CN108287149A - A kind of surface plasmon resonance, preparation method and quantitative detecting method - Google Patents
A kind of surface plasmon resonance, preparation method and quantitative detecting method Download PDFInfo
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Abstract
It includes step that the present invention, which discloses a kind of surface plasmon resonance, preparation method and quantitative detecting method, preparation method,:A, two-dimentional antimony alkene nano material is soluble in water, obtain the antimony aqueous solution of a concentration of 0.01 10mg/ml;B, surface plasmon resonance chip is immersed in antimony aqueous solution, soaking time is 0.01 48 hours;C, by the gold nanorods solution of the single stranded DNA modification of step B treated surface plasmon resonance chip is immersed in base complementrities, soaking time is 0.01 24 hours, obtains the surface plasmon resonance based on antimony alkene.The present invention can overdelicate detection miRNA, and have ultralow detectable limit.The present invention can solve the problems, such as to reduce miRNA detectable limit difficulties at present.Detection method is simple, economical, efficient, stable, has a good application prospect.
Description
Technical field
The present invention relates to technical field of biological more particularly to a kind of surface plasmon resonance, preparation sides
Method and quantitative detecting method.
Background technology
MiRNA is single-stranded microRNA, and length is 20-24 base, and it includes Apoptosis, breeding, organ to control
Generation, development, tumour generation and hematopoiesis etc..After 1993 find for the first time, miRBase databases have issued 9539 altogether at present
Kind miRNA, wherein miRNA155 and miRNA21 etc. have high expression when tumour occurs, and miRNA is considered tumorigenic
Marker object, the ultra-sensitivity about miRNA155 and miRNA21 detect research, have to the diagnosis of the diseases such as tumour important
Meaning.Surface plasmon resonance (SPRbiosensor or Biosensors Based on Surface Plasmon Resonance device) has
In real time monitoring, the advantages that label-free, detection cycle is short, stability is good, protein and the label-free detection of DNA may be implemented, but
It is, since golden film is as the factors such as SPR chips are limited to the fixed amount of bioprobe molecule, seriously to constrain SPR technique pair
The ultra-sensitivity of the small molecules such as miRNA detects.
The technical method of main detection miRNA is mainly at present:It is RNA blottings, PCR (PCR), micro-
Array chip etc., main problem of the existing technology are:(1) fluorescent molecular is needed to mark, program is cumbersome, (2) detection cycle
Long, (3) detection sensitivity is low.
Therefore, the existing technology needs to be improved and developed.
Invention content
In view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of surface plasmon resonance sensings
Device, preparation method and quantitative detecting method, it is intended to which the sensitivity of miRNA detection modes is low in the prior art, the period is long, program for solution
The problems such as cumbersome.
Technical scheme is as follows:
A kind of preparation method of the surface plasmon resonance based on antimony alkene, wherein including step:
A, two-dimentional antimony alkene nano material is soluble in water, obtain the antimony aqueous solution of a concentration of 0.01-10mg/ml;
B, surface plasmon resonance chip is immersed in antimony aqueous solution, soaking time is that 0.01-48 is small
When;
C, the single stranded DNA of step B treated surface plasmon resonance chip is immersed in base complementrities is repaiied
In the gold nanorods solution of decorations, soaking time is 0.01-24 hours, obtains the surface plasmon resonance sensing based on antimony alkene
Device.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein before the step A also
Including:
Ball milling is carried out to bulk sb alkene nano material in advance and ultrasound is removed, then is dried in vacuo to obtain two-dimentional antimony alkene and be received
Rice material.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein in the step B, leaching
After the completion of bubble, the antimony aqueous solution not adsorbed is removed using deionized water.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein in the step C, leaching
After the completion of bubble, deionized water is then used to remove the gold nanorods solution for the single stranded DNA modification that do not adsorb.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein the base complementrity
The preparation method of the gold nanorods solution of single stranded DNA modification is as follows:
The single strand dna aqueous solution that disulfide bond is modified is added in gold nanorods solution, stands overnight, is then added
Phosphate buffer solution, after standing a period of time, traditional vacuum concentration removes the single strand dna not linked.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein finally will be after concentration
Solution is dispersed in phosphate buffered saline solution.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein the chip material system
Preparation Method is as follows:It is coated with golden film in glass or quartz surfaces.
The preparation method of the surface plasmon resonance based on antimony alkene, wherein the golden film thickness is
30-60 nanometers.
A kind of surface plasmon resonance based on antimony alkene, wherein use any one of them preparation side as above
Method is made.
A kind of quantitative detecting method of miRNA, wherein including step:
In multiple sample cells, prepare the surface plasmon resonance as described above based on antimony alkene;
The target miRNA of various concentration is configured, and will be put into respective sample pond;
Using the Biosensors Based on Surface Plasmon Resonance device test surfaces plasmon resonance curve of spectrum, formant is determined
The variation of position, and quantitative detection is carried out to target miRNA according to the variation.
Advantageous effect:The present invention in surface plasmon resonance chip surface by assembling antimony alkene, using Jenner
Rice stick signal amplification, can overdelicate detection miRNA, by analyze surface plasmon resonance sensor light set a song to music
The variation of the formant of line quantitatively detects the concentration of miRNA, and it is 10 to have ultralow detectable limit, the lowest detection limit-17M。
The result shows that the present invention can solve the problems, such as to reduce miRNA detectable limit difficulties at present.Detection method is simple, passes through
It helps, is efficient, stablizing, having a good application prospect.
Description of the drawings
Fig. 1 is the atomic force microscope images (AFM) of the two-dimentional antimony alkene nano material of the present invention.
Fig. 2 is various concentration (10 of the present invention-17-10-11M the corresponding SPR spectroscopy curve graph of miRNA solution).
Fig. 3 is various concentration (10 of the present invention-17-10-11M miRNA solution testing result figures).
Specific implementation mode
A kind of surface plasmon resonance of present invention offer, preparation method and quantitative detecting method, to make this hair
Bright purpose, technical solution and effect are clearer, clear, and the present invention is described in more detail below.It should be appreciated that herein
Described specific embodiment is only used to explain the present invention, is not intended to limit the present invention.
A kind of preparation method of surface plasmon resonance based on antimony alkene provided by the present invention comprising step
Suddenly:
It is S1, two-dimentional antimony alkene nano material is soluble in water, obtain the antimony aqueous solution of a concentration of 0.01-10mg/ml;
S2, surface plasmon resonance chip is immersed in antimony aqueous solution, soaking time is that 0.01-48 is small
When;
S3, by the single stranded DNA of step S2 treated surface plasmon resonance chip is immersed in base complementrities
In the gold nanorods solution of modification, soaking time is 0.01-24 hours, obtains the surface plasmon resonance sensing based on antimony alkene
Device.
In the present invention, antimony alkene nano material has chemical stability is high, hydrophilic, polarity is high etc. as two-dimension nano materials
Advantage, antimony alkene nano material with single stranded DNA there is stronger interaction, binding force to be far longer than double-stranded DNA.
The present invention carries out sensitive inspection using two-dimensional material antimony alkene as the sensitive layer of SPR chips, using SPR technique to miRNA
It surveys, there is ultralow detectable limit, be able to detect that a concentration of 10-17MmiRNA concentration changes.
Antimony alkene is assembled in SPR chip surfaces by the present invention, and the signal amplification based on gold nanorods is made using ssDNA
For probe molecule, when, there are when target molecule miRNA, SPR spectroscopy curve moves to left, and realizes ultralow detectable limit in analytical solution
Detect miRNA.
Specifically, further include before the step S1:
Ball milling is carried out to bulk sb alkene nano material in advance and ultrasound is removed, then is dried in vacuo to obtain two-dimentional antimony alkene and be received
Rice material.
Specifically usable ball mill carries out ball milling to bulk sb alkene nano material and obtains powdery antimony alkene nano material, after ball milling
Ultrasonic stripping is carried out to powdery antimony alkene nano material using probe sonication method.Then sheet single layer or few layer antimony are obtained by centrifugation
Alkene solution obtains two-dimentional antimony alkene nano material powder by being dried in vacuo.
In the step S1, two-dimentional antimony alkene nano material is soluble in water, obtain antimony aqueous solution;Ultrasound is removed
Two-dimentional antimony alkene nano material afterwards is soluble in water, obtains the antimony aqueous solution of favorable dispersibility.Its concentration is preferably 0.01-
10mg/ml, such as can be 1mg/ml.
In the step S2, surface plasmon resonance chip is immersed in antimony aqueous solution, when immersion
Between be 0.01-48 hours.For example, surface plasmon resonance chip can be put into SPR sample cells, then antimony alkene is water-soluble
Liquid is passed through SPR sample cells, to carry out immersion treatment.After the completion of immersion, the antimony alkene not adsorbed is removed using deionized water
Aqueous solution removes extra antimony aqueous solution.
In the step S3, the single stranded DNA for surface plasmon resonance chip being immersed in base complementrity is repaiied
In the gold nanorods solution of decorations.Specifically, the gold nanorods solution that the single stranded DNA of base complementrity is modified is passed into equipped with surface
The SPR sample cells of plasmon resonance sensor chip, to carry out immersion treatment.After the completion of immersion, deionized water is used
The gold nanorods solution for removing the single stranded DNA modification that do not adsorb, that is, the gold nanorods for removing extra single stranded DNA modification are molten
Liquid.
In above-mentioned steps S2 and step S3, used deionized water is preferably handled by deoxygenation.
Further, the preparation method of the gold nanorods solution of the single stranded DNA modification of the base complementrity is as follows:
The single strand dna aqueous solution that disulfide bond is modified is added in gold nanorods solution, stands overnight, is then added
Phosphate buffer solution, after standing a period of time, traditional vacuum concentration removes the single strand dna not linked.The list of base complementrity
Chain DNA links gold nanorods, has compared with strong interaction with antimony alkene, is largely adsorbed on antimony alkene surface.
The number of traditional vacuum concentration can be multiple, such as three times, to the solution (i.e. concentrate) after being concentrated,
To remove the single strand dna not linked.
Finally the solution after concentration is dispersed in phosphate buffered saline solution, is preserved.
Preferably, the chip material preparation method is as follows:It is coated with golden film in glass or quartz surfaces.The present invention utilizes
Antimony alkene is adsorbed on golden film surface, and single stranded DNA has very strong interaction, the single stranded DNA for linking gold nanorods a large amount of with antimony alkene
It is adsorbed on antimony alkene surface.Sensing process is exactly to form double-strand by base complementrity using miRNA and single stranded DNA, makes to inhale originally
The gold nanorods and single stranded DNA for being attached to antimony alkene surface are detached from antimony alkene surface, reduce the single stranded DNA connection gold nanorods of base complementrity
Quantity on antimony alkene surface.
Preferably, the golden film thickness is 30-60 nanometers, for example, the golden film thickness is 45 nanometers.
The present invention also provides a kind of surface plasmon resonances based on antimony alkene, using preparation side as described above
Method is made.
The present invention also provides a kind of quantitative detecting methods of miRNA comprising step:
In multiple sample cells, prepare the surface plasmon resonance as described above based on antimony alkene;
The target miRNA of various concentration is configured, and will be put into respective sample pond;
Using the Biosensors Based on Surface Plasmon Resonance device test surfaces plasmon resonance curve of spectrum, formant is determined
The variation of position, and quantitative detection is carried out to target miRNA according to the variation.For example, from low to high according to target miRNA concentration
Sequence, target miRNA is passed through successively in the sample cell equipped with surface plasmon resonance, and impregnate 0.01-24
Hour, such as 5 hours;To bent using Biosensors Based on Surface Plasmon Resonance device test surfaces plasmon resonance spectrum
Line determines the variation of resonance peak.
The present invention uses the interaction of antimony alkene and the probe ssDNA of base complementrity, application surface plasmon resonance skill
Art detects the miRNA of various concentration, by the linear change of SPR spectroscopy curve resonance angle, realizes the ultra-sensitivity to miRNA
Detection.
The present invention makes full use of antimony alkene two-dimension nano materials and the stronger interactions of probe ssDNA, as miRNA and probe
After hydridization forms double-strand, SPR spectroscopy curve movement is made to reduce, realization quantitatively detects miRNA.Sensor according to the present invention leads to
It crosses and is linked using gold nanorods and probe ssDNA, increase the sensitivity of target miRNA and reduce detectable limit.
One specific example is as follows:
1) prepare two-dimentional antimony alkene nano material
Ball milling is carried out to bulk sb alkene material using ball mill, ultrasonic stripping is carried out to it using probe sonication method, then
Sheet single layer or few layer antimony alkene solution are obtained by centrifugation, antimony alkene nano material powder is obtained by vacuum drying, is configured to not
With the antimony alkene solution of concentration, antimony alkene test chart is referring to Fig. 1, antimony alkene atomic force microscope phenogram.
2) prepared by the gold nanorods solution of the single stranded DNA modification of base complementrity
The gold nanorods that the single strand dna aqueous solution of 250 μ l, the modification of 40 μM of disulfide bond are added to 3ml, 6nM are molten
It in liquid, stands overnight, 3ml phosphate buffer solutions is then added, after standing 2h, traditional vacuum is concentrated into 0.9ml, and traditional vacuum is dense
Contracting three times, removes the single strand dna not linked, finally concentrate is dispersed in phosphate buffered saline solution.
3) assembling of the antimony aqueous solution on SPR chip golden films surface
The antimony aqueous solution of 1mg/ml is passed through SPR sample cells, was impregnated by 5 hours, then passes to the deionization of deoxygenation
Water, removal are not adsorbed on the extra antimony alkene of chip surface.
4) absorption of the gold nanorods with single stranded DNA on antimony alkene surface
By high concentration (10-11-10-3M, such as 10-5M the gold nanorods solution of the single stranded DNA modification of base complementrity) is passed through
SPR sample cells were impregnated by 0.5 hour, and the deionized water of deoxygenation is then used to remove the gold nanorods that do not adsorb and single-stranded
DNA。
5) miRNA molecule of the Mismatching of detection various concentration
By a concentration of 10-17-10-11The miRNA solution of M each leads into SPR sample cells according to low concentration to high concentration, respectively
SPR spectroscopy curve and kinetic curve are tested, the corresponding SPR spectroscopy curve of various concentration analyzes SPR spectroscopy curve referring to Fig. 2
Resonance angle situation of change realizes the ultra-sensitivity detection of various concentration miRNA molecule referring to Fig. 3.
The method of the present invention detection miRNA molecule method is simple, high sensitivity, and operability is strong.As a kind of novel
MiRNA molecule biosensor, shows high sensitivity and extremely low detectable limit, and the lowest detection limit is 10- 17M。
In conclusion the present invention by surface plasmon resonance chip surface assemble antimony alkene, using Jenner
Rice stick signal amplification, can overdelicate detection miRNA, by analyze surface plasmon resonance sensor light set a song to music
The variation of the formant of line quantitatively detects the concentration of miRNA, and it is 10 to have ultralow detectable limit, the lowest detection limit-17M。
The result shows that the present invention can solve the problems, such as to reduce miRNA detectable limit difficulties at present.Detection method is simple, passes through
It helps, is efficient, stablizing, having a good application prospect.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations should all belong to the guarantor of appended claims of the present invention
Protect range.
Claims (10)
1. a kind of preparation method of the surface plasmon resonance based on antimony alkene, which is characterized in that including step:
A, two-dimentional antimony alkene nano material is soluble in water, obtain the antimony aqueous solution of a concentration of 0.01-10mg/ml;
B, surface plasmon resonance chip is immersed in antimony aqueous solution, soaking time is 0.01-48 hours;
C, by the single stranded DNA modification of step B treated surface plasmon resonance chip is immersed in base complementrities
In gold nanorods solution, soaking time is 0.01-24 hours, obtains the surface plasmon resonance based on antimony alkene.
2. the preparation method of the surface plasmon resonance according to claim 1 based on antimony alkene, feature exist
In the step A further includes before:
Ball milling is carried out to bulk sb alkene nano material in advance and ultrasound is removed, then is dried in vacuo to obtain two-dimentional antimony alkene nanometer material
Material.
3. the preparation method of the surface plasmon resonance according to claim 1 based on antimony alkene, feature exist
In in the step B, after the completion of immersion, the antimony aqueous solution that does not adsorb is removed using deionized water.
4. the preparation method of the surface plasmon resonance according to claim 1 based on antimony alkene, feature exist
In, in the step C, after the completion of immersion, the gold nanorods for the single stranded DNA modification that then do not adsorbed using deionized water removing
Solution.
5. the preparation method of the surface plasmon resonance according to claim 1 based on antimony alkene, feature exist
In the preparation method of the gold nanorods solution of the single stranded DNA modification of the base complementrity is as follows:
The single strand dna aqueous solution that disulfide bond is modified is added in gold nanorods solution, stands overnight, phosphoric acid is then added
Buffer solution, after standing a period of time, traditional vacuum concentration removes the single strand dna not linked.
6. the preparation method of the surface plasmon resonance according to claim 5 based on antimony alkene, feature exist
In finally the solution after concentration is dispersed in phosphate buffered saline solution.
7. the preparation method of the surface plasmon resonance according to claim 1 based on antimony alkene, feature exist
In the chip material preparation method is as follows:It is coated with golden film in glass or quartz surfaces.
8. the preparation method of the surface plasmon resonance according to claim 7 based on antimony alkene, feature exist
In the golden film thickness is 30-60 nanometers.
9. a kind of surface plasmon resonance based on antimony alkene, which is characterized in that any using such as claim 1~8
Preparation method described in is made.
10. a kind of quantitative detecting method of miRNA, which is characterized in that including step:
In multiple sample cells, prepare the surface plasmon resonance as claimed in claim 9 based on antimony alkene;
The target miRNA of various concentration is configured, and will be put into respective sample pond;
Using the Biosensors Based on Surface Plasmon Resonance device test surfaces plasmon resonance curve of spectrum, resonance peak is determined
Variation, and quantitative detection is carried out to target miRNA according to the variation.
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Application publication date: 20180717 |