CN104297219A - A high-sensitivity triphosadenine detecting method - Google Patents
A high-sensitivity triphosadenine detecting method Download PDFInfo
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- CN104297219A CN104297219A CN201310297780.6A CN201310297780A CN104297219A CN 104297219 A CN104297219 A CN 104297219A CN 201310297780 A CN201310297780 A CN 201310297780A CN 104297219 A CN104297219 A CN 104297219A
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- atriphos
- particle
- atp
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Abstract
A high-sensitivity triphosadenine detecting method is disclosed. The method comprises following steps of: (1) preparing aminated magnetic nanometer particles; (2) preparing an immobilized triphosadenine detecting enzyme system; (3) pretreating the prepared triphosadenine detecting enzyme system, and removing residue triphosadenine in the system; and (4) detecting the triphosadenine by utilization of an MPI-B chemiluminescence detector. The triphosadenine residue in a reagent background is removed by utilization of a nanometer technology so as to reduce the luminescent value of the reagent itself, thus improving detection sensitivity of the reagent.
Description
Technical field
The present invention relates to a kind of detection method, particularly relate to a kind of highly sensitive atriphos detection method.
Background technology
ATP(atriphos) biloluminescence method is based on fire fly luminescence principle, utilizes luciferase-fluorescein system, detects the one method fast and effectively of atriphos (ATP) quantity.ATP bioluminescence ratio juris is that luciferase (luciferase) is under magnesium ion exists, catalysis luciferin (luciferin) and ATP form the fluorescein that intermediate rear oxidation becomes oxidized form, produce AMP(adenylic acid), and chemical energy is become luminous energy, discharge photon.
In certain ATP concentration range, this reacts the light intensity discharged and becomes good linear relationship with the ATP quantity existed in system.Because atriphos (ATP) is present in all active somatic cells, and the bacterium in each growth period is all also containing more constant ATP amount, therefore this can as a kind of indicator of active somatic cell, detect ATP quantity by ATP biloluminescence method, and extrapolate the quantity of bacterium in system thus.Compared with traditional plating method, this method can obtain result fast, and operator is without the need to technology, method is easy to be understandable, is more easily accepted by general population, and plating method required time is longer, usually need to cultivate 24-48 hour, just can obtain net result, and higher to the technical requirement of operator, complicated operation.
Along with research is goed deep into, existing result shows that ATP biloluminescence method has high sensitivity, can detect 10
-17molATP is even lower, is about equivalent to the ATP amount of five bacteriums, greatly meets the requirement that HACCP detects microorganism fast.In recent years, the method is adopted by fields such as food industry, pharmaceuticals industry and health surveillance system, as detected raw material milk microorganism and body cell concentration by ATP biloluminescence method, monitor the health status of micro organism quantity and research milk cow in raw material milk thus to the impact of milk.In the production run of this external food, to apparatus sanitation, environmental health, the management of personal hygiene, prevents the secondary pollution of product.In addition, this method also can be used for the mensuration of SDS in broiler chickens living contaminants, the determination of activity etc. of brewer's yeast.
ATP bioluminescence technique is quick based on it, sensitive, easy advantage, will become the effective ways of a kind of fast and convenient detection micro organism quantity and detection food processing environment cleanliness factor.
Because luciferase common on market and fluorescein are all by technique for gene engineering fermenting and producing gained, even if so through strict operation, may also can remain by the ATP containing trace in the enzyme liquid of standard, therefore cause the luminous intensity detecting enzyme liquid itself higher, the background value of reagent own is higher, is unfavorable for improving luminous detection sensitivity.In this case, most kit instructions requires after configuring detection enzyme liquid, and at room temperature standing a period of time remains with the ATP consumed wherein, but places at normal temperatures for a long time, this will certainly reduce the activity detecting enzyme, finally causes detection sensitivity to reduce.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of highly sensitive atriphos detection method, it utilizes nanometer technology to remove the remaining ATP(atriphos of reagent background), reduce the luminous value of reagent itself, thus improve the detection sensitivity of reagent.
The present invention solves above-mentioned technical matters by following technical proposals: a kind of highly sensitive atriphos detection method, and it is characterized in that, it comprises the following steps:
Step one, preparation amination magnetic nano-particle;
Step 2, preparation immobilization atriphos detects enzyme system;
Step 3, detects enzyme system to the atriphos configured and carries out pre-service, atriphos residual in scavenger enzyme system;
Step 4, uses MPI-B chemiluminescence detector to carry out atriphos mensuration.
Preferably, described step one specifically comprises the following steps: by 10g 1,6-hexane diamine, 5g anhydrous sodium acetate, 4g FeCl
36H
2o is as source of iron constant temperature 50 DEG C of vigorous stirring in 15ml ethylene glycol; After completion of the reaction, above-mentioned solution is transferred in Teflon reactor, at 200 DEG C, heat 10h; Treat that above-mentioned reaction terminates, to the magnetic nano-particle externally-applied magnetic field in reactor, incline the solution be not attracted by the magnetic force simultaneously, adds appropriate amount of deionized water to after magnetic nano-particle cleaning 2 ~ 3 times, with washes of absolute alcohol 2 ~ 3 times, the magnetic nano-particle of 25nmMNPs finally can be obtained.
Preferably, described step 2 specifically comprises the following steps: get 25% glutaraldehyde and mix with the ratio of 4:1 with amination magnetic nano-particle, 2 hours are hatched at constant temperature 30 DEG C, treat that glutaraldehyde and amination magnetic nano-particle are in conjunction with complete, by the PBS buffer solution for cleaning 2 to 3 times of pH7.4, add the PBS damping fluid of pH7.4, under 0 DEG C of ice bath, reaction makes triphosphoric acid bisphosphatase be combined with the magnetic nano-particle connecting carboxyl for 6 hours, concentration is 4U/mL, triphosphoric acid bisphosphatase is fixed on magnetic nano-particle, use washed with de-ionized water immobilised enzymes, wash away loose triphosphoric acid bisphosphatase, being fixed triphosphoric acid bisphosphatase.
Preferably, described step 3 specifically comprises the following steps: immobilization triphosphatase is added the luciferase-luciferin system configured, guarantee that immobilization triphosphoric acid bisphosphatase ratio is 6:7, isothermal vibration reaction 20min at 30 DEG C, now remaining in immobilization triphosphoric acid bisphosphatase decomposing solution in solution ATP generates AMP and Pi; After question response terminates, under constant temperature 4 DEG C of environment, externally-applied magnetic field, is separated immobilization apyrase in solution, pretreated ATP is detected enzyme system and slowly tilts to pour out, for subsequent detection.
Positive progressive effect of the present invention is: the present invention utilizes nanometer technology to remove the remaining ATP(atriphos of reagent background), reduce the luminous value of reagent itself, thus improve the detection sensitivity of reagent.
Accompanying drawing explanation
Fig. 1 does not use the pretreated ATP of immobilization triphosphoric acid bisphosphatase to detect reagent and use immobilization triphosphoric acid bisphosphatase pretreated ATP to detect the schematic diagram that reagent compares same group of standard A TP gradient solution testing result.
Fig. 2 does not use the pretreated ATP of immobilization triphosphoric acid bisphosphatase to detect reagent and use immobilization triphosphoric acid bisphosphatase pretreated ATP to detect the schematic diagram that reagent compares same group of Escherichia coli bacteria suspension testing result.
Fig. 3 does not use the pretreated ATP of immobilization triphosphoric acid bisphosphatase to detect reagent and use immobilization triphosphoric acid bisphosphatase pretreated ATP to detect the schematic diagram that reagent compares same group of miscellaneous bacteria bacteria suspension testing result.
Embodiment
Present pre-ferred embodiments is provided, to describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.
As shown in Figure 1, the highly sensitive atriphos detection method of the present invention comprises the following steps:
Step one, preparation amination magnetic nano-particle;
Step 2, preparation immobilization atriphos detects enzyme system;
Step 3, detects enzyme system to the atriphos configured and carries out pre-service, atriphos residual in scavenger enzyme system;
Step 4, uses MPI-B chemiluminescence detector to carry out atriphos mensuration.
Step one specifically comprises the following steps: by 10g 1,6-hexane diamine, 5g anhydrous sodium acetate, 4g FeCl
36H
2o is as source of iron constant temperature 50 DEG C of vigorous stirring in 15ml ethylene glycol; After completion of the reaction, above-mentioned solution is transferred in Teflon reactor, at 200 DEG C, heat 10h; Treat that above-mentioned reaction terminates, to the magnetic nano-particle externally-applied magnetic field in reactor, incline the solution be not attracted by the magnetic force simultaneously, adds appropriate amount of deionized water to after magnetic nano-particle cleaning 2 ~ 3 times, with washes of absolute alcohol 2 ~ 3 times, the magnetic nano-particle of 25nmMNPs finally can be obtained.
Step 2 specifically comprises the following steps: get 25% glutaraldehyde with amination magnetic nano-particle with 4:1(v/m) ratio mix, 2 hours are hatched at constant temperature 30 DEG C, treat that glutaraldehyde and amination magnetic nano-particle are in conjunction with complete, by the PBS buffer solution for cleaning 2 to 3 times of pH7.4, add the PBS damping fluid of pH7.4, under 0 DEG C of ice bath, reaction makes triphosphoric acid bisphosphatase be combined with the magnetic nano-particle connecting carboxyl for 6 hours, concentration is 4U/mL, triphosphoric acid bisphosphatase is fixed on magnetic nano-particle, use washed with de-ionized water immobilised enzymes, wash away loose triphosphoric acid bisphosphatase, being fixed triphosphoric acid bisphosphatase.
Step 3 specifically comprises the following steps: immobilization triphosphatase is added the luciferase-luciferin system configured, guarantee that immobilization triphosphoric acid bisphosphatase ratio is 6:7(m/v), isothermal vibration reaction 20min at 30 DEG C, now remaining in immobilization triphosphoric acid bisphosphatase decomposing solution in solution ATP generates AMP and Pi; After question response terminates, under constant temperature 4 DEG C of environment, externally-applied magnetic field, is separated immobilization apyrase in solution, pretreated ATP is detected enzyme system and slowly tilts to pour out, for subsequent detection.
Embodiment 1
Bioassay standard ATP gradient solution
Preparation 1 × 10
-2the ATP solution of mol/L, stores liquid as ATP.Use 25 mmol/L, pH7.4 Tris-HCl damping fluid (containing 1 mmol/L MgCl2) prepares reactant liquor (containing 1 × 10
-6mol/L luciferase and 3 × 10
-4mol/L luciferin), adopt immobilization triphosphoric acid bisphosphatase to carry out pre-service to reactant liquor, measuring ATP concentration is respectively 1 × 10
-7, 5 × 10
-8, 1 × 10
-8, 5 × 10
-9, 1 × 10
-9, 5 × 10
-10, 1 × 10
-10mol/L.Get the ATP solution of 10 μ L variable concentrations, add in 500 μ L reactant liquors, measure the luminous value of 60 s after reacting 15 s, the ATP of each concentration all repeats 3 times.
According to Fig. 1 display, conventionally, ATP is used to detect reagent analysis standard A TP gradient solution, when analyzing the ATP detecting low content, it is linearly not obvious, the ATP dynamic range of this curve at 10Fmol to 100Fmol, when ATP amount be less than 10Fmol time, luminous intensity is suitable with blank intensity, be not easily distinguishable, just because of the restriction of detectability and sensitivity, make ATP biloluminescence method for the higher occasion of some purity requirements, as hospital operating room, toilet etc. cannot use.And adopt the pretreated ATP of immobilization triphosphoric acid bisphosphatase to detect reagent, in the scope that ATP content is 0Fmol to 100Fmol, there is good linear relationship, R
2=0.9942.And there are higher slope K=4.7364 than untreated control group, even more the ATP of low content also can accurately make a distinction, are conducive to ATP biloluminescence method and apply in the place that purity requirements is higher.
Embodiment 2
Measure the amount of ATP in Escherichia coli bacteria suspension
Escherichia coli titer: choose single bacterium (E.Coli.) and put into 1ml beef-protein medium incubated overnight, gets the E. coli broth 1ml after cultivation and is transferred in 40ml beef-protein medium, rotating speed 120r min
-1, cultivate 2h.Get 0.5ml nutrient solution and put into sterilizing 4.5ml physiological saline, be mixed with the E. coli broth that dilution gradient is 10 times, and method prepared and diluted degree is 10 according to this
2with 10
3e. coli broth doubly.Measure the ATP in the Escherichia coli titer of each extension rate respectively.
The determination step of Escherichia coli bacteria liquid is: get 10ul Escherichia coli bacteria liquid, be expelled to and soaked on the swab head of ATP extraction agent, swab is put into and stirs for several times by the pretreated luciferase of immobilization triphosphoric acid bisphosphatase-fluorescein mixed enzyme solution, ATP on swab head is eluted completely, and react with enzyme solutions, then detection bottle is put into MPI-B detection cell and detect.
According to Fig. 2 display, conventionally, ATP is used to detect reagent analysis Escherichia coli bacteria suspension, when analyzing the low cell content bacteria suspension of detection, it is linearly not obvious, when cell content is less than 350 cell/mL, luminous intensity is suitable with blank intensity, is not easily distinguishable.And adopt the pretreated ATP of immobilization triphosphoric acid bisphosphatase to detect reagent, effectively can reduce the background value of reagent, when cell content is 15 cell/mL, still have good linear, R
2=0.9989.
Embodiment 3
Measure ATP amount in plastc ring
In this experiment, choose modal three kinds of bacterial classifications in daily life, Escherichia coli, staphylococcus aureus, bacillus subtilis, as three large constituents of miscellaneous bacteria bacteria suspension.
Miscellaneous bacteria titer: picking one ring Escherichia coli put into 1ml beef-protein medium incubated overnight, gets the E. coli broth 1ml after cultivation and is transferred in 40ml beef-protein medium, rotating speed 120r min
-1, cultivate 2h.OD to be cultured to
600take out for subsequent use when being about 0.3.Cultivate staphylococcus aureus and bacillus subtilis bacterium liquid in the same way.From above-mentioned three kinds of bacterium liquid, respectively get 1ml, be placed in sterilized blank cuvette, fully mix, to make in miscellaneous bacteria bacterium liquid three kinds of bacterial cell content reach the ratio of desirable 1:1:1.Get 0.5ml nutrient solution and put into sterilizing 4.5ml physiological saline, be mixed with the miscellaneous bacteria nutrient solution that dilution gradient is 10 times, and method prepared and diluted degree is 10 according to this
2with 10
3miscellaneous bacteria nutrient solution doubly.Measure the ATP in the miscellaneous bacteria titer of each extension rate respectively.
The determination step of miscellaneous bacteria bacterium liquid is: get 10ul miscellaneous bacteria bacterium liquid, be expelled to and soaked on the swab head of ATP extraction agent, swab is put into and stirs for several times by the pretreated luciferase of immobilization triphosphoric acid bisphosphatase-fluorescein mixed enzyme solution, ATP on swab head is eluted completely, and react with enzyme solutions, then detection bottle is put into MPI-B detection cell and detect.
According to Fig. 3 display, conventionally, ATP is used to detect reagent analysis miscellaneous bacteria bacteria suspension, when analyzing the low cell content bacteria suspension of detection, it is linearly not obvious, when cell content is less than 371 cell/mL, luminous intensity is suitable with blank intensity, is not easily distinguishable.And adopt the pretreated ATP of immobilization triphosphoric acid bisphosphatase to detect reagent, while reduction detects reagent background value, also effective slope that must improve its typical curve, is conducive to detecting of low cell content.
When applying ATP biloluminescence method inspected object surface cleanliness, what detect is not single culture, but the miscellaneous bacteria flora be made up of various bacteria, the present embodiment, being intended to illustrate when using ATP bioluminescent detection technological assessment body surface sanitary condition, adopting the pre-service of immobilization triphosphoric acid bisphosphatase to detect reagent, reduction background value can be reached too, improve the effect of detection sensitivity, this is also of great importance in actual applications for this technology.
Those skilled in the art can carry out various remodeling and change to the present invention.Therefore, present invention covers the various remodeling in the scope falling into appending claims and equivalent thereof and change.
Claims (4)
1. a highly sensitive atriphos detection method, it is characterized in that, it comprises the following steps:
Step one, preparation amination magnetic nano-particle;
Step 2, preparation immobilization atriphos detects enzyme system;
Step 3, detects enzyme system to the atriphos configured and carries out pre-service, atriphos residual in scavenger enzyme system;
Step 4, uses MPI-B chemiluminescence detector to carry out atriphos mensuration.
2. highly sensitive atriphos detection method as claimed in claim 1, it is characterized in that, described step one specifically comprises the following steps: by 10g 1,6-hexane diamine, 5g anhydrous sodium acetate, 4g FeCl
36H
2o is as source of iron constant temperature 50 DEG C of vigorous stirring in 15ml ethylene glycol; After completion of the reaction, above-mentioned solution is transferred in Teflon reactor, at 200 DEG C, heat 10h; Treat that above-mentioned reaction terminates, to the magnetic nano-particle externally-applied magnetic field in reactor, incline the solution be not attracted by the magnetic force simultaneously, adds appropriate amount of deionized water to after magnetic nano-particle cleaning 2 ~ 3 times, with washes of absolute alcohol 2 ~ 3 times, the magnetic nano-particle of 25nmMNPs finally can be obtained.
3. highly sensitive atriphos detection method as claimed in claim 1, it is characterized in that, described step 2 specifically comprises the following steps: get 25% glutaraldehyde and mix with the ratio of 4:1 with amination magnetic nano-particle, 2 hours are hatched at constant temperature 30 DEG C, treat that glutaraldehyde and amination magnetic nano-particle are in conjunction with complete, by the PBS buffer solution for cleaning 2 to 3 times of pH7.4, add the PBS damping fluid of pH7.4, under 0 DEG C of ice bath, reaction makes triphosphoric acid bisphosphatase be combined with the magnetic nano-particle connecting carboxyl for 6 hours, concentration is 4U/mL, triphosphoric acid bisphosphatase is fixed on magnetic nano-particle, use washed with de-ionized water immobilised enzymes, wash away loose triphosphoric acid bisphosphatase, being fixed triphosphoric acid bisphosphatase.
4. highly sensitive atriphos detection method as claimed in claim 1, it is characterized in that, described step 3 specifically comprises the following steps: immobilization triphosphatase is added the luciferase-luciferin system configured, guarantee that immobilization triphosphoric acid bisphosphatase ratio is 6:7, isothermal vibration reaction 20min at 30 DEG C, now remaining in immobilization triphosphoric acid bisphosphatase decomposing solution in solution ATP generates AMP and Pi; After question response terminates, under constant temperature 4 DEG C of environment, externally-applied magnetic field, is separated immobilization apyrase in solution, pretreated ATP is detected enzyme system and slowly tilts to pour out, for subsequent detection.
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