CN109540860A - A kind of biological sensor and its preparation method and application detecting kanamycins - Google Patents
A kind of biological sensor and its preparation method and application detecting kanamycins Download PDFInfo
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Abstract
The present invention relates to biosensor technology fields, in particular to the biological sensor based on hybridization chain reaction amplification.In order to solve the problems, such as that the above method specificity for detecting kanamycins in the prior art and sensitivity are all relatively low, at high cost.The fluorescence resonance energy transfer of circulation amplification and fluorophor and quencher, homogeneous reaction mixed liquor are realized in the cooperation that endonuclease IV is hybridized chain reaction by the present invention with chain.Preparation method: gold nanoparticle is prepared；Walker and Track is modified to gold nanoparticle surface；The nano-Au solution of label is mixed with homogeneous reaction solution；Hybridize chain reaction, fluorescence detection；The Idiotype identification of nucleic acid Aptamer is utilized, is detected using high specific of the nucleic acid Aptamer to object kanamycins；Amplified using hybridization chain reaction, realizes the effect of signal amplification.
Description
Technical field
The present invention relates to biosensor technology fields, in particular to hybridizing chain reaction amplification Fluorometric assay card, that is mould
The biological sensor of element, further relates to preparation method and application.
Background technique
Kanamycins is wide spectrum aminoglycosides antibiotics, a kind of water-soluble sulfate is intended for, to many leather
Blue negative bacteria includes that stranguria syndrome, salmonella and Pulmonary Tuberculosis Infection are effective in cure, and the form as the second line antibiotic is answered extensively
Medicine is shared for people and animals.Kanamycins can excessively cause ototoxicity and renal toxicity.Kanamycins can accumulate in animal body, lead to
Cross food chain transmitting.Therefore, the kanamycins in animal derived food is potentially dangerous to human health.
Currently, there are commonly high performance liquid chromatography, Capillary Electrophoresis, immunoassays in the analysis method of kanamycins
Deng.Chromatography needs complicated instrument and cumbersome sample pretreatment, and immunoassay is quick, sensitive, but there is also some defects,
If false positive is high, performance is unstable, antibody used is easy failure in preservation, it is necessary to establish more reliable quick analysis side
Method.
It Aptamer(Aptamer is) that one kind can be with higher affinity and all kinds of target molecules, the single-stranded widow that specifically combines
Nucleotide (DNA, RNA, modification RNA).Aptamer is based on single-chain nucleic acid structure and space conformation in conjunction with various target molecules
Diversity, it itself can be adapted to by pairing between complementary bases certain in chain and electrostatic interaction, hydrogen bond action etc.
Property fold, form some stable three-D space structures, be bound on its target molecule, to usually be shown to its target molecule non-
The affinity of Chang Gaodu.
Summary of the invention
In order to which the method for solving to detect kanamycins in the prior art is cumbersome, the pre-treatment of sample or concentration process are numerous
Trivial, expensive equipment is difficult to universal problem, and the present invention provides a kind of high specificities, target substance kind affinity height, combined
The bio-sensing for the hybridization chain reaction amplification Fluorometric assay kanamycins that class is more, molecular weight is smaller and synthesis process is easy
Device, meanwhile, additionally provide preparation method.
It is a kind of detect kanamycins biological sensor, including homogeneous reaction liquid, Aptamer, nano-Au solution,
Walker and Track DNA sequence dna；
The homogeneous reaction liquid, comprising: aqua sterilisa, object, HAP1, HAP2,10 × buffer, restriction nuclease inscribe
Enzyme IV, signal probe；
The Aptamer, signal probe, HAP1, HAP2, Walker and Track DNA sequence dna, sequence is respectively:
Walker sequence is as shown in SEQ No.1；
Track sequence is as shown in SEQ No.2；
HAP1 sequence is as shown in SEQ No.3；
HAP2 sequence is as shown in SEQ No.4；
Aptamer sequence is as shown in SEQ No.5；
Signal probe sequence is as shown in SEQ No.6；
4th base T of the signal probe modifies quencher Dabcyl, and the 9th base T modifies fluorophor FAM,
7th base A is modified with a tetrahydrofuran site.
5 ' terminal modified-SH of the Walker and Track DNA sequence dna；5 ' ends the of the Track DNA sequence dna
There is a tetrahydrofuran site after 23 base sequences.
Final concentration of 0.01 nM of the nano-Au solution.
The final concentration of 0.1-0.6 nM of HAP1 in the homogeneous reaction liquid；The final concentration of restriction endonuclease IV
For 20-140 U；Final concentration of 0.2-1.4 μM of signal probe；The final concentration of 0.1-0.6 nM of HAP2.
The concentration of object is more than or equal to 5 pM in the homogeneous reaction liquid.
The preparation method of above-mentioned biological sensor, comprising the following steps:
(1) gold nanoparticle is prepared；
(2) nano-Au solution of preparation modification: by Aptamer, Walker and Track modification to gold nanoparticle surface；
(3) hybridize chain reaction: by the nano-Au solution of modification and homogeneous reaction solution hybrid reaction；
(4) fluorescence detection.
The step (2), comprising the following steps:
S1 is by Walker and Aptamer in after the ratio hybridization of 1:1 and Track according to the ratio uniform of 1:20 is blended in one
It rises, forms substrate probe；
S2 is added substrate probe and places at 4 DEG C after mixing into nano-Au solution；
50 μ L PB buffers are added according to the speed of 1 μ L/min in S3, and after mixing evenly, 27 μ L are added in the speed of 1 μ L/min
PBS buffer solution stirs evenly, and places 48h at 4 DEG C；
DNA chain on S4 sterilizing water elution is unmarked.
The concentration ratio of nanogold and substrate probe is 1:5000 in the S2.
The condition of step (3) the hybridization chain reaction are as follows: temperature is 37 DEG C, reaction time 2h.
A kind of kanamycins for being used to detect in animal derived food using above-mentioned biological sensor.
6 DNA chain are used altogether in the present invention, sequence is respectively:
Walker:5 '-SH- TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTAGC CTCAACC ATCATC--
3’
Aptamer : 5’- TGG G GG TTG AGG CTA AGC CGA C -3’
Track:5 '-SH- TTTTTTTTTTCACTTGATGATGGXTG AGA ATA AAG TGT TTA AGT A -- 3 '
HAP1:5 '-AGG AGTGT TTA AGT TGA AGA ATA G TA CTT AAA CAC TTT ATT CT TGA
GGA -3’
HAP2:5 '-AGG AGTCTAT TCT TCA ACT TAA AC T AGA ATA AGG TGT TTA AGT TGA
GGA-3’
Signal probe:5 '-TCC T(Dabcyl) CA AXT (FAM) CCT
Terminal modified-the SH in the 5 ' of Walker is modified Walker to nanometer gold surface, italicized item therein by Au-S covalent bond
Sequence can be complementary with the italicized item sequence of Aptamer.
Terminal modified-the SH in the 5 ' of Track modified Track to nanometer gold surface by Au-S covalent bond, wherein Track
The sequence of underscore part can be complementary with the sequence of underscore part with Walker, and contains one among Track
Tetrahydrofuran site (site TAP), can be cut by restriction endonuclease IV, release thickened portion (Primer).Primer can be beaten
The thickened portion in HAP1 is opened, therewith sequence complementary pairing, the italicized item of HAP1 can open the italicized item of HAP2, therewith
The thickened portion of sequence complementary pairing, HAP2 can be with the thickened portion sequence complementary pairing of HAP1.In this process, HAP1
It is all tilted respectively with the 5 ' of HAP2 and 3 ' ends, and complementary pairing can be hybridized with Signal probe, wherein Signal
The 4th base T of probe modifies quencher Dabcyl, and the 9th base T modifies fluorophor FAM, and is modified with one
A tetrahydrofuran site (site TAP), can be cut by restriction endonuclease IV, release fluorophor with quencher, to generate
Fluorescence.
The detection of kanamycins is realized in homogeneous phase solution in the present invention, is realized by way of hybridizing chain reaction
Signal amplification, to realize the highly sensitive detection of kanamycins, and obtains lower Monitoring lower-cut.
The reaction occurred in homogeneous mainly has: Walker and Aptamer carries out base pair complementarity and forms duplex structure.When
In the presence of having kanamycins, Aptamer is combined with kanamycins, while Walker is discharged.The Walker released
Base pair complementarity can be carried out with the part of Track, be sheared in the presence of restriction endonuclease IV, Primer piece
Section is shear off from double-strand, while releasing the Track chain of remainder, Walker again can further with another
Track combines the step for repeating.In addition, the Primer generated can open HAP1, HAP1 can open HAP2, and HAP2 can be with
HAP1 is opened, is continued with this.Stick up 5 ' and 3 ' can be carried out with Signal probe base pair complementarity be formed it is double
Chain, endonuclease IV can shear the tetrahydrofuran base position (site TAP) among Signal probe and make Signal
Probe is broken and opens with HAP1, HAP2 points, to generate fluorescence signal, separated HAP1, HAP2 can be with other Signal
Probe carries out base pair complementarity again, realizes circulation amplification.
The detection mode of the invention is Fluorometric assay, utilizes luminoscope.Before testing, first passing through Au-S key will
Walker and Track is modified to gold nanoparticle surface, and homogeneous reaction solution is mixed with nano Au particle has been marked, and secondly will
Object bacteria, HAP1, HAP2 and Signal probe are added to homogeneous phase solution, are eventually adding endonuclease IV, are incubated at 37 DEG C
2h completes hybridization chain reaction amplification process.Then it is 486nm with luminoscope setting excitation wavelength, detects fluorescence at 518 nm
Intensity.
The present invention is based on the specific recognition of nucleic acid Aptamer and object, Walker is anti-in the hybridization of nanometer gold surface
It answers, the hybridization chain reaction amplification of the cooperation of endonuclease IV and the fluorescence resonance energy of fluorophor and quencher
Amount transfer constructs aptamer biosensors.The advantages that sensor has detection speed fast, and detection limit is low, and specificity is high, can
To make up the shortcomings and deficiencies of the existing detection method of kanamycins, realize to its fast and accurate quantitative detection.
Beneficial effects of the present invention:
1, high specific detects
The Idiotype identification of nucleic acid Aptamer is utilized, utilizes being implemented in combination with to object for Aptamer and kanamycins
High specific detection；The cleavage site (AP site) of endonuclease IV is utilized, realizes positional dissection；
2, ultrasensitiveness detects
Using hybridization chain reaction, it is exaggerated fluorescence signal, improves the sensitivity of detection, is realized to object kanamycins
Ultrasensitiveness detection；
3, it reacts mild, easy to operate, is suitble to industrialization
The reaction condition of the sensor is mild, and reaction speed is fast；Due to using fluorescence method, detection method is easy to operate, detects
Period is short；The main process of testing principle is to improve reaction speed in homogeneous middle realization, reduce the complicated journey of operation
Degree, realizes the quick of object, simply, sensitive to detect；Preparation method is simple, and performance is stablized, the repeatability of fluorescence detection
It is good, the practical application of the detection of kanamycins and biosensor industrialization suitable for food safety and water body；It makes simultaneously
The process costs of the biosensor are low, the inexpensive requirement suitable for industrialization.
Detailed description of the invention
Fig. 1 is the schematic diagram of the experiment；
Fig. 2 is 1 testing result figure of embodiment；
Fig. 3 is 2 testing result figure of embodiment；
Fig. 4 is 3 testing result figure of embodiment；
Fig. 5 is the standard curve that 4 sensor of embodiment detects kanamycins.
Specific embodiment
Invention is further explained combined with specific embodiments below.
Embodiment 1
A kind of preparation method for the biological sensor detecting kanamycins, comprising the following steps:
(1) gold nanoparticle is prepared；
(2) nano-Au solution of preparation modification: by Aptamer, Walker and Track modification to gold nanoparticle surface；
(3) hybridize chain reaction: by the nano-Au solution of modification and homogeneous reaction solution hybrid reaction；
(4) fluorescence detection.
The step (1), preferably steps are as follows for the preparation manipulation of nano Au particle:
(1) required instrument is installed, 200mL ultrapure water is added into three-necked flask and (is careful not to allow three-necked flask decline ash charge
Dirt).
(2) 500uL(0.04g/mL is taken) HAuCl4 in the centrifuge tube of individual packaging, takes 500uL and 200mL with liquid-transfering gun
In ultrapure water, agitating and heating, mixing speed 450 turns left the right side, until boiling.
(3) under stirring conditions, the citric acid three sodium solution of 3mL 1% is taken to rapidly join in solution, it is molten in a few minutes
Liquid color becomes claret from light yellow, continues after heating 15min, removes heat source, be slowly cooled to room temperature, saves as 4 DEG C
It is spare.
(4) it takes 60uL gold nano grain solution in microcolorimetric ware, uses UV-2550 ultraviolet-uisible spectrophotometer pair
It carries out light absorption spectral scan, according to molar extinction coefficient 8.78 × 10 of the wavelength at 519nm8M-1cm-1 calculates gold
The concentration of nanoparticles solution is about 0.3nM..
The step (2) specific steps are as follows:
(1) by Walker and Aptamer in after the ratio hybridization of 1:1 and Track according to the ratio uniform of 1:20 is blended in one
It rises, forms substrate probe.
(2) it takes 1 mL nano-Au solution in centrifuge tube, is centrifuged 10 min, while it is spare to be centrifuged two pipes.It is centrifuged to supernatant
Liquid is colorless and transparent, removes supernatant, and 300 μ L aqua sterilisas, which are added, makes nano-Au solution be concentrated into 3 nM.Move into 1 mL vial
In, it is sealed with masking foil.
(3) after being placed at room temperature for 30 min, the modification that 150 μ L concentration the are 30 μM substrate probe of-SH, (nanometer is added
The concentration ratio of gold and substrate chain is 1:5000) after mixing, 24 h are placed at 4 DEG C.
(4) it is slowly added to 50 μ L PB buffers several times, magneton (the previous day is impregnated with chloroazotic acid) is added and stirs 10 min
Afterwards, 27 μ L PBS buffer solution are continuously added.Take out magneton, 4 DEG C of 48 h of placement.
(5) nano-Au solution marked is transferred in centrifuge tube, aqua sterilisa is added to 1 mL, is centrifuged 10 min, goes
Except supernatant.The centrifugation of 1 mL aqua sterilisa is added, this process is repeated twice (in order to elute the DNA chain on unmarked.
The key step of the reaction process of the step (3) is as follows: by aqua sterilisa, object (4 μ L), modification receive
Rice gold solution (6 μ L), HAP1(2 μ L), HAP2(2 μ L), 10 × buffer (buffer) (4 μ L), restriction nuclease inscribe
Enzyme IV(3 μ L) (concentration is respectively 20U, 40U, 60U, 80U, 100U, 200U), signal probe (6 μ L), it is added to and prepares in advance
In the EP pipe of good sterilizing.30s is shaken, is put into 37 DEG C of insulating box and is incubated for 2 h；
Step (2) process is that the solution (10 μ L) after reaction is diluted to 100 μ L, is examined at 518nm with luminoscope
Survey fluorescence.
Luminoscope excitation wavelength is set as 486nm, launch wavelength 518nm, detection range 490nm-600nm, reads glimmering
Change in optical signal detects object.
The preparation method for the solution used in the above process:
1, ultrapure water is both needed to carry out high-temperature sterilization processing.Specific method is that ultrapure water is individually positioned in conical flask, is then used
Masking foil and newspaper are sealed.In high-pressure sterilizing pot 120 DEG C at a temperature of sterilize 20 min.
10 2, × buffer (buffer) be to be bought with polymerase, can be used directly.
As a result see Fig. 2, it can be seen from the figure that with the increase of endonuclease IV amount, the fluorescence intensity tested
Constantly enhancing, after enzyme amount reaches 100U, fluorescence intensity is basically unchanged.Illustrate that enzyme reparation recycles required endonuclease IV amount
For 100U.
Embodiment 2
A kind of preparation method for the biological sensor detecting kanamycins, comprising the following steps:
(1) gold nanoparticle is prepared；
(2) nano-Au solution of preparation modification: by Aptamer, Walker and Track modification to gold nanoparticle surface；
(3) hybridize chain reaction: by the nano-Au solution of modification and homogeneous reaction solution hybrid reaction；
(4) fluorescence detection.
Step (1), (2), (4) are the same as embodiment 1.
The key step of the reaction process of the step (3) is as follows: by aqua sterilisa, object (4 μ L), modification receive
Rice gold solution (6 μ L), HAP1(2 μ L) (final concentration is respectively 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM), 10
× buffer (buffer) (4 μ L), restriction endonuclease IV(3 μ L), signal probe (6 μ L), be added to preparatory standard
In the EP pipe for the sterilizing got ready.30s is shaken, is put into 37 DEG C of insulating box and is incubated for 2 h.
Luminoscope excitation wavelength is set as 486nm, launch wavelength 518nm, detection range 490nm-600nm, reads glimmering
Change in optical signal detects object.
As a result see Fig. 3, it can be seen from the figure that the fluorescence intensity tested first enhances with the increase of HAP1 amount, after
It remains unchanged, after HAP1 amount reaches 0.4nM, fluorescence intensity is basically unchanged.Illustrate that HAP1 amount needed for hybridizing chain reaction is
0.4 nM。
Embodiment 3
A kind of preparation method for the biological sensor detecting kanamycins, comprising the following steps:
(1) gold nanoparticle is prepared；
(2) nano-Au solution of preparation modification: by Aptamer, Walker and Track modification to gold nanoparticle surface；
(3) hybridize chain reaction: by the nano-Au solution of modification and homogeneous reaction solution hybrid reaction；
(4) fluorescence detection.
Step (1), (2), (4) are the same as embodiment 1.
The key step of the reaction process of the step (3) is as follows: by aqua sterilisa, object (4 μ L), modification receive
Rice gold solution (6 μ L), HAP1(2 μ L), HAP2(2 μ L), 10 × buffer (buffer) (3 μ L), restriction nuclease inscribe
Enzyme IV(3 μ L), signal probe (6 μ L) (final concentration is respectively 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4
μM), it is added in the EP pipe of preprepared sterilizing.30s is shaken, is put into 37 DEG C of insulating box and is incubated for 2 h.
Luminoscope excitation wavelength is set as 486nm, launch wavelength 518nm, detection range 490nm-600nm, reads glimmering
Change in optical signal detects object.
As a result Fig. 4 is seen, it can be seen from the figure that the fluorescence intensity tested first increases with the increase of signal probe amount
By force, it remains unchanged after, after signal probe amount reaches 1 μM, fluorescence intensity is basically unchanged.Illustrate letter needed for hybridizing chain reaction
Number probe amount is 1 μM.
Embodiment 4
A kind of preparation method for the biological sensor detecting kanamycins, comprising the following steps:
(1) gold nanoparticle is prepared；
(2) nano-Au solution of preparation modification: by Aptamer, Walker and Track modification to gold nanoparticle surface；
(3) hybridize chain reaction: by the nano-Au solution of modification and homogeneous reaction solution hybrid reaction；
(4) fluorescence detection.
Step (1), (2), (4) are the same as embodiment 1.
The key step of the reaction process of the step (3) is as follows: by aqua sterilisa, object (4 μ L) (final concentration point
Wei 0,5pM, 10pM, 50pM, 100pM, 500pM, 1nM, 5nM, 10nM), the nano-Au solution (6 μ L) of modification, HAP1(2 μ
L), HAP2(2 μ L), 10 × buffer (buffer) (3 μ L), restriction endonuclease IV(2 μ L), signal probe (6
μ L), it is added in the EP pipe of preprepared sterilizing.30s is shaken, is put into 37 DEG C of insulating box and is incubated for 2 h.
Luminoscope excitation wavelength is set as 486nm, launch wavelength 518nm, detection range 490nm-600nm, reads glimmering
Change in optical signal detects object.
Testing result is as shown in figure 5, it will be seen that glimmering when kanamycins concentration is in 5 pM to 10000 pM in figure
Light value is continuously increased, and stable reaction carries out.Kanamycins concentration in 5 pM to 10000 pM, pair of kanamycins concentration
Number, matched curve proportional with the size of fluorescence intensity level: A=113.5*logC+226(A is fluorescence intensity level, and C is
The concentration of kanamycins), meanwhile, we continue on the basis of the concentration of 5 pM to lower Concentration Testing, work as concentration through detection
When lower than 5 pM, the relationship of fluorescence intensity and concentration is no longer complies with matched curve rule, i.e. absorption peak in figure most just
Therefore this method can be obtained in high point Monitoring lower-cut is 5 pM.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention and should not be limited by the examples,
Its any change made without departing from the spirit and principles of the present invention, modification, combination, substitution, simplification should be equivalent
Alternative is included within the scope of the present invention.
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Claims (10)
1. a kind of biological sensor for detecting kanamycins, which is characterized in that including homogeneous reaction liquid, Aptamer, nanometer
Gold solution, Walker and Track DNA sequence dna；
The homogeneous reaction liquid, comprising: aqua sterilisa, object, HAP1, HAP2,10 × buffer, restriction nuclease inscribe
Enzyme IV, signal probe；
The Aptamer, signal probe, HAP1, HAP2, Walker and Track DNA sequence dna, sequence is respectively:
Walker sequence is as shown in SEQ No.1；
Track sequence is as shown in SEQ No.2；
HAP1 sequence is as shown in SEQ No.3；
HAP2 sequence is as shown in SEQ No.4；
Aptamer sequence is as shown in SEQ No.5；
Signal probe sequence is as shown in SEQ No.6；
4th base T of the signal probe modifies quencher Dabcyl, and the 9th base T modifies fluorophor FAM,
7th base A is modified with a tetrahydrofuran site.
2. biological sensor according to claim 1, which is characterized in that Walker the and Track DNA sequence
5 ' terminal modified-SH of column；There is a tetrahydrofuran site after 5 ' the 23rd base sequences in end of the Track DNA sequence dna.
3. biological sensor according to claim 1, which is characterized in that the nano-Au solution it is final concentration of
0.01 nM。
4. biological sensor according to claim 1, which is characterized in that the end of HAP1 in the homogeneous reaction liquid
Concentration is 0.1-0.6 nM；The final concentration of 20-140 U of restriction endonuclease IV；The final concentration of 0.2- of signal probe
1.4μM；The final concentration of 0.1-0.6 nM of HAP2.
5. biological sensor according to claim 1, which is characterized in that object in the homogeneous reaction liquid
Concentration is more than or equal to 5 pM.
6. a kind of preparation method of biological sensor described in claim 1, which comprises the following steps:
(1) gold nanoparticle is prepared；
(2) nano-Au solution of preparation modification: by Aptamer, Walker and Track modification to gold nanoparticle surface；
(3) hybridize chain reaction: by the nano-Au solution of modification and homogeneous reaction solution hybrid reaction；
(4) fluorescence detection.
7. preparation method according to claim 6, which is characterized in that the step (2), comprising the following steps:
S1 is by Walker and Aptamer in after the ratio hybridization of 1:1 and Track according to the ratio uniform of 1:20 is blended in one
It rises, forms substrate probe；
S2 is added substrate probe and places at 4 DEG C after mixing into nano-Au solution；
50 μ L PB buffers are added according to the speed of 1 μ L/min in S3, and after mixing evenly, 27 μ L are added in the speed of 1 μ L/min
PBS buffer solution stirs evenly, and places 48h at 4 DEG C；
DNA chain on S4 sterilizing water elution is unmarked.
8. preparation method according to claim 7, which is characterized in that the concentration of nanogold and substrate probe in the S2
Than for 1:5000.
9. preparation method according to claim 6, which is characterized in that the condition of step (3) the hybridization chain reaction
Are as follows: temperature is 37 DEG C, reaction time 2h.
10. a kind of kanamycins for being used to detect in animal derived food using biological sensor described in claim 1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110441277A (en) * | 2019-08-15 | 2019-11-12 | 济南大学 | A kind of biosensor and its preparation method and application detecting ampicillin |
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| CN111521808A (en) * | 2020-05-26 | 2020-08-11 | 济南大学 | Biosensor for detecting polychlorinated biphenyl and preparation method thereof |
| CN111521808B (en) * | 2020-05-26 | 2022-11-25 | 济南大学 | Biosensor for detecting polychlorinated biphenyl and preparation method thereof |
| CN111876412A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Fluorescent aptamer probe and detection method and application thereof |
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