000 ~ 10000) in pure water to its dialysis, removed remaining shitosan in lasting 2 ~ 6 days, obtain carbon nano-particles, carbon nano-particles prepared by 0.5 ~ 2 g with after the nitric acid of 1.0 mL 63% and 1.0 mL dimethyl formamides mix under 100 degrees centigrade reaction 8 h, then, product utilization 0.22 μ M ultra filtration membrane is filtered, by under the centrifugal 10 min(14000 rmp conditions of filtrate), and with second distillation water washing three times, then being dispersed in redistilled water and being prepared into concentration is 2.0 mg/mL solution,
1.3 carbon nano-particles is modified the magnetic bead preparation, the imidazole buffer (pH is 6.8) that adds the carboxylated carbon nano-particles of 10 ~ 150 μ L and 2000 μ L 0.1 M in little centrifuge tube, reaction 30 min under 37 ℃, EDC, the NHS and the 50 μ L amination magnetic beads that add 1000 μ L to contain 0.1 M continue reaction 12 h again, then with phosphate buffer solution, wash three times, be settled to 500 μ L, thereby make the fit sensor of chemiluminescence.
2. the method based on carboxylated carbon nano-particles and the fit sensor detection of DNA interaction chemiluminescence lysozyme content of utilizing claim 1 preparation, is characterized in that: in ABEI-apt solution, add Mg
2+Ion, then add the lysozyme of variable concentrations, incubation reaction; Get this solution of part, add carbon nano-particles to modify in magnetic bead solution after incubation reaction, after magnetic resolution, supernatant carries out chemiluminescence detection, according to chemiluminescence signal, obtains the quantitative information of lysozyme.
3. the application of electrochemical luminous sensor according to claim 1 in detecting lysozyme content.
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