CN108085370A - A kind of construction method of individual particle bioprobe and application thereof - Google Patents
A kind of construction method of individual particle bioprobe and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of construction methods of individual particle bioprobe and application thereof, include the following steps：Gold and silver core-shell nano cube (Au@AgNCs) colloidal sol and tsDNA solution are prepared respectively, and the Au@AgNCs are fixed on indium tin oxide conductive film glass surface；The tsDNA solution is added drop-wise to the indium tin oxide conductive film glass surface for being fixed with Au@AgNCs, after being incubated at room temperature, with milli-Q water, and is dried up with nitrogen, obtains the individual particle LSPR probes based on tsDNA.For prepared individual particle probe grain size between 50~60nm, detection sensitivity reaches 1aM, and LSPR scattering spectrums peak is located at 540~560nm.Compared with prior art, the present invention has following advantageous effect：1st, compared with silver nanocubes, there are Au@Ag NCs similar plasma characteristics and structure more preferably to stablize；2nd, compared with the single strand dna of one-dimentional structure and the hair clip type DNA molecular of two-dimensional structure, the tsDNA of three-dimensional structure has many advantages, such as preferably rigidity, structural stability and is easy to multifunction.
Description
Technical field
The present invention relates to a kind of individual particle LSPR based on tsDNA (tetrahedral structure DNA), (local surface plasma is common
Shake) construction method of probe and application thereof, belong to technical field of biological.
Background technology
MicroRNAs is RNAs of a kind of length in the endogenous non-protein coding of 20~24 nucleotide, usually in morning
Phase develops, and very important effect is played in the biological processes such as cell Proliferation, differentiation, apoptosis.The unconventionality expression of MicroRNAs
It is happened in precancerous malignant cell, with many cancers (such as：Lung, liver cell, large intestine and breast cancer etc.) it is related.
MicroRNA-21 (miR-21) is present in the cancerous tissue of above-mentioned many mankind, the expression especially in lung squamous cell carcinoma cancers
It is as many as 2 times in normal structure.Therefore, microRNAs can be as biomarker for the diagnosis of early-stage cancer and pre-
After treat.Develop a kind of highly sensitive analysis method tracing detection microRNAs for early-stage cancer diagnosis for biology and
Diagnostics is vital.
In many biosystems, the research of single molecules level can reveal that intermolecular interaction, dynamics and
The slight change of conformation.Feasible method currently used for single molecules level detection usually requires fluorescence molecule and marks, however glimmering
Phenomena such as photobleaching, easily occurs for light probe and signal-to-noise ratio is relatively low.In recent years, due to its it is unique dependent on size, pattern,
The optical property of component and microenvironment, plasma cause extensive research interest in chemistry and bio-sensing field.These
Sensor is based on noble metal nano particles (such as：Gold nano grain and silver nano-grain) LSPR characteristics, pass through the LSPR of particle
Peak (λmax) it is mobile as detection signal.LSPR sensors are used to measure the intermolecular interaction of metal nanoparticle surface
Cause the concern of researchers.Many different methods for fixing biological molecules to metal nanoparticle surface are also wide
General report.Also, the signal obtained from single metal nano particle can provide more detailed information.Therefore, Wo Menxi
It hopes based on single nanoparticle level to detect the interaction between the biomolecule of metal nanoparticle surface.
However, since microRNAs contents are low, using the single metal nano particle shape modified based on simple ssDNA
Into the plasma nano biosensor of probe molecule structure 1fM can only be arrived to the detection of miR-21 limit.
The content of the invention
Technical problem：The defects of the purpose of the present invention is being directed in the prior art, provides a kind of individual particle bioprobe
Construction method and application thereof, compared with silver nanocubes, Au@Ag NCs have similar plasma characteristics
And structure is more preferably stablized；Compared with the single strand dna of one-dimentional structure and the hair clip type DNA molecular of two-dimensional structure, three-dimensional knot
The tsDNA of structure has many advantages, such as preferably rigidity, structural stability and is easy to multifunction.
Technical solution：A kind of construction method of individual particle bioprobe of the present invention is the individual particle LSPR based on tsDNA
The construction method of probe, includes the following steps：
Au@AgNCs colloidal sols and tsDNA solution are prepared respectively, and the Au@AgNCs are fixed on indium tin oxide conductive film
Glass surface；
The tsDNA solution is added drop-wise to the indium tin oxide conductive film glass surface for being fixed with Au@AgNCs, at room temperature into
It after row is incubated 2~6h, is washed with ultra-pure water, and is dried up with nitrogen, obtained the individual particle LSPR based on tsDNA and visit
Pin.
Preferably, the preparation method of the Au@AgNCs colloidal sols includes the following steps：
Synthesize nanometer gold seeds；
The nanometer gold seeds with hexadecyltrimethylammonium chloride is reacted, obtains gold size solution；
After the gold size solution and ascorbic acid are reacted at 40~80 DEG C, silver nitrate is added in, is obtained after reaction
Au@AgNCs colloidal sols.
Preferably, the Au@AgNCs are in the fixing means of indium tin oxide conductive film glass surface：
By the surface clean of indium tin oxide conductive film glass it is clean after, be immersed in Au@AgNCs colloidal sols, stand 1~5min
After take out, cleaned with ultra-pure water and dried up with nitrogen, you can.
Preferably, the preparation method of the tsDNA solution includes the following steps：
Each single stranded DNA (ssDNA) is dissolved, the absorption at 260nm is measured under ultraviolet specrophotometer, and with reference to every
The molar extinction coefficient of ssDNA, determines its concentration；
Four ssDNA are mixed in equal proportion in TM buffer solutions, after adding in three (2- carboxyethyls) phosphines, are transferred to polymerase chain
In formula reaction instrument, after keeping the temperature 10~15min at 90~98 DEG C, 0~5 DEG C is cooled to rapidly, obtains tsDNA solution.
Detection and core of a kind of individual particle LSPR probes based on tsDNA prepared such as foregoing construction method in miR-21
Purposes in sour restriction endonuclease (KpnI and StuI) Activity determination.
Preferably, concrete operations of the individual particle LSPR probes based on tsDNA when detecting miR-21 are：
MiR-21 solution is added drop-wise to probe molecule surface, after reacting 2~5h at room temperature, with ultrapure water, is used in combination
Nitrogen dries up, and carries out the measurement of LSPR scattering spectrums.
Preferably, the individual particle LSPR probes based on tsDNA detection endonuclease (KpnI and
Concrete operations when StuI) are：
The endonuclease enzyme solutions of 2~5 μ L and enzyme cutting buffering liquid and ultra-pure water are mixed to get the anti-of 100~200 μ L
Answer liquid.
The reaction solution is added drop-wise to probe molecule surface, after 20~50 DEG C are reacted 10~30min, is rushed with ultra-pure water
It washes, and is dried up with nitrogen, carry out the measurement of LSPR scattering spectrums.
The basic principle of the present invention is：Using tsDNA as stent, the individual particle LSPR of structure " from bottom to top " is visited
Pin, distance, orientation of probes between nanoscale internal control manufacturing probe.Here, the individual particle Au@Ag NC based on tsDNA modifications
Probe devises plasma nano biosensor for single molecules level miR-21 and endonuclease (KpnI and StuI)
Activity determination.Testing principle for target molecule miR-21 is as shown in Figure 1, when the generation of target molecule miR-21 and tsDNA molecules is miscellaneous
Reaction is handed over, target molecule miR-21 can substitute the hydrone on Au@Ag NCs surfaces, since the refractive index (RI) of RNA molecule is more than water
The RI of molecule causes the increase of Au@Ag NCs surface RI, so as to cause Au@Ag NC-tsDNA probe molecule LSPR scattering peaks
Red shift realizes the detection to target molecule miR-21 in this, as signal.Fig. 2 is given based on Au@Ag NC-tsDNA17-miR-21
The schematic diagram of the endonuclease activity detection of nano-complex.It can be seen from the figure that when add in endonuclease KpnI or
After StuI, tsDNA17Side can be destroyed, tsDNA at this time17The position of DNA molecular will be substituted by the hydrone of surrounding on side,
Cause the reduction of particle surface RI, the LSPR of probe molecule is caused to scatter λmaxGeneration blue shift realizes the inspection to endonuclease
It surveys.
Advantageous effect：Compared with prior art, the present invention has following advantageous effect：
1st, compared with AgNCs, there are Au@Ag NCs similar plasma characteristics and structure more preferably to stablize；
2nd, the tsDNA of three-dimensional structure has very compared with the ssDNA of one-dimentional structure and the hair clip type DNA of two-dimensional structure
Strong rigidity can be in upright state in particle surface；Space orientation ability is strong, can improve substance mass transfer rate；Also,
It can accurately control the distance between probe, improve the joint efficiency of target molecule and capture probe and to be easy to multifunction etc. excellent
Point.Direct-connected DNA is replaced with the tsDNA of three-dimensional structure, and by the design to tsDNA, is corresponded to respectively on its 3 upright side
DNA sequence dna and cleavage site corresponding with endonuclease (KpnI and StuI) with miR-21 complementations, can realize simultaneously
Detection to miR-21 and endonuclease.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the Au@AgNC-tsDNA of the present invention17The principle schematic of probe molecule identification target molecule miR-21；
Fig. 2 is the present invention based on Au@Ag NC-tsDNA17The endonuclease activity detection of-miR-21 nano-complexes
Schematic diagram
Fig. 3 is the electrophoretic analysis figure of the tsDNA that embodiment 1 is prepared in the present invention；
Fig. 4 is the TEM pictures of Au@AgNCs prepared by embodiment 2 in the present invention；
Fig. 5 is the HAADF-STEM picture and elemental scan figure of single Au@AgNCs prepared by embodiment 2 in the present invention
Piece；
Fig. 6 is the uv absorption spectra of Au@Ag NCs prepared by embodiment 2 in the present invention；
Fig. 7 is the tsDNA that embodiment 3 designs in the present invention17Structure diagram；
Fig. 8 is the Au@Ag NCs surface modifications tsDNA that embodiment 3 obtains in the present invention17Front and rear (a) details in a play not acted out on stage, but told through dialogues photo and
(b) typical LSPR scattering spectrum figures；
Fig. 9 is the Au@Ag NC-tsDNA that embodiment 3 obtains in the present invention17(a) details in a play not acted out on stage, but told through dialogues photo and (b) statistics LSPR
Scattering spectrum figure；
Figure 10 is the Au@Ag NC-tsDNA obtained using embodiment in the present invention 317Probe molecule identification target molecule miR-
The typical LSPR scattering spectrum figures of (a) details in a play not acted out on stage, but told through dialogues photo and (b) before and after 21；
Figure 11 is Au@Ag NC-tsDNA in Figure 1017SEM in situ figures before and after probe molecule identification target molecule miR-21；
Figure 12 is Au@Ag NC-tsDNA7, Au@Ag NC-tsDNA17With Au@Ag NC-tsDNA26Three kinds of probe molecules exist
Identify that 1pM target molecules miR-21 causes the red shift amount at particle LSPR peaks under the same terms；
Figure 13 is Au@Ag NC-tsDNA17The corresponding LSPR scatterings light of probe molecule identification target molecule miR-21 different times
The LSPR scattering spectrums of particle when spectrogram, wherein curve 1 to 7 are reaction 0,30,60,90,120,150,180min respectively；
Figure 14 is Au@Ag NC-tsDNA17The LSPR of the target molecule miR-21 correspondences of probe molecule identification various concentration
The concentration that scattering spectrum figure, wherein curve 1 to 11 correspond to miR-21 respectively is 0,1,101, 102, 103, 104, 105, 106, 107,
108With 109aM；
Figure 15 is Au@Ag NC-tsDNA17Probe molecule LSPR scattering spectrums knots modification and the line of target molecule miR-21 concentration
Sexual intercourse figure；
Figure 16 is (a) Au@Ag NC-tsDNA17Probe molecule identifies the target molecule miR-21 (1aM to 1nM) of various concentration
The moving curve at LSPR peaks in different times；(b)Au@Ag NC-tsDNA17Probe molecule identifies single target molecule miR-21
LSPR peaks time performance graph；
Figure 17 is Au@Ag NC-tsDNA17The specific detection ability of probe molecule；
Figure 18 is Au@Ag NC-tsDNA17Detectability of the probe molecule in complex system；
Figure 19 is tsDNA17In the front and rear electrophoretic analysis figure of endonuclease KpnI and StuI effect；
Figure 20 is Au@Ag NC-tsDNA17- miR-21 nano-complexes are before and after endonuclease KpnI and StuI effect
The DFM images of typical LSPR scattering spectrums figure and particle；
Figure 21 is Au@Ag NC-tsDNA17Probe molecule is to the selectivity of endonuclease KpnI and StuI.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Primary drug involved in the present invention is as follows：
Silver nitrate (AgNO3, 99.9%), gold chloride (HAuCl4·3H2O, 99%), ascorbic acid (AA, 99%), boron hydrogen
Change sodium (NaBH4, 99%), cetyl trimethylammonium bromide (CTAB, 98%), hexadecyltrimethylammonium chloride (CTAC) purchase
From Sigma-Aldrich companies.Ethylenediamine tetra-acetic acid (EDTA), trishydroxymethylaminomethane (Tris), coke diethyl phthalate
(DEPC) purchased from Shanghai Aladdin Reagent Company, other reagents are the pure purchase of analysis in the limited public affairs of Chinese medicines group chemical reagent
Department.Tetrahedron DNA self assemblies solution is TM buffer solutions (20mM Tris, 50mM MgCl2, pH 8.0), polyacrylate hydrogel electrophoresis
Buffer solution is 1 × TBE (89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.3).Solution is all used in MicroRNA in experiment
What the water handled with DEPC was prepared.
Above-mentioned drug and reagent do not add processing as do not added other explanations.Experimental water is ultra-pure water, pure by Milli-Q
Prepared by water instrument (Milliproe Co., Ltds), resistivity is 18M Ω cm.DNA, MicroRNA and endonuclease used
Enzyme is all synthesized and purified by Dalian treasured biotech firm, and sequence is as shown in table 1.
Key instrument involved in the present invention is as follows：Uv-visible absorption spectra is divided light in Shimadzu (Japan) UV-3600
It is tested on degree meter；Scanning electron microscopic picture is shot using S-4800 (Japan) scanning electron microscope (SEM)；Transmission electron microscope picture
Piece is shot using JEM 2010 (Japan) transmission electron microscope (TEM)；Constant temperature blending instrument is Thermomixer comfort
(Eppendorf, Germany)；Polyacrylamide gel electrophoresis instrument (Bio-Rad, USA)；Details in a play not acted out on stage, but told through dialogues picture and individual particle scattering light
Spectrum use is equipped with dark field condenser (0.8<NA<0.95) Nikon Ti-U types inverted microscopes (Japan) shooting.Exciting light
Light source is 100W halogen lamp, and photo is gathered by the very color digital image-forming CCD of Nikon DS-fi, and spectrum is by being equipped with PIXIS
The Acton SP2300i types monochromator acquisition of the spectrum CCD of 400BR-excelon, raster density 300L/mm, glitter ripple
A length of 500nm is provided by Princeton Instruments.Details in a play not acted out on stage, but told through dialogues picture and individual particle scattering spectrum are put by 60 times
Big microcobjective acquisition (NA=0.8), all 20 seconds of acquisition time.
Embodiment 1
The present embodiment is related to a kind of preparation method of tsDNA, includes the following steps：
The design of tsDNA is with base pair complementarity, with as few as possible secondary structure and G/C content 50% with first-class item
Part is principle, and it is respectively tri- kinds of various sizes of tsDNA of 7bp, 17bp and 26bp to devise the length of side.The synthesis of tsDNA, preparation side
Method can refer to " A DNA nanostructure-based biomolecular probe carrier platform for
Electrochemical biosensing, Pei H, Lu N, Wen Y, et al.Advanced Materials, 2010,22
(42):4754-4758 ", specific method are as follows：
First, each ssDNA is dissolved, the absorption at 260nm is measured under ultraviolet specrophotometer, precious biology is public from Dalian
Department obtains the molar extinction coefficient of every ssDNA, determines the actual concentrations of every ssDNA；
2nd, four ssDNA for forming tsDNA are blended in equal proportion in TM buffer solutions, add in final concentration of 1~10mM
Three (2- carboxyethyls) phosphines, be made into final concentration of 1~10 μM of mixed liquor；
3rd, the mixed liquor prepared is put into Polymerized human serum albumin and continues 10~15min for 90~98 DEG C, then rapidly
It is cooled to 0~5 DEG C, you can obtain tsDNA solution.
The formation of tsDNA is verified using polyacrylate hydrogel electrophoresis (PAGE).Experimental method is as follows：First, will be formed
TsDNA and the contrast structure that is formed of single-stranded, two chains and three chains be loaded in the PAGE of 1 × tbe buffer liquid preparation,
It is run under 60V voltages；Then glue is dyed into 10~30min in 1% gel-red aqueous solutions；Finally in G:It is shot in Box,
It preserves.
Table 1
Composition chain (4) equal proportion of the tsDNA of every suit is mixed respectively, annealing, form tsDNA.It is coagulated with polypropylene
Gel electrophoresis verify whether tetrahedron nanostructured forms.The length of side is set forth as 17bp, 7bp and 26bp in Fig. 3 a, 3b and 3c
TsDNA formed glue figure, it can be seen from the figure that the tsDNA of each size can be synthesized successfully, and yield all exists
More than 85%.From same glue it can be seen from the figure that, tsDNA is compared with other several DNA structures for not being added completely into four chains
Due to 3-D solid structure, being subject to the resistance of bigger, migration rate is most slow；It can be seen by comparing the glue figure of difference
Go out, the size of tsDNA is smaller, and the position where migration corresponds to the Marker of 20bp more on the front.Glue figure result is also shown that
Sulfydryl modification does not influence the formation of DNA tetrahedral structures, the assembling available for Au@Ag NCs surfaces.
Embodiment 2
The present embodiment is related to the preparation method that a kind of surface is fixed with the indium tin oxide conductive film substrate of glass of Au@AgNCs,
Include the following steps：
First, the synthesis of nanometer gold seeds：We prepare the gold of 20~40nm of diameter using a common seed mediated growth method
Seed.In order to prepare diameter 1~5nm gold nano grains, the fresh 5~15mM boron hydrogen for first preparing 0.2~0.8mL ice water
Change sodium solution to be quickly added with stirring to 5~10mM gold chlorides of 50~150mM CTAB and 0.2~0.5mL of 5~15mL
In mixed solution, obtain brown solution and 2~5h is kept the temperature in 20~30 DEG C of water-baths to ensure that it is complete that extra sodium borohydride decomposes
It is for use afterwards.Then, gold seeds solution is made by the following method, and 50~200mM ascorbic acid solutions of 2~5mL are added to 5
It is obtained in the gold chloride mixed solution of 100~200mM CTAC solution of~15mL and 0.2~0.5mM of 5~10mL colourless
Bright solution is stirring evenly and then adding into 0.2~0.5mL and dilutes 2~6 times of 1~5nm gold nano grain solution, the color of solution by
Water white transparency becomes shiny red, for use after 0.5~2.5h of heat preservation in 20~40 DEG C of water-baths.Then, by the 50 of 2~5mL~
0.2~0.5mM gold chlorides of the ascorbic acid solution of 200mM, 10~200mM CTAC solution of 5~15mL and 5~10mL are molten
Liquid mixes, and then adds in 10~15nm gold seeds solution that 1~5mL had previously been prepared thereto, the color of solution is by water white transparency
Slowly become red, 1~3h is kept the temperature in 20~40 DEG C of water-baths, for the growth of Au@Ag NCs.The colloidal sol of gained is using Japan
The UV-3600 ultraviolet-visible spectrophotometers of Shimadzu Corporation are characterized, and the strong suction of aurosol is had at 520~540nm
Peak is received to occur.It is that aurosol prepared by 25~35nm (100 particles of statistics) is gold seeds that its grain size is found after TEM characterizations,
It is stored in brown bottle, is preserved in refrigerator under the conditions of 0~5 DEG C.
2nd, the synthesis of Au@Ag NCs：Diameter is synthesized in the method for the Au@Ag NCs of 40~60nm mainly by following several
A step.By 100~200mM CTAC of the gold seeds solution of 2~5mL, the 20~40nm previously prepared and 5~10mL in 20mL
5~20mL gold size solution is made into reaction bulb, thereto add in 2~5mL 50~200mM ascorbic acid solutions stirring 1~
50~70 DEG C of heat preservations after 5min, then, by the silver nitrate solution of 5~20mM of 0.5~2.0mL with 0.5~2.0mL/15min's
Rate is added drop-wise to by fluid filling pump in 50~70 DEG C of reaction bulbs of constant temperature, and the color of solution becomes orange rear yellowing by red.
The Au@Ag NCs solution of gained is dispersed to after 4000~7000rpm 5min are washed 2~3 times in 1~3mL ultra-pure waters, is adopted
It is characterized with the UV-3600 ultraviolet-visible spectrophotometers of Japanese Shimadzu Corporation, Au@Ag is had at 450~500nm
Absworption peak strong NCs occurs.It is about 55nm (100 particles of statistics) that its diameter is found after TEM characterizations, and the colloidal sol of preparation is
Au@Ag NCs, are stored in brown bottle, preserve under the conditions of 0~5 DEG C in refrigerator, be usually finished in one month.
3rd, the cleaning of substrate of glass：The indium tin oxide conductive film glass used in experiment uses liquid detergent respectively before use
Solution, deionized water, acetone, absolute ethyl alcohol, ultra-pure water are cleaned by ultrasonic, and are dried up with nitrogen.Finally, by indium tin oxide conductive film
Glass, which is positioned over 10~30min of irradiation in UV ozone washer, makes indium tin oxide conductive film glass surface activation, generates a large amount of
Siloxy in favor of carrying out the fixed experiment of next step Au@Ag NCs.
4th, the fixation and analysis of Au@Ag NCs：Au@Ag NCs are fixed to cleaning by us using the method for physical absorption
Clean indium tin oxide conductive film glass surface, specific method of modifying are as follows：The indium tin oxide conductive film glass that will be cleaned up
In the Au@Ag NCs colloidal sols that the diameter previously prepared for being immersed in 40~60 times of dilution is about 55nm, by indium oxide after 1~5min
Tin electropane, which takes out, removes extra Au@Ag NCs solution with its surface of ultrapure water, and is dried up with nitrogen, you can system
Obtain the equally distributed detection interfaces of Au@Ag NCs.Indium tin oxide conductive film glass is fixed on details in a play not acted out on stage, but told through dialogues inverted microscope platform,
Visible light source collects on nano-particle the i.e. scattering spectrum of observable individual particle by dark field condenser.
The TEM photos of the Au@Ag NCs colloidal sols prepared in the present embodiment are as shown in figure 4, it can be seen from the figure that synthesis
Au@Ag NCs patterns, size uniformity, diameter about 55nm.Further, Fig. 5 a confirm the nucleocapsid and member of Au@Ag NCs
Element distribution.Fig. 5 b~5d give single Au@Ag NC element Surface scan figures, it can be seen from the figure that Ag is covered in the outer of Au cores
Surface, the Elemental redistribution of the two are not overlapped, it was confirmed that the core-shell structure of Au@Ag NCs.Its ultraviolet-visible light modal data such as Fig. 6
Shown, absworption peak occurs being located at 478nm, shows at this wavelength, and plasma resonance phenomenon most easily occurs for Au@Ag NCs surfaces.
Embodiment 3
The present embodiment is related to a kind of method of modifying of tsDNA to Au@Ag NCs, includes the following steps：
The tsDNA solution for taking the 1pM embodiments 1 of 100~200 μ L synthetic is added drop-wise to embodiment 2 and fixes Au@Ag NCs
Indium tin oxide conductive film glass surface, be incubated at room temperature 2~6h.Then extra tsDNA solution is removed with ultra-pure water
It goes, and is dried up with nitrogen, you can the single Au@Ag NC LSPR probes based on tsDNA are made.
Size according to the tetrahedron length of side is named, three kinds of difference 7bp, and the tetrahedron of the 17bp and 26bp length of sides is respectively
tsDNA7, tsDNA17And tsDNA26.Due to having very strong covalent effect between silver and sulphur, with the tsDNA of the 17bp length of sides17For
Example, as shown in fig. 7, in tsDNA17Three vertex on modify sulfydryl, wherein a line (l1) vacate one section of ssDNA molecule and be used for
MiR-21 molecules are captured, in two other side (l2And l3) on separately design the recognition site of endonuclease KpnI and StuI.
In order to verify that mercapto-modified tsDNA can be assembled in Au@Ag NCs surfaces, with tsDNA17Exemplified by, use dark field light
Microscope stage is composed to observe its assembling on Au@Ag NCs surfaces.Au@Ag NCs surface modifications are set forth in Fig. 8 a and 8b
tsDNA17Front and rear details in a play not acted out on stage, but told through dialogues photo and typical LSPR scattering spectrums figure.It can be seen from the figure that tsDNA is not being added in17It
Before, the LSPR scatterings peak position (λ of Au Ag NCsmax) at 505nm, the LSPR scattering light of particle is in blueness；Adding in 1pM's
tsDNA17Afterwards, the LSPR scatterings λ of Au@Ag NCsmaxThe apparent red shift of generation passes through tsDNA to 10017Au@Ag after modification
The LSPR scatterings λ of NCsmaxIt is counted, finds the λ of particlemaxTo 556nm or so (as illustrated in figures 9 a and 9b), and particle
LSPR scattering light becomes green, shows tsDNA17It can be fixed on the surface of Au@Ag NCs, detection and core for miR-21
The research of sour restriction endonuclease (KpnI and StuI) Activity determination.
Embodiment 4
The present embodiment is related to a kind of detection of the individual particle LSPR probes to miR-21 based on tsDNA, includes the following steps：
The miR-21 of 100~200 μ L various concentrations is added drop-wise to the single Au@Ag NC based on tsDNA of above-mentioned preparation
LSPR probes react 2~5h at room temperature.Then ultrapure water is used, and is dried up with nitrogen, for the survey of LSPR scattering spectrums
Amount.The reagent used to miRNA detections is prepared with the processed Milli-Q water of DEPC in experiment, and experimental implementation all exists
It is carried out in super-clean bench.
Experimental result is made of following three parts：
First, the LSPR behaviors of miR-21 are detected
Choose the Au@Ag NC-tsDNA that LSPR scatterings initial peak is located at 556nm17Probe molecule is research object, is investigated
The LSPR scattering light colors of particle and scattering λ before and after add in target molecule miR-21maxVariation.Figure 10 gives typically
Au@Ag NC-tsDNA17Details in a play not acted out on stage, but told through dialogues photo and LSPR scatterings λ before and after probe molecule identification target molecule miR-21maxSpectrogram.From figure
In as can be seen that in the presence of no target molecule miR-21, the LSPR scattering spectrums and scattering light color of Au@Ag NCs are protected substantially
It holds constant；In the presence of the target molecule miR-21 of 1pM, the LSPR scattering light colors of Au@Ag NCs change to yellow green by green,
LSPR scatters λmaxBy original 556nm red shifts to 587nm, the red shift of 31nm has occurred.Meanwhile the Au@to being chosen in Figure 10 a
Ag NC-tsDNA17Probe molecule has carried out the analysis (as shown in figure 11) of SEM in situ, in Au@Ag NC-tsDNA17Probe point
In son and target molecule miR-21 crossover process, the pattern of Au@Ag NCs does not change.It can be pushed away by above-mentioned experimental result
Break and, Au@Ag NC-tsDNA17After probe molecule identification target molecule miR-21, hybridization forms DNA double helical structure, DNA molecular
Partial moisture in gap is discharged, and causes the increase of Au@Ag NCs surface refractive indexs, and particle LSPR is caused to scatter light color
Variation and its LSPR scattering λmaxRed shift, so as to fulfill the detection to target molecule miR-21.
Report in document, the density of DNA probe molecule identify that the process of target molecule has serious shadow for it
It rings.In order to obtain optimal detection performance, applicants studied various sizes of tsDNA to the detection result of miRNA.Mechanism is such as
Shown in Fig. 1, we select tsDNA respectively7, tsDNA17And tsDNA26Modify single Au@Ag NC surface constructions Au@Ag
NC-tsDNA7, Au@Ag NC-tsDNA17With Au@Ag NC-tsDNA26Three kinds of individual particle LSPR probe molecules have investigated three kinds of spies
The hybridization of pin molecule and target molecule miR-21.Its result is as shown in figure 12, and under the same conditions, above-mentioned three kinds of probe molecules are in list
A Au@Ag NC surfaces hybridize with 1pM target molecules miR-21, the corresponding LSPR scattering spectrum red shift amount (Δs for causing particle
λmax-red) it is respectively 15nm, 31nm and 25nm, wherein Au@Ag NC-tsDNA17Cause particle with target molecule miR-21 hybridization
LSPR scattering Δs λmax-redMaximum, detection result is most apparent, and the selection for single-particle miRNA nano-sensor probe molecules provides
Foundation.
Result of study based on front, we select the tsDNA for 17bp using the length of side17The single Au@Ag NC of modification make
To identify the probe of target molecule miR-21.In order to study Au@Ag NC-tsDNA17Probe molecule is with target molecule miR-21 single
The dynamic process of Au@Ag NC surfaces hybridization, we have recorded particle LSPR scattering spectrums λ in real timemaxVariation.Figure 13 gives
Au@Ag NC-tsDNA are gone out17The corresponding LSPR scattering spectrums figure of probe molecule identification target molecule miR-21 different time sections.From
It can be seen from the figure that, in the presence of no miR-21, the LSPR scattering spectrums λ of Au@Ag NCmaxIt is held essentially constant；When with
In the presence of 1pM target molecules miR-21, the LSPR scattering spectrums λ of Au@Ag NCmaxIt can occur as time increases apparent red
It moves, and Δ λmax-redQuickly increase in the starting stage, when reacted between reach 2h after, reaction rate tends towards stability, reaction
Δ λ after 3hmax-redReach definite value 31nm, it is believed that reaction terminates substantially.
2nd, miR-21 is quantitatively detected
By investigating metal nanoparticle LSPR scattering spectrums λmaxAnd the variation of scattering light color, it can be very sensitive right
Target molecule is detected, so being measured in this way to a series of target molecule miR-21 of various concentrations.Figure 14 gives
Au@Ag NC-tsDNA are gone out17Probe molecule identifies the corresponding LSPR scattering spectrums figures of target molecule miR-21 of various concentration.From
In as can be seen that the LSPR scatterings Δ λ of particlemax-redIncrease with the increase of target molecule miR-21 concentration, detect dynamic model
It encloses from 1aM to 1nM, across 10 orders of magnitude, when detection is down to 1aM, the LSPR scattering Δs λ of particlemax-redFor 4nm, noise
Than more than 5.0 (Figure 15).Based on single Au@Ag NC-tsDNA17The plasma nano biosensor of probe molecule structure
Detection sensitivity be better than the biosensor of many individual particles level reported in the past.
Choose initial LSPR scatterings λmaxAu@Ag NC-tsDNA at 556nm17Probe molecule is as research object, profit
With LSPR spectrometers to Au@Ag NC-tsDNA17The process of the target molecule miR-21 of probe molecule identification various concentration has carried out reality
Shi Guangpu is tracked.As shown in Figure 16 (a), after the target molecule miR-21 (1aM to 1nM) of various concentration is added in, Au@Ag NC-
tsDNA17The LSPR scatterings λ of probe moleculemaxDifferent degrees of red shift occurs, increases with the increase of target molecule miR-21 concentration
Add, maximum reaches as many as 40nm.As previously mentioned, Δ λmax-redQuickly increase in the starting stage, when reacted between reach 2h
Afterwards, reaction rate tends towards stability, Δ λmax-redReach more than the 95% of maximum, it is believed that reaction terminates substantially.Figure 16
(b) Au@Ag NC-tsDNA are given17The target molecule miR-21 of probe molecule identification 1pM is dissipated in the LSPR of 110min to 120min
Penetrate λmaxTime performance graph.It can be seen from the figure that due to target molecule miR-21 and Au@Ag NC-tsDNA17Probe molecule
The collision effect at interface causes the LSPR of particle to scatter λmax(it is less than 0.2nm) within the specific limits to beat；As Au@Ag NC-
tsDNA17When hybridization reaction occurs for probe molecule and single target molecule miR-21, caused respectively at 113min and 117min
The LSPR scatterings λ of grainmaxAn about 0.4nm red shift.
3rd, single Au@Ag NC-tsDNA17Detectability in the specific detection ability and complex system of probe molecule
Specificity and selectivity to target molecule detection are to weigh the important indicator of a probe molecule.Figure 17 and Figure 18 points
Au@Ag NC-tsDNA are not given17Probe molecule is to the specific detection ability of target molecule miR-21 and in complex system
Detectability.As can be seen from Figure 17, as Au@Ag NC-tsDNA17Probe molecule and the target molecule of same concentrations (1pM)
When miR-21, the miRNA of single base mispairing and random miRNA have an effect, random miRNA scatters λ to particle LSPRmax
Influence almost can be ignored；Au@Ag NC-tsDNA17Probe molecule identification target molecule miR-21 causes particle LSPR to dissipate
Penetrate Δ λmax-redIt is Δ λ caused by the miRNA of single base mispairingmax-redAs many as 2 times.The result shows that Au@Ag NC-
tsDNA17Probe molecule has specific detection ability to target molecule miR-21.In addition, tsDNA has good anti-protein adsorption
With the degradation-resistant ability in serum, it can be used for the detection of complex system target molecule.As shown in figure 18, as this Au@Ag
NC-tsDNA17Probe molecule detection hyclone (FBS) and during the inner target molecule miR-21 of human serum (HS), will not both increase the back of the body
Scape signal, will not lossing signal molecule.Comparative result find, in 50% hyclone and in 50% human serum with it is pure molten
The signal of the 1pM target molecules miR-21 measured in liquid is compared, the LSPR scattering Δs λ of particlemax-redFluctuation is within 10%.Show
Au@Ag NC-tsDNA17Probe molecule can be with the target molecule in Selective recognition complex system, to being applied to the inspection of clinical sample
Survey brings hope.
Embodiment 5
The present embodiment is related to a kind of individual particle LSPR probes based on tsDNA to endonuclease (KpnI and StuI) activity
Detection, include the following steps：
The enzyme solutions of 2~5 μ L and enzyme cutting buffering liquid and ultra-pure water are mixed to get to the reaction solution of 100~200 μ L, then
The detecting probe surface of the Au@Ag NCs of above-mentioned tsDNA modifications is added dropwise to, after 25~40 DEG C are reacted 10~30min, use is ultrapure
Water rinses, and is dried up with nitrogen, carries out the measurement of LSPR scattering spectrums.
In order to realize with single Au@Ag NC-tsDNA17Plasma nano biosensor as probe molecule it is more
Functionality, we are applied to the detection of endonuclease KpnI and StuI activity.Core is verified with polyacrylate hydrogel electrophoresis
Whether sour restriction endonuclease KpnI and StuI is to tsDNA17There is shear action.Figure 19 gives tsDNA17In endonuclease KpnI and
The front and rear electrophoretic analysis figure of StuI effects.It can be seen from the figure that after endonuclease KpnI or StuI effect
tsDNA17Migration rate be compared to it is fast before using, and by endonuclease KpnI and StuI simultaneously act on after tsDNA17,
2 bar segments are cut into, successfully illustrate endonuclease KpnI and StuI to tsDNA17All there is apparent shear action.
We scatter λ using LSPR spectrometers and details in a play not acted out on stage, but told through dialogues inverted microscope (DFM) with initial LSPRmaxPositioned at the Au@Ag NC- of 587nm
tsDNA17- miR-21 nano-complexes are research object, as shown in figure 20, are existed in no endonuclease KpnI or StuI
When, Au@Ag NC-tsDNA17The LSPR scatterings λ of-miR-21 nano-complexesmaxIt is held essentially constant (I)；In endonuclease
In the presence of KpnI or StuI, Au@Ag NC-tsDNA17The LSPR scatterings λ of-miR-21 nano-complexesmaxCertain indigo plant has occurred
Move (Δ λmax-blue), it is respectively 8nm (II, Δ λmax-blue-miR-21@KpnI=8nm) and 10nm (III, Δ λmax-blue-miR-21@StuI
=10nm), LSPR scattering light colors change to yellow green by orange；When endonuclease KpnI and StuI are existed simultaneously, Au@
Ag NC-tsDNA17The LSPR scattering light colors of-miR-21 nano-complexes change to green, LSPR scatterings λ by orangemaxBy original
The 587nm come is blue shifted to 569nm, and blue shift (IV, Δ λ of 18nm has occurredmax-blue-miR-21@KpnI@StuI=18nm).Illustrate to work as and divide
Not Jia Ru endonuclease KpnI or StuI when, tsDNA17Side (l2Or l3) can be destroyed respectively；It is added in when simultaneously in nucleic acid
During enzyme cutting KpnI and StuI, tsDNA17Two side (l2And l3) can be destroyed simultaneously, tsDNA at this time17Side (l2Or l3) on DNA
The position of molecule will be substituted by the hydrone of surrounding, cause the reduction of the RI near particle, so that the LSPR of probe molecule
Scatter λmaxGeneration blue shift.
In order to further verify with single Au@Ag NC-tsDNA17Plasma nano bio-sensing as probe molecule
Device can specificity detection endonuclease KpnI and StuI activity, in addition we have selected two kinds of endonucleases
HindIII and SalI is as control.Figure 21 gives Au@Ag NC-tsDNA17- miR-21 nano-complexes identify different nucleic acid
LSPR scatters λ before and after restriction endonuclease KpnI, StuI, HindIII and SalImaxVariation.It can be seen from the figure that in same reaction
Under the conditions of, with Au@Ag NC-tsDNA17The LSPR scatterings λ of-miR-21 nano-complexes in itselfmaxIt compares, identifies endonuclease
After KpnI or StuI, Au@Ag NC-tsDNA17The LSPR scatterings λ of-miR-21 nano-complexesmaxCertain indigo plant has occurred respectively
It moves, and after identifying endonuclease HindIII or SalI, Au@Ag NC-tsDNA17The LSPR scatterings of-miR-21 nano-complexes
λmaxThere is no apparent variation occurs.These results indicate that the plasma nano biosensor of structure is to surveying endonuclease
Enzyme KpnI and StuI have good selectivity.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (7)
1. a kind of construction method of individual particle bioprobe, which is characterized in that this method comprises the following steps：
1) prepares Au@AgNCs colloidal sols and tetrahedral structure DNA solution respectively, and the Au@AgNCs are fixed on tin indium oxide
Electropane surface；
2) the tetrahedral structure DNA solution is added drop-wise to the indium tin oxide conductive film glass surface for being fixed with Au@AgNCs, room by
Temperature is lower be incubated 2~6h after, with milli-Q water, and dried up with nitrogen, obtain the individual particle LSPR probes based on tsDNA.
2. the construction method of individual particle bioprobe as described in claim 1, which is characterized in that the system of the Au@AgNCs colloidal sols
Preparation Method includes the following steps：
1.1) synthesizes nanometer gold seeds；
1.2) reacts the nanometer gold seeds with hexadecyltrimethylammonium chloride, obtains aurosol；
1.3) after is reacted the aurosol and ascorbic acid at 40~80 DEG C, silver nitrate is added in, is obtained after reaction
Au@AgNCs colloidal sols.
3. the construction method of individual particle bioprobe as described in claim 1, which is characterized in that the Au@AgNCs are fixed on table
The method of face indium tin oxide conductive film glass is：
By the surface clean of indium tin oxide conductive film glass it is clean after, be immersed in Au@AgNCs colloidal sols, stand 1~5min after take
Go out, cleaned with ultra-pure water and dried up with nitrogen, you can.
4. the construction method of individual particle bioprobe as described in claim 1, which is characterized in that the tetrahedral structure DNA is molten
The preparation method of liquid includes the following steps：
Each single stranded DNA is dissolved, the absorption under ultraviolet specrophotometer at measure 260nm, and every single stranded DNA of reference
Molar extinction coefficient determines its concentration；
Four single stranded DNAs are mixed in equal proportion in TM buffer solutions, after adding in three (2- carboxyethyls) phosphines, are transferred to polymerase chain
It reacts in instrument, after continuing 10~15min at 90~98 DEG C, is cooled to~5 DEG C rapidly, obtains tsDNA solution.
5. individual particle bioprobe prepared by a kind of construction method as described in the appended claim 1 MicroRNA-21 detection with
And the purposes in endonuclease activity detection, which is characterized in that the endonuclease includes KpnI and StuI.
6. purposes as claimed in claim 5, which is characterized in that the individual particle bioprobe is being detected in MicroRNA-21
Concrete operations during miR-21 are：
MiR-21 solution is added drop-wise to the surface of probe molecule, after reacting 2~5h at room temperature, with ultrapure water, and uses nitrogen
Air-blowing is done, and carries out the measurement of LSPR scattering spectrums.
7. purposes as claimed in claim 5, which is characterized in that the individual particle bioprobe is specific in endonuclease
It operates and is：
The endonuclease enzyme solutions of 2~5 μ L and enzyme cutting buffering liquid and ultra-pure water are mixed to get to the reaction solution of 100~200 μ L,
The reaction solution is added drop-wise to probe molecule surface, after 20~50 DEG C are reacted 10~30min, with ultrapure water, and
It is dried up with nitrogen, carries out the measurement of LSPR scattering spectrums.
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