CN109283147A - It is a kind of detect kanamycins homogeneous bio-sensing method and its application - Google Patents
It is a kind of detect kanamycins homogeneous bio-sensing method and its application Download PDFInfo
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Abstract
It is a kind of detect kanamycins homogeneous bio-sensing method and its application, pass through tetra- serobila DNA(S1 of G-) it is active with the double-strand hybridization reaction inhibition DNA enzymatic of kanamycins aptamer (S2), and the S1 of the specific binding release corresponding number between kanamycins and its aptamer, and combine hemin to form DNA enzymatic and carry out catalyzed coloration signal transduction；In addition, the hairpin structure formed between kanamycins and its aptamer can be recycled after introducing complementary strand S3 and hybridizing by the catalysis target analytes of exonuclease III come the sensitivity of improvement method；Using the Catalytic color reaction of the DNA enzymatic of aptamer specific recognition release, the quantitative relationship between color product absorbance and kanamycins analyte concentration is constructed；Operation of the present invention is convenient, and low in cost, high sensitivity, stability is good, has fine application value for the quick detection of yapamicin relict in the complex dielectrics such as food, water body.
Description
Technical field
The present invention relates to bioassay technique field, it is specifically a kind of detection kanamycins homogeneous bio-sensing method and
It is applied.
Background technique
Kanamycins is a kind of aminoglycoside high-efficiency broad spectrum antibiotic, uncommon to most enterobacteriaceae lactobacteriaceaes such as large intestine angstrom
Bacterium, Klebsiella, Enterobacter, Proteus, Shigella, Salmonella, citrobacter category, Pu Luofeideng bacterium
Belong to, yersinia's genus and haemophilus influenzae, Brucella, Neisseria meningitidis, NEISSERIA GONORRHOEAE etc. have good antibacterial
Effect, thus it is widely used in the treatment of humans and animals various Gram-positives and gram positive bacterial infection disease.However,
Being excessively used for kanamycins not only can cause dysacousis, tinnitus or even reaction of renal toxicity and drug allergy etc. serious
Side effect, but also serious drug resistance can be generated.In recent years, food caused by the abuse of antibiotic, water body, antibiosis in soil
Element remains great threat that is exceeded and its producing by the enrichment of food chain in human body to human health, and it is general to cause society
All over concern.Therefore, many countries and regions including China all propose the antibiotic residues such as kalamycin in food
Stringent limitation standard.
Currently, national standard generally uses Liquid Chromatography-Mass Spectrometry to carry out the detection of yapamicin relict, still
This method expensive equipment, testing cost is high, and can only generally carry out in laboratory, is difficult to meet in practical application real-time, scene
The needs of detection.To solve this problem, people act on the bio-identifications such as antigen (antibody), DNA and electrochemistry, ratio in recent years
The analysis methods such as color combine, and have developed the bio-sensing method that largely can be used for that antibiotic is quick, facilitates detection.But this
A little methods are typically all to be based on out-phase analytical model and construct, and generally require very complicated solid phase interface modification and multistep
Incubation, separation and washing operation, or even also to amplify in conjunction with nano material signal to improve its sensitivity for analysis, thus not only divide
The analysis time is longer, cumbersome, and many more manipulations can also influence the repeatability of method to a certain extent, improves analysis cost,
Extremely it is unfavorable for it to be widely used.Therefore, develop a kind of easy to operate, and have high sensitivity, accuracy good, low in cost etc.
The intelligent bio-sensing new method of excellent performance has a very important significance.
Summary of the invention
Cumbersome, higher cost that the purpose of the present invention is to the methods of current detection kalamycin, time is longer,
The problems such as repeatability is bad, provide it is a kind of can in simple, quick test sample kalamycin content homogeneous bio-sensing method,
This method is easy to operate, low in cost, high sensitivity, and stability is good, and analysis time is shorter, and the requirement to operator is low, right
Kalamycin residual scene, quick detection have good application value in the complex dielectrics such as food, water body.
A kind of homogeneous bio-sensing method of detection kanamycins (Kana) of the invention, comprising the following steps:
(1) prepared by tetra- serobila characteristic sequence of G- and kanamycins aptamer heteroduplex
It is 10mM pH=7.4 3 (methylol) aminomethane buffer solution that 990 μ L concentration are added into PV pipe, and the buffering is molten
In liquid containing 200mM NaCl, 10mM KCl, mass fraction is 1% DMSO and mass fraction is 0.05% Triton X-100,
10 μM of kanamycins aptamer (S2) solution of 5 μ L, 10 μM of G-, tetra- serobila DNA chain (S1) and 5 μ L are added thereto again,
The base sequence of the tetra- serobila DNA chain (S1) of G- is 5 '-GGG TAG GGC GGG TTG GGA ACC TCA AGA CCA
CTT GGA CAT TTT-3 ', the base sequence of the kanamycins aptamer (S2) are 5 '-TGT CCA AGT GGT
It is stand-by that aptamers heteroduplex complex solution is obtained after CTT GAG GTT TTTT-3 ', room temperature concussion reaction 45min；
(2) homogeneous reaction measures the content of kanamycins (Kana) in standard solution
6 parts of above-mentioned each 100 μ L of aptamers heteroduplex complex solution are taken, are separately added into thereto containing kalamycin (Kana)
Concentration gradient be 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL and 10ng/mL 20mM pH
50 μ L of=7.4 Tris-HCl buffer solution contains 100mM NaCl, 2mM MgCl in the buffer solution2With 5mM KCl, then to
5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences (S3), 5 μ L 5U/ μ L exonucleases are added in every part of solution
III(Exo III) and 1 μM of 5 μ L hemin (hemin), the kanamycins aptamer partial complementarity sequence
(S3) base sequence in is 5 '-AAA AAA CCT GAC ACT AC-3 '；After 37 DEG C of vortexs mix reaction 70min, temperature is increased
Degree is down to 25 DEG C after keeping 5min to 65 DEG C；Then 70 μ L are added into every part of solution and contain 0.4mM TMB, 0.4mM H2O2's
The citrate buffer solution of pH=5.0 contains 95.8mM Na in the buffer solution2HPO4, 52.1mM sodium citrate and 40mM
After KCl, chromogenic reaction 5min, 30 μ L 2M H are added in continuation thereto2SO4Color development stopping reaction；Utilize UV-vis spectroscopy
The absorbance value of photometric determination solution, with the quantitative relationship established between absorbance and kanamycins concentration；
(3) in sample kanamycins (Kana) content detection
It takes suitable sample and carries out pre-treatment, the hybridization of 100 μ L aptamers is added in the 50 μ L of sample solution that takes that treated thereto
Double-stranded complex solution, 5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences (S3), 5 μ L 5U/ μ L exonucleases
III(Exo III) and 1 μM of 5 μ L hemin (hemin), the kanamycins aptamer partial complementarity sequence
(S3) base sequence in is 5 '-AAA AAA CCT GAC ACT AC-3 '；After 37 DEG C of vortexs mix reaction 70min, temperature is increased
Degree is down to 25 DEG C after keeping 5min to 65 DEG C；Then 70 μ L are added into sample solution and contain 0.4mM TMB, 0.4mM H2O2's
The citrate of pH=5.0 delays solution, contains 95.8mM Na in the buffer solution2HPO4, 52.1mM sodium citrate and 40mM KCl,
After chromogenic reaction 5min, 30 μ L 2M H are added in continuation thereto2SO4Color development stopping reaction；Utilize spectrophotometry
The absorbance value of meter measurement sample solution, calculates kanamycins according to the quantitative relationship between absorbance and kanamycins concentration
(Kana) content.
The present invention also provides a kind of homogeneous bio-sensing method for detecting kanamycins kanamycins in test sample
Application in content.
The working principle of the invention is: passing through tetra- serobila DNA chain (S1) of G- and kanamycins (Kana) aptamer
(S2) the DNA double chain hybridization reaction between can inhibit tetra- serobila of G- contained by S1 mutually to tie with hemin (hemin) very well
Conjunction forms DNA enzymatic, and the specificity knot between S2 and kanamycins (Kana) analyte as kanamycins aptamer
The S1 that can discharge corresponding number again is closed, to combine the catalyzed coloration for forming DNA enzymatic by S1 and hemin (hemin)
Reaction carries out colorimetric signal transduction；In addition, combining the class hair clip formed between kanamycins (Kana) and its aptamer S2
Structure can introduce after S2 partial complementarity chain S3 hybridized, and pass through exonuclease III(Exo III) endonuclease reaction release
It releases kanamycins (Kana) and S3 carries out the amplification of target analytes cycle signal, and then effectively improve the sensitive of analysis method
Degree；It is discharged using kanamycins and its aptamer specific recognition and exonuclease III enzymatic reaction free
The Catalytic color reaction of DNA enzymatic can construct the quantitative relationship between color product absorbance and kanamycins analyte concentration.
The aptamer that the present invention uses is not only more preferable with stability compared with conventional antibodies, cost is more cheap etc. excellent
Point, and between aptamer and kanamycins analyte the effect of better specific recognition can very high guarantee this method exist
Fine selectivity in complex matrices analysis；React the Catalytic color reaction of the DNA enzymatic of release and the enzymatic of exonuclease III
Circular response signal amplification makes this method possess very high sensitivity for analysis, avoids nanometer complicated in conventional method
The non-specific adsorption that may cause in material preparation and its signal amplification process influences；Importantly, whole identification reactions,
Cycle signal amplification and DNA enzymatic form a step all in homogeneous phase solution and carry out, and not only operate very simple, but also ensure that very well
The repeatability of method, thus there is superiority very outstanding and practical value compared with conventional method.
The bio-sensing method that the present invention constructs can be convenient, simply for the highly selective inspection of kalamycin content
It surveys, there is high sensitivity, the excellent performances such as the range of linearity is wide, detection time is shorter, testing cost is cheap, it can be in 0.1pg/mL-
It realizes in 10ng/mL concentration range to the quantitative response and Accurate Determining of kalamycin, it is numerous to overcome operated in accordance with conventional methods very well
Trivial, higher cost, the defects of time is longer, repeatability is bad etc..
Detailed description of the invention
Fig. 1 is detection schematic diagram of the invention.
Specific embodiment
Embodiment 1
A kind of homogeneous bio-sensing method of the detection kanamycins of the present embodiment, comprising the following steps:
(1) prepared by tetra- serobila characteristic sequence of G- and kanamycins (Kana) aptamer heteroduplex
It is 10mM pH=7.4 3 (methylol) aminomethane buffer solution that 990 μ L concentration are added into PV pipe, and the buffering is molten
In liquid containing 200mM NaCl, 10mM KCl, mass fraction is 1% DMSO and mass fraction is 0.05% Triton X-100,
10 μM of kanamycins (Kana) aptamers (S2) of 5 μ L, 10 μM of G-, tetra- serobila DNA chain (S1) and 5 μ L are added thereto again
Solution, base sequence is 5 '-GGG TAG GGC GGG TTG GGA ACC TCA AGA in the tetra- serobila DNA chain (S1) of G-
CCA CTT GGA CAT TTT-3 ', the base sequence in the kanamycins aptamer (S2) are 5 '-TGT CCA AGT
It is stand-by that aptamers heteroduplex complex solution is obtained after GGT CTT GAG GTT TTTT-3 ', room temperature concussion reaction 45min；
(2) homogeneous reaction measures the content of kanamycins in standard solution
6 parts of above-mentioned each 100 μ L of aptamers heteroduplex complex solution are taken, are separately added into thereto containing kalamycin (Kana)
Concentration gradient be 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL and 10ng/mL 20mM pH
50 μ L of=7.4 Tris-HCl buffer solution contains 100mM NaCl, 2mM MgCl in the buffer solution2With 5mM KCl, then to
5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences (S3), 5 μ L 5U/ μ L exonucleases are added in every part of solution
III(Exo III) and 1 μM of 5 μ L hemin (hemin), the kanamycins aptamer partial complementarity sequence
(S3) base sequence in is 5 '-AAA AAA CCT GAC ACT AC-3 '；After 37 DEG C of vortexs mix reaction 70min, temperature is increased
Degree is down to 25 DEG C after keeping 5min to 65 DEG C；Then 70 μ L are added into every part of solution and contain 0.4mM TMB, 0.4mM H2O2's
The citrate buffer solution of pH=5.0 contains 95.8mM Na in the buffer solution2HPO4, 52.1mM sodium citrate and 40mM
After KCl, chromogenic reaction 5min, 30 μ L 2M H are added in continuation thereto2SO4Color development stopping reaction；Utilize UV-vis spectroscopy
The absorbance value of photometric determination solution is obtained with the quantitative relationship established between absorbance and kanamycins (Kana) concentration
The working curve of kanamycins standard solution, see the table below 1.
1 kanamycins standard solution working curve of table
| Detectable substance | The range of linearity (ng/mL) | Linearly dependent coefficient | Detection limit (pg/mL) |
| Kanamycins | 0.0001-10 | 0.998 | 0.045 |
The detection of kanamycins content in 2 powdered milk sample of embodiment
According to the working curve that embodiment 1 obtains, the content of kanamycins in powdered milk sample is detected.It is molten to weigh commercially available milk powder 1g
Solution contains 100mM NaCl, 2mM MgCl in 5 mL 20mM pH, 7.4 Tris-HCl buffer solution in the buffer solution2
With 5mM KCl, dissolved 100 μ L of milk power solution is taken, the acetic acid that mass fraction is 20% is added thereto and is adjusted to pH=4.6,
It is centrifuged off the protein and fat solidified in sample after 20min, then handles sample solution with 0.22 μm of membrane filtration, and will
Sample solution is adjusted to pH=7.4 again；The 50 μ L of sample solution that takes that treated, then 100 μ L aptamers heteroduplexes are added thereto
Complex solution, 5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences (S3), 5 μ L 5U/ μ L exonuclease III
The hemin (hemin) of (Exo III) and 1 μM of 5 μ L, the kanamycins aptamer partial complementarity sequence
(S3) base sequence in is 5 '-AAA AAA CCT GAC ACT AC-3 '；After 37 DEG C of vortexs mix reaction 70min, temperature is increased
Degree is down to 25 DEG C after keeping 5min to 65 DEG C；Then 70 μ L are added into sample solution and contain 0.4mM TMB, 0.4mM H2O2
The citrate buffer solution of pH=5.0,95.8mM Na is contained in the buffer solution2HPO4, 52.1mM sodium citrate and 40mM
After KCl, chromogenic reaction 5min, 30 μ L 2M H are added in continuation thereto2SO4Color development stopping reaction；Utilize UV-vis spectroscopy
The absorbance value of photometric determination powdered milk sample solution, according to determining between the absorbance in embodiment 1 and kanamycins concentration
Magnitude relation calculates the content of kanamycins (Kana).
Testing result shows to detect in milk sample without yapamicin relict.It continues up to state in powdered milk sample and be added not
With the kanamycins standard solution of concentration, recovery testu is carried out, experimental result is shown in the following table 2.
The recovery testu result of 2 powdered milk sample solution of table
| Serial number | Additional amount (μM) | Average yield (μM) | RSD(%, n=5) | Average recovery rate (%) |
| 1 | 1 | 1.05 | 2.8 | 105.0 |
| 2 | 0.1 | 0.107 | 2.5 | 107.0 |
| 3 | 0.01 | 0.0096 | 3.3 | 96.0 |
As can be seen from Table 2, the relative standard deviation (RSD) of 2 test result of the present embodiment is 2.5-3.3%, recovery of standard addition
96.0-107.0% illustrates the accuracy with higher of the analysis method of the present embodiment and precision.
The detection of kanamycins content in 3 honey sample of embodiment
According to the working curve that embodiment 1 obtains, the content of kanamycins in honey sample is detected.It is molten to weigh commercially available honey 2g
Solution contains 100mM NaCl, 2mM MgCl in the 4mL 20mM Tris-HCl buffer solution of pH=7.4 in the buffer solution2With
5mM KCl；Sample solution is handled with 0.22 μm of membrane filtration again；Filtered 50 μ L of sample solution is taken, 100 are added thereto
μ L aptamers heteroduplex complex solution, 5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences (S3), 5 μ L 5U/ μ
L exonuclease III(Exo III) and 1 μM of 5 μ L hemin (hemin), the kanamycins aptamer
Base sequence in partial complementarity sequence (S3) is 5 '-AAA AAA CCT GAC ACT AC-3 '；37 DEG C of vortexs mix reaction
After 70min, temperature is increased to 65 DEG C, is down to 25 DEG C after keeping 5min；Then 70 μ L are added into sample solution and contain 0.4mM
TMB、0.4mM H2O2The citrate buffer solution of pH=5.0, the buffer solution contains 95.8mM Na2HPO4, 52.1mM lemon
Lemon acid sodium and 40mM KCl, after chromogenic reaction 5min, 30 μ L 2M H are added in continuation thereto2SO4Color development stopping reaction；It utilizes
Ultraviolet-visible spectrophotometer measures the absorbance value of solution, according between the absorbance in embodiment 1 and kanamycins concentration
Quantitative relationship calculate kanamycins (Kana) content.
Testing result shows to detect in honey sample without yapamicin relict.
Claims (2)
1. a kind of homogeneous bio-sensing method for detecting kanamycins, it is characterised in that the following steps are included:
(1) prepared by tetra- serobila characteristic sequence of G- and kanamycins aptamer heteroduplex
It is 10mM pH=7.4 3 (methylol) aminomethane buffer solution that 990 μ L concentration are added into PV pipe, and the buffering is molten
In liquid containing 200mM NaCl, 10mM KCl, mass fraction is 1% DMSO and mass fraction is 0.05% Triton X-100,
10 μM of kanamycins aptamer solution of 5 μ L, 10 μM of G-, tetra- serobila DNA chain and 5 μ L, the G- tetra- is added thereto again
The base sequence of serobila DNA chain is 5 '-GGG TAG GGC GGG TTG GGA ACC TCA AGA CCA CTT GGA CAT
TTT-3 ', the base sequence of the kanamycins aptamer are 5 '-TGT CCA AGT GGT CTT GAG GTT TTTT-
It is stand-by that aptamers heteroduplex complex solution is obtained after 3 ', room temperature concussion reaction 45min；
(2) homogeneous reaction measures the content of kanamycins in standard solution
6 parts of above-mentioned each 100 μ L of aptamers heteroduplex complex solution are taken, are separately added into the concentration containing kalamycin thereto
Gradient is pH=7.4 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL and 10ng/mL 20mM
50 μ L of Tris-HCl buffer solution contains 100mM NaCl, 2mM MgCl in the buffer solution2It is then molten to every part with 5mM KCl
5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences, 5 μ L 5U/ μ L exonuclease III and 5 μ L are added in liquid
1 μM of hemin, the base sequence in the kanamycins aptamer partial complementarity sequence are 5 '-AAA AAA
CCT GAC ACT AC-3'；After 37 DEG C of vortexs mix reaction 70min, temperature is increased to 65 DEG C, is down to 25 DEG C after keeping 5min；
Then 70 μ L are added into every part of solution and contain 0.4mM TMB, 0.4mM H2O2The citrate buffer solution of pH=5.0, this is slow
It rushes and contains 95.8mM Na in solution2HPO4, after 52.1mM sodium citrate and 40mM KCl, chromogenic reaction 5min, continue to it
30 μ L 2M H of middle addition2SO4Color development stopping reaction；Using the absorbance value of ultraviolet-visible spectrophotometer measurement solution, to build
Quantitative relationship between vertical absorbance and kanamycins concentration；
(3) in sample kanamycins content detection
It takes suitable sample and carries out pre-treatment, the hybridization of 100 μ L aptamers is added in the 50 μ L of sample solution that takes that treated thereto
Double-stranded complex solution, 5 μ L, 2 μM of kanamycins aptamer partial complementarity sequences, 5 μ L 5U/ μ L exonuclease III
And the hemin of 1 μM of 5 μ L, the base sequence in the kanamycins aptamer partial complementarity sequence are 5 '-
AAA AAA CCT GAC ACT AC-3'；After 37 DEG C of vortexs mix reaction 70min, temperature is increased to 65 DEG C, is dropped after keeping 5min
To 25 DEG C；Then 70 μ L are added into sample solution and contain 0.4mM TMB and 0.4mM H2O2The citrate of pH=5.0 delay it is molten
Liquid contains 95.8mM Na in the buffer solution2HPO4, after 52.1mM sodium citrate and 40mM KCl, chromogenic reaction 5min, after
It is continuous that 30 μ L 2M H are added thereto2SO4Color development stopping reaction；Utilize the suction of ultraviolet-visible spectrophotometer measurement sample solution
Shading value calculates the content of kanamycins according to the quantitative relationship between absorbance and kanamycins concentration.
2. a kind of homogeneous bio-sensing method for detecting kanamycins kanamycins in test sample as described in claim 1
Application in content.
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| CN113406028A (en) * | 2021-06-29 | 2021-09-17 | 湖北师范大学 | Kanamycin homogeneous biosensing method based on nanogold aggregation and application thereof |
| CN113419057A (en) * | 2021-05-31 | 2021-09-21 | 江苏科技大学 | DNA/Ni-Fe LDO cubic network structure-based ultra-sensitive kanamycin detection method |
| CN114113359A (en) * | 2021-05-07 | 2022-03-01 | 佛山市南海北沙制药有限公司 | Central control detection method of 7-ACA derivative |
| CN114199859A (en) * | 2021-09-08 | 2022-03-18 | 湖南农业大学 | Method for detecting biological molecules based on G4 aptamer |
| CN114460072A (en) * | 2022-02-11 | 2022-05-10 | 江南大学 | Colorimetric detection method for kanamycin based on nano enzyme and application thereof |
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