CN103399005B - Interact based on carboxylated carbon nano-particles and DNA and measure the method for lysozyme - Google Patents
Interact based on carboxylated carbon nano-particles and DNA and measure the method for lysozyme Download PDFInfo
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Abstract
A detection method for lysozyme content, belongs to chemiluminescence aptamer sensor technical field, for the detection of lysozyme content in cell and biological sample.Utilize carboxylated carbon nano-particles to modify amination magnetic bead, obtain carbon nano-particles and modify magnetic bead; Modify lysozyme is fit with chemical illuminating reagent ABEI, preparation mark is fit.Under lysozyme exists, mark and to be fitly combined with lysozyme, then the solution after lysozyme-mark fit effect is modified magnetic bead with carbon nano-particles mix, after magnetic resolution, supernatant is at H
2o
2and Co
2+produce chemiluminescence under existence, thus achieve the detection of lysozyme.The inventive method is simple, cost is low.
Description
Technical field
The invention belongs to the method field measuring lysozyme, be specially a kind of interaction based on carboxylated carbon nano-particles and DNA and measure the method for lysozyme.
Background technology
Lysozyme (Lysozyme, Lyz) is also called kytoplasm enzyme (Muramidase) or N-acetyl murein polysaccharide hydrolase (N-axetylmuramide glycanhydrolase).Because the cell membrane that lysozyme can be hydrolyzed gram-positive bacteria blocks the effect that murein polysaccharide reaches destruction cell membrane, therefore there is the effect of sterilization.Lysozyme is requisite in animals and humans immune system, and in different tissues, its content is different, and when disease occurs Yu under normal circumstances, content is different.In tissue, lysozyme content reduces and easily causes numerous disease.Therefore lysozyme content detects and is significant.
The classic method sensitivity of current mensuration lysozyme is not high, complicated operation, is not easy satisfied actual testing requirement.Fit (aptamers, aptamer) is that technology screening out for the phyletic evolution (Systematic Evolution of Ligands by Exponential Enrichment, SELEX) of utilization index enrichment aglucon.It can carry out the combination of high affinity, high specific with target protein etc., and its specificity can compare favourably with antigen-antibody.Simultaneously fit stable, cheap, easily synthesis, renewable, thus one of powerful becoming protein analysis in recent years.Utilize fit identification to carry out lysozyme detection method at present to mainly contain enzyme and cut [the Xu Hun such as signal amplification technique, electrochemiluminescence aptamer technologies, DNA enzymatic technology, 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 the testing requirement to lysozyme in various degree, but the sensitivity of method are not high, complicated operation.So must develop 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 detecting the chemiluminescence aptamer sensor of lysozyme, and a kind of method adopting described chemiluminescence sensor to detect lysozyme is provided.
The present invention is realized by following measures: based on carboxylated carbon nano-particles and DNA interaction chemiluminescence aptamer sensor, it is characterized in that comprising the following steps:
(1) utilize glutaraldehyde method to be cross-linked by amidized lysozyme DNA aptamer apt and chemical illuminating reagent N-(4-ammonia butyl) the different luminol of-N-ethyl (ABEI), prepare mark fit (ABEI-apt);
(2) carboxylated carbon nano-particles (cCNP) is prepared with microwave process for synthesizing;
(3) utilize amino carboxylated carbon nano-particles and amination magnetic bead are carried out be cross-linked with the effect of carboxyl prepare carbon nano-particles and modify magnetic bead (cCNP-MB);
(4) utilization mark is fit modifies magnetic bead formation chemiluminescence aptamer sensor with carbon nano-particles.
The chemiluminescence aptamer sensor set up that utilizes of the present invention detects lysozyme method, it is characterized in that:
Mg is added in ABEI-apt solution
2+ion, then adds 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 portable injection chemiluminescence method to carry out chemiluminescence detection, take lysozyme concentration as horizontal ordinate, chemiluminescence signal intensity is ordinate, drawing standard curve; Measure the chemiluminescence signal intensity of unknown sample, obtain the content of lysozyme in sample according to typical curve, thus realize carrying out quantitative measurement to lysozyme.
Described portable injection chemiluminescence stream as shown in Figure 1.A: sample/blank solution (after magnetic resolution supernatant); B: current-carrying-buffer solution; C:CoCl
2solution; D:H
2o
2; V: introduction valve; F: chemiluminescence flow cell; PMT: photomultiplier; BPCL: chemiluminescent analyzer; PC: computing machine.
Chemiluminescence aptamer sensor and the assay method of the present invention's development have following features:
By the Competition between electrostatic repulsion and pi-pi accumulation effect, utilization mark is fit and carboxylated carbon nano-particles non-covalent bond self assembly principle establishes a kind of chemiluminescence aptamer sensor, utilize lysozyme to cause in solution the change marking aptamer concentrations, thus utilize chemiluminescence to realize lysozyme assay.
Only need a DNA chain just to establish the chemiluminescence aptamer sensor of lysozyme, this design have simply, advantage fast.In addition, experimentally chemiluminescence signal (Fig. 5) can be found out, when sensor of the present invention is for detecting fibrin ferment, ATP, BSA, codeine, cocaine and immunoglobulin G, its luminous intensity is well below the chemiluminescence signal detecting lysozyme, and this illustrates that this sensor has very high selectivity to detect lysozyme.Therefore, the chemiluminescence sensor that the present invention relates to embodies good development prospect in the research method building detection protein.
Accompanying drawing explanation
Fig. 1 is portable injection chemiluminescence flow circuit diagram of the present invention.
Fig. 2 is specific experiment schematic diagram of the present invention.
Fig. 3 is lysozyme and marks fit binding time (Time) to chemiluminescence signal (Δ I
cL) affect situation map.
Fig. 4 is chemiluminescence signal and lysozyme concentration (C
lyz) typical curve.
Fig. 5 is the testing result to sample (Sample) lysozyme (a), fibrin ferment (b), ATP (c), BSA (d), 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 describing further, but protection scope of the present invention be not limited to these embodiments.All do not deviate from the present invention's design change or equivalent substituting include within protection scope of the present invention.
A kind of chemiluminescence sensor detecting lysozyme that the present invention relates to, modifies amination magnetic bead (carbon nano-particles modification magnetic bead), with it for capturing carrier by carboxylated carbon nano-particles; Modify lysozyme is fit with chemical illuminating reagent ABEI, preparation mark is fit.Under the existence of lysozyme, mark and to be fitly combined with lysozyme, obtain lysozyme-mark aptamer solutions; Again lysozyme-mark aptamer solutions and carbon nano-particles are modified magnetic bead effect.The mark do not reacted with lysozyme is fit, and by the Competition between electrostatic repulsion and pi-pi accumulation effect, be adsorbed on carbon nano-particles and modify magnetic bead surfaces, recycling magnetic separation technique, isolates solution, the lysozyme of only residue reaction in solution-mark fit; This lysozyme-mark fit at hydrogen peroxide, Co
2+produce chemiluminescence (h υ) under existence, realize the detection of lysozyme with this.
The present invention is realized by following measures: a kind of preparation method detecting the chemiluminescence sensor of lysozyme, comprises the following steps:
(1) preparation of ABEI-apt:
The preparation of described ABEI-apt, preferably, utilizes glutaraldehyde as cross linker that amidized DNA aptamer (apt) is carried out reaction with ABEI and has prepared ABEI-apt.Detailed process is as follows:
(a) 20 μ L 10
-6~ 10
-4the amidized DNA aptamer of M adds in the phosphate buffer solution of 5% glutaraldehyde, Keep agitation 2h;
B () adds 1 ~ 10mg ABEI solution again, at room temperature stir and spend the night, to obtain final product.
(2) preparation of carboxylated carbon nano-particle:
The preparation of described carboxylated carbon nano-particles, preferably, detailed process is as follows:
Prepared by (a) carbon nano-particles:
Preferably, 0.1 ~ 1.0g shitosan is dissolved in 10mL water, puts into micro-wave oven (900W) and heat 5 ~ 15 minutes (min) and change to solution colour.Above-mentioned solution is removed impurity with the centrifugal 30min of 13000rpm, then uses bag filter (molecular cut off 3000 ~ 10000) to dialyse to it in pure water, continue to remove remaining shitosan in 2 ~ 6 days, obtain carbon nano-particles.
B () utilizes carbon nano-particles to prepare carboxylated carbon nano-particles:
Preferably, under 100 degrees Celsius, 8 hours are reacted after the nitric acid of 0.5 ~ 2 gram of carbon nano-particles and 1.0mL 63% and 1.0mL dimethyl formamide being mixed.Then, filtered by product utilization 0.22 μM of ultra filtration membrane, by centrifugal for filtrate 10min (under 14000rmp condition), and with second distillation water washing three times, being then dispersed in redistilled water and being prepared into concentration is 2.0mg/mL solution, to obtain final product.
(3) carbon nano-particles modifies the preparation of magnetic bead:
Described carbon nano-particles modifies the preparation of magnetic bead, and detailed process is as follows:
Preferably, the imidazole buffer (pH is 6.8) of the carboxylated carbon nano-particles of 10 ~ 150 μ L and 2000 μ L 0.1M is added in little centrifuge tube, react 30min at 37 DEG C, then add 1000 μ L and continue reaction 12h containing EDC, NHS of 0.1M and 50 μ L amination magnetic beads.Then wash three times with phosphate buffer solution, be settled to 500 μ L.Obtained carbon nano-particles modifies magnetic bead.
Utilize DNA recognition reaction, Competition between electrostatic repulsion and pi-pi accumulation effect, build chemiluminescence aptamer sensor, principle as shown in Figure 2.
The detection of lysozyme:
Measure lysozyme with described chemiluminescence aptamer sensor, detailed process is as follows:
Preferably, in ABEI-apt solution, add the Mg of 10.0mM
2+ion, then adds the lysozyme of variable concentrations, 37 DEG C of isothermal reaction 15min.Get 1 ~ 5 this solution of μ L, add carbon nano-particles and to modify in magnetic bead solution after 58 DEG C of reaction 15min, get supernatant and carry out chemiluminescence detection.
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, chemiluminescence intensity is maximum.So it is top condition that experiment selects pH to be 11.5.
(2) H of detection system
2o
2concentration.Preferably, H is worked as
2o
2time concentration is 1.5mM, chemiluminescence intensity is maximum.So H is selected in experiment
2o
2concentration is 1.5mM is top condition.
(3) Co of detection system
2+concentration.Preferably, Co is worked as
2+when concentration is 0.1mM, chemiluminescence intensity is maximum.So Co is selected in experiment
2+concentration is 0.1mM is top condition.
Lysozyme with mark fit action time.Specific features is as follows:
Preferably, within the scope of 1 ~ 15min, the increase of chemiluminescence signal intensity lysozyme action time and strengthening, until reach maximum value during 15min, after being greater than 15min, change is little.Therefore experiment determines that the Best Times combined is 15min.
Embodiment: interact based on carboxylated carbon nano-particles and DNA and measure the method for lysozyme.
1. experimental section
1.1 instruments and reagent
IFFM-E type Flow Injection Analysis/Chemiluminescence instrument (Xi'an Rui Mai Analytical Instrument Co., Ltd); Lmax II/II384 chemiluminescent analyzer (MD company of the U.S.); THZ-82A gas bath constant temperature oscillator (Medical Instruments factory of Quan Tan city).
Chemical illuminating reagent (ABEI) is purchased from Aladdin Reagent Company; Amination magnetic bead (preferred, diameter 2 ~ 3 μm) doubly thinks 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 the equal available from Sigma of immunoglobulin G; N-hydroxy-succinamide (NHS), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) is 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 aptamer (apt) is carried out reaction with ABEI and prepare ABEI-apt.Detailed process is as follows: (1) 20 μ L 10
-4the amidized DNA aptamer of M adds in the phosphate buffer solution of 5% glutaraldehyde, Keep agitation 2h; (2) add 10mg ABEI solution again, at room temperature stir and spend the night.
1.2.2 the preparation of carboxylated carbon nano-particles:
(1) carbon nano-particles preparation:
0.5g shitosan is dissolved in 10mL water, puts into micro-wave oven (900W) and heat 9.5min and change to solution colour.Above-mentioned solution is removed impurity with the centrifugal 30min of 13000rpm, then uses bag filter (molecular cut off 8000) to dialyse to it in pure water, continue to remove remaining shitosan in 4 days, obtain carbon nano-particles.
(2) carbon nano-particles is utilized to prepare carboxylated carbon nano-particles:
Under 100 degrees Celsius, 8 hours are reacted after the nitric acid of 2 grams of carbon nano-particles and 1.0mL 63% and 1.0mL dimethyl formamide being mixed.Then, filtered by product utilization 0.22 μM of ultra filtration membrane, by centrifugal for filtrate 10min (under 14000rmp condition), and with second distillation water washing three times, being then dispersed in redistilled water and being prepared into concentration is 2.0mg/mL solution.
1.2.3 carbon nano-particles modifies the preparation of magnetic bead:
In little centrifuge tube, add the imidazole buffer (pH is 6.8) of the carboxylated carbon nano-particles of 150 μ L and 2000 μ L 0.1M, at 37 DEG C, react 30min, then add 1000 μ L and continue reaction 12h containing EDC, NHS of 0.1M and 50 μ L amination magnetic beads.Then wash three times with phosphate buffer solution, be settled to 500 μ L.Obtained carbon nano-particles modifies magnetic bead.
The detection of lysozyme:
The Mg of 10.0mM is added in ABEI-apt solution
2+ion, then adds the lysozyme of variable concentrations, 37 DEG C of isothermal reaction 15min.Get 5 these solution of μ L, add carbon nano-particles and to modify in magnetic bead solution after 58 DEG C of reaction 15min, get supernatant and carry out chemiluminescence detection.
Chemiluminescence detection tests 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, another two conveying CoCl
2(Co
2+) and H
2o
2solution.Inject with six-way valve sample thief, each sampling volume is 100 μ L, and the sample injected mixes first intersection along with current-carrying, and then this potpourri is again at the 3rd intersection and CoCl
2+ H
2o
2in the mixing of flow cell leading portion, there is chemical reaction and produce chemiluminescence in mixed solution, utilizes photomultiplier to detect luminous signal, thus determine the concentration of surveyed solution according to the intensity of chemiluminescence signal, carry out the content of lysozyme in working sample with this.
1.3 results and discussion
(1) pH of detection system.When pH is 11.5, chemiluminescence intensity is maximum.So it is top condition that experiment selects pH to be 11.5.
(2) H of detection system
2o
2concentration.Work as H
2o
2time concentration is 1.5mM, chemiluminescence intensity is maximum.So H is selected in experiment
2o
2concentration is 1.5mM is top condition.
(3) Co of detection system
2+concentration.Work as Co
2+when concentration is 0.1mM, chemiluminescence intensity is maximum.So Co is selected in experiment
2+concentration is 0.1mM is top condition.
(4) lysozyme with mark fit action time.As shown in Figure 3, within the scope of 1 ~ 15min, the increase of chemiluminescence signal intensity lysozyme action time and strengthening, until reach maximum value during 15min, after being greater than 15min, change is little.Therefore experiment determines that the Best Times combined is 15min.
Under the preferred conditions, variable concentrations lysozyme becomes some linear with between chemiluminescence signal, obtains the typical curve, the range of linearity and the linear equation that detect lysozyme.When the concentration of lysozyme is 1.0 × 10
-10~ 1.0 × 10
-8time between M, the chemiluminescence signal of system increases along with the increase of lysozyme concentration (Fig. 4).Equation of linear regression is Δ I
cL=691.45C+78.2 (Δ I
cLfor the chemiluminescence signal of 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 good reappearance.
Select the comparison that fibrin ferment, ATP, BSA, codeine, cocaine and immunoglobulin G detect lysozyme as detection chemiluminescence aptamer sensor.As shown in Figure 5: be 2.0 × 10 by method detectable concentration
-9m lysozyme, lysozyme, codeine, immunoglobulin G and BSA, and 1.0 × 10
-5m ATP, cocaine, and chemiluminescence signal is compared.Can find out, the chemiluminescence signal detecting fibrin ferment, ATP, BSA, codeine, cocaine and immunoglobulin G, well below the chemiluminescence signal (Fig. 5) detecting lysozyme, illustrates that this chemiluminescence aptamer sensor has sufficiently high selectivity to survey lysozyme.
As can be seen from above-described embodiment, by the Competition between electrostatic repulsion and pi-pi accumulation effect, utilization mark is fit and carboxylated carbon nano-particles non-covalent bond self assembly principle constructs chemiluminescence aptamer sensor, establishes and detects lysozyme method.Utilize a DNA chain to realize lysozyme assay, method is simple.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; the change made under other any deviates from Spirit Essence of the present invention and principle, modification, replacement, combination, simplification; comparatively the substitute mode of equivalence, is included within protection scope of the present invention.
Claims (3)
1., based on a preparation method for carboxylated carbon nano-particles and DNA interaction chemiluminescence aptamer sensor, feature comprises the following steps:
1.1 ABEI-apt preparations, 20 μ L 10
-6~ 10
-4add in the phosphate buffer solution of 5% glutaraldehyde in the amidized DNA aptamer solution of M, Keep agitation 2h, then add 1 ~ 10mg chemical illuminating reagent ABEI, at room temperature stir and spend the night, to obtain final product;
1.2 carboxylated carbon fabrications, 0.1 ~ 1.0g shitosan is dissolved in 10mL water, put into microwave-oven-heating 5 ~ 15min to change to solution colour, solution is removed impurity with the centrifugal 30min of 13000rpm, it is dialysed in pure water with the bag filter that molecular cut off is 3000 ~ 10000 again, continue to remove remaining shitosan in 2 ~ 6 days, obtain carbon nano-particles, under 100 degrees Celsius, 8h is reacted after the nitric acid of the carbon nano-particles prepared by 0.5 ~ 2g and 1.0mL 63% and 1.0mL dimethyl formamide mix, then, product utilization 0.22 μm of ultra filtration membrane is filtered, by filtrate centrifugal 10min under 14000rmp condition, and with second distillation water washing three times, then being dispersed in redistilled water and being prepared into concentration is 2.0mg/mL solution,
1.3 carbon nano-particles modify magnetic bead preparation, the carboxylated carbon nano-particles of 10 ~ 150 μ L is added and 2000 μ L 0.1M pH are the imidazole buffer of 6.8 in little centrifuge tube, 30min is reacted at 37 DEG C, add EDC, NHS and 50 μ L amination magnetic beads continuation reaction 12h that 1000 μ L contain 0.1M again, then three times are washed with phosphate buffer solution, be settled to 500 μ L, thus obtained chemiluminescence aptamer sensor.
2. the method detecting lysozyme content based on carboxylated carbon nano-particles and DNA interaction chemiluminescence aptamer sensor utilizing the preparation method described in claim 1 to prepare, is characterized in that: in ABEI-apt solution, add Mg
2+ion, then adds the lysozyme of variable concentrations, incubation reaction; Get this solution of part, add carbon nano-particles and to modify in magnetic bead solution after incubation reaction, get supernatant after magnetic resolution and carry out chemiluminescence detection, obtain the quantitative information of lysozyme according to chemiluminescence signal.
3. the chemiluminescence aptamer sensor that preparation method according to claim 1 obtains is detecting the application in lysozyme content.
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