CN107858403B - A kind of trace target object detecting method based on single molecular fluorescence sensing - Google Patents
A kind of trace target object detecting method based on single molecular fluorescence sensing Download PDFInfo
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- CN107858403B CN107858403B CN201711047656.9A CN201711047656A CN107858403B CN 107858403 B CN107858403 B CN 107858403B CN 201711047656 A CN201711047656 A CN 201711047656A CN 107858403 B CN107858403 B CN 107858403B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Abstract
The invention discloses a kind of trace target object detecting methods based on single molecular fluorescence sensing.The detection method includes the following steps: that (1) prepares the standard solution containing object；(2) assist probes are fixed on slide, aptamer is added, assist probes hybridize with aptamer；(3) standard solution is added on slide, object is detached from it from assist probes in conjunction with aptamer；(4) signal probe is added on slide, with the assist probes being released occurs for signal probe of short duration hybridize；(5) slide is placed on the objective table of utilizing total internal reflection fluorescence microscope and is detected, single molecular fluorescence of the record fluorescence state change frequency greater than 15 times is counted, and using it as ordinate, makes standard curve by abscissa of target concentration in standard solution；(6) sample to be tested is repeated into step (3)-(5), single molecular fluorescence of the record fluorescence state change frequency greater than 15 times is counted, according to standard curve up to the concentration of object in sample to be tested.
Description
Technical field
The present invention relates to a kind of trace target object detecting methods, and in particular to a kind of trace based on single molecular fluorescence sensing
Target object detecting method belongs to trace object detection field.
Background technique
Trace analysis refers to the analysis method for extremely low concentration object.Pharmaceutical Analysis, medical diagnosis on disease, environmental analysis,
Food Safety Analysis, public safety field have extensive trace analysis demand.For example, in the morbidity early stage of some diseases,
Some biomarkers can be generated in blood, and the concentration of these markers is usually very low.
Trace analysis is the key areas of contemporary analytical chemistry, and the technology that can be used for trace analysis has reached over one hundred kind.Needle
To the trace detection of small molecule compound and metal ion, more traditional analysis method is chromatograph-mass spectrometer coupling and atom light
Spectrometry.Such methods have great advantages in reliability, repeatability, but when the longer sample pre-treatments of needs and analysis
Between and instrument cost it is higher.Since different types of compound has different optical property and chargeability, some compounds
Sensitivity can not meet trace analysis demand.Bio-sensing method is as a kind of emerging detection technique, in recent years in clinic
There are many achievements in the fields such as diagnosis, environmental analysis, food analysis.Single molecular fluorescence sensing is on single molecular fluorescence microscope
It constructs molecular recognition and signal generates primitive.Since single molecular fluorescence microscope can provide the spatial and temporal resolution of superelevation,
Can be in individual molecule level detection and quantitative objective object, the sensitivity with superelevation.Unimolecule microscope mainly includes copolymerization
Focusing microscope and total internal reflectance microscope, the latter's unimolecule flux with higher are suitable for exploitation detection architecture.Complete interior anti-
Under the excitation mode penetrated, due to the presence of evanescent field, the fluorescent molecule only within the 100nm of interface is excited, thus shape
At monolayer, solution fluorescence background is substantially reduced, but non-specific adsorption of the fluorescer at interface is often examined to unimolecule
Survey brings stronger fluorescence background.And can not only sensitivity and specificity be made to drop by simple control experiment background correction
It is low, and will affect repeatability and reliability.Therefore, more specific signal is matched on single molecule fluorescence detection platform to produce
Raw and molecular recognition system is the key that promote single molecular fluorescence sensing in the expressive force in trace analysis field.
Summary of the invention
The object of the present invention is to provide a kind of trace target object detecting method based on single molecular fluorescence sensing, the detections
Method can detect a plurality of types of trace objects in agricultural product.
Trace target object detecting method provided by the present invention based on single molecular fluorescence sensing, includes the following steps:
(1) at least three kinds of standard solution containing object are prepared；
(2) assist probes are fixed on slide, and aptamer is added, the assist probes are adapted to the nucleic acid
Body hybridization；
(3) be added the standard solution on Xiang Suoshu slide, the object make in conjunction with the aptamer its from
It is detached from the assist probes；To discharge the part assist probes, signal probe can be received；
(4) signal probe is added on Xiang Suoshu slide, the auxiliary being released in the signal probe and step (3) is visited
Of short duration hybridization occurs for needle, to generate characteristic fluorescence signal；
(5) slide is placed on the objective table of utilizing total internal reflection fluorescence microscope and is detected, record fluorescence state variation
Single molecular fluorescence of the number greater than 15 times is counted, using the concentration of object described in the standard solution as abscissa, with fluorescence
Single molecular fluorescence points of the state change frequency greater than 15 times are ordinate, make standard curve；
(6) sample to be tested is repeated into step (3)-(5), record fluorescence state change frequency is greater than 15 single molecular fluorescence points
Number arrives the concentration of object described in the sample to be tested according to the standard curve；
Existing partial complementarity sequence is less than the assist probes and institute between the signal probe and the assist probes
Existing partial complementarity sequence between aptamer is stated, when ensuring that there is no the object, the signal probe will not
Fluorescence signal is generated in conjunction with the assist probes.
In above-mentioned detection method, in step (1), a variety of standard solution can be configured as needed, such as 8 kinds.
In above-mentioned detection method, complementary base number is 9~10 between the signal probe and the assist probes
It is a.
In above-mentioned detection method, complementary base number is 13~15 between the aptamer and the assist probes
It is a.
In above-mentioned detection method, 5 ' end label Cy3 fluorophors of the signal probe.
It is affine by biotin-between the assist probes and the slide in step (2) in above-mentioned detection method
The interaction connection of element；
Biotin is marked on the assist probes；
Avidin is modified on the slide.
In above-mentioned detection method, in step (2), the condition of the hybridization is as follows:
Temperature is 20~27 DEG C, and the time is 10~30 minutes；
It is stable hybridize between the assist probes and the aptamer.
In above-mentioned detection method, in step (3), condition of the object in conjunction with the aptamer is as follows:
Temperature is 20~27 DEG C, and the reaction time is 60~120min；
By the combination of the object and the aptamer, make the aptamer from the assist probes
It is detached from, to activate the bound fraction of the assist probes Yu the signal probe.
In above-mentioned detection method, in step (4), " of short duration hybridization " mode refers to double-strandednucleic acid in its unwinding temperature
The balance of hybridization with unwinding is nearby presented in degree.When two chains are in hybridized state, fluorescent marker chain can enter total internal reflection fluorescent
Microscopical excites scope；Conversely, fluorescent marker chain can escape out swashing for utilizing total internal reflection fluorescence microscope when two chain unwindings
Range is sent out, the fluorescent value detected in reaction system in total internal reflectance microscope can generate variation.
In above-mentioned detection method, the object can be nicotinoids pesticide, such as Acetamiprid and Polychlorinated biphenyls；
The sample to be tested can be fresh and live agricultural product；
The detection method can achieve 30fM (11.9pg/L) for the detection limit of Acetamiprid, (such as Polychlorinated biphenyls
PCB-77 (3,3 ', 4,4 '-tetrachloro biphenyl)) detection limit can achieve 50fM (14.6pg/L).
In above-mentioned detection method, in step (5), the time of the detection is at least 10min.
In above-mentioned detection method, in step (1), in the standard solution, the concentration of the object is 0~
5000fM。
The of short duration hybridization of DNA is introduced single molecular fluorescence and senses system by the present invention, by reasonably designing, so that object point
Son can activate the of short duration hybridisation events of DNA, i.e. activation finger pattern signal label.Detection method can be in single molecules level
Object directly is pointed out, the sensitivity and specificity of single molecular fluorescence sensing can be increased substantially.
Detection method can rapidly and accurately detect contaminant molecule in fresh and live agricultural product, and to target
Object has the selectivity and sensitivity of height.Meanwhile detection method can effectively exclude background interference, can be applicable in
In complicated and diversified environment；And related matched reagent is easily prepared, easy to operate, and repeatability is high, is easily generalized to amateur
Personnel's operation.
Detailed description of the invention
Fig. 1 is the schematic diagram of the target stains object detecting method sensed the present invention is based on single molecular fluorescence.
Fig. 2 is the schematic diagram that the present invention detects single molecular fluorescence using total internal reflectance microscope.
Fig. 3 be with/without object in the presence of, single short fluorescent DNA probe and the substrate table for being modified with dynamic nucleic acid probe
The single molecular fluorescence track that face interaction generates.
Fig. 4 be with/without object in the presence of, the single molecular fluorescence geometric locus of output, height-low Poison state change frequency system
Meter figure.
Fig. 5 is the mark-on contaminant molecule concentration in chicken matrix in the embodiment of the present invention 1 and is only thought nucleic acid breath
The linear relationship of the single molecular fluorescence point of hybridization.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Detection method is one kind using aptamer as Recognition unit and using nucleic acid breath hybridization as signal base
The contaminant molecule detection method of member, specifically, comprising the following steps:
A, microslide is surface-functionalized
In order to realize the fixation of nucleic acid probe, first have to modify microscopy surface.Utilize three ethoxy of 3- aminopropyl
Base silane (APTES) carries out amination to surface of glass slide, and then the polyethylene glycol with succinimide valerate label is added
(mPEG), succinimide valerate label and the polyethylene glycol (bio-PEG) of biotin double labelling are reacted with amino, by poly- second
Glycol modification is used to fixed nucleic acid probe in surface of glass slide；
B, dynamic nucleic acid probe and short fluorescent DNA probe are designed according to object
Assist probes and aptamer chain are single-stranded DNA sequence, and assist probes are generally 25~30 bases, and nucleic acid is suitable
The length of ligand depends on detection object.Assist probes can be with base sequence partial complementarity on aptamers chain；At room temperature, it assists
Probe hybridizes with aptamer stabilization, is formed " dynamic nucleic acid probe "；Wherein assist probes are marked with biotin, can pass through
Biotin-avidin (for example, Streptavidin) interaction, which is coupled at, is marked with the polyethyleneglycol modified micro- of biotin
On mirror slide；There are when object in system, aptamer can be detached from, assist probes in conjunction with object with assist probes
Base all exposure, to have activated the part (Fig. 1) hybridized with short fluorescent DNA probe；
Signal probe is one section of 5 ' nucleotide sequence of the end with Cy3 label, forms of short duration hybridize with capture probe；One
As, the signal probe base number complementary with assist probes is 9~11, less than base of the assist probes in conjunction with aptamers
Number ensure that short fluorescent DNA probe will not generate signal in conjunction with assist probes there is no when object；
C, the double-strandednucleic acid that assist probes and aptamer are formed is fixed on the microslide modified；
D, object is added, object can be in conjunction with the aptamer on assist probes, and makes it from assist probes
It is detached from；To discharge assist probes, signal probe can be received；
E, signal probe is added, signal probe can be formed with the assist probes being released of short duration hybridizes balance.Due to complete
The microscopical excitation angle of internal reflection forms evanescent field in surface of glass slide, the molecule ability only in surface of glass slide 100nm
It is excited.Therefore, when signal probe is in conjunction with assist probes, fluorescence enhancement；It is glimmering when signal probe and assist probes dissociate
Light weakens.It can recorde a large amount of individual molecule phosphor dots (Fig. 2), and available one using the enhanced CCD of electronics (EMCCD)
The change in fluorescence curve of each unimolecule point in the section time.
According to the present invention, if fluorescent value present regularity variation, this unimolecule be considered as object (target
Object is by taking Acetamiprid as an example) assist probes (Fig. 3 (A)) that release if not changing at existing regularity are considered as short fluorescence
Change in fluorescence caused by DNA probe and the non-specific interaction on surface (Fig. 3 (B)).There to be different fluorescence state variations time
Several molecules are counted, and draw histogram, if it exists object, are activated since nucleic acid breath hybridizes, then most of molecule
Change (Fig. 4 (A)) with multiple fluorescence state, if it does not exist object, then most of molecule does not have multiple fluorescence state variation (figure
4(B)).Molecule of the fluorescence state variation more than 15 times is considered as nucleic acid breath hybridization single molecular fluorescence point, and then is confirmed to be mesh
Mark object.
According to the present invention, being identified as signal probe, (fluorescence state is handed over the of short duration single molecular fluorescence point hybridized of assist probes
Change more than 15 times) quantity there are linear relationships with measured target object.
Embodiment 1: single molecular fluorescence sensing detection Acetamiprid and PCB-77 are used
(1) micro- eyeglass surface modification
Disposable glass slide is 20 minutes ultrasonic in the KOH solution of 1M, it washes with water.In 14% hydrogen peroxide/ammonium hydroxide
In boil 20 minutes or more, wash with water, then in methyl alcohol ultrasound 15 minutes, cleaned with acetone.It is put at dry practice head cartridge bottom
Certain deionized water keeps moist environment.The slide of above-mentioned drying is placed on the shelf of pipette tips box.
The sodium bicarbonate solution for preparing 0.1M, as PEG buffer.Contain 250mg/ml mPEG with this buffer,
The solution of 25mg/ml biotin-PEG, drips on glass slide immediately, and every 120 μ L are covered, Bu Yaoyou with another glass slide
Bubble.It reacts 3 hours, washes with water and with being dried with nitrogen at room temperature, the surface being modified is placed on the frame of pipette tips box upward
On son.The sodium bicarbonate solution for preparing 1M, as DST buffer.The DST solution for preparing 28.6mg/ml, drips immediately in glass slide
On, every 120 μ L are covered with another glass slide, not there is bubble.Half an hour is reacted at room temperature.Wash with water glass slide
With coverslip and with being dried with nitrogen.It will be cut at the top of the Eppendorf pipette tips of 200 μ L, be placed on the coverslip table modified
Then face is carefully coated in the joint on pipette tips and surface with Epoxy glue, fixes it.
(2) probe is fixed captures with object
The T of 100 μ L is drawn with liquid-transfering gun50Buffer (10mM Tris-HCl, 50mM NaCl and 1mM EDTA, pH=8),
It is added dropwise on the slide that surface is covered with biotin-PEG, is sucked after rinse slide.
The Avidin (pH=8) of the 1mg/mL of 50 μ L is drawn with liquid-transfering gun, is added dropwise on the slide after rinse, is incubated for
It is sucked after 10min, and cleans slide twice with 1 × PBS of 50 μ L.
On the slide of cladding Avidin, the capture probe that 100 μ L concentration are 1pM biotin labeling is added dropwise, is incubated for 10min
After suck, and clean slide twice with 1 × PBS of 50 μ L.
The aptamer chain that 100 μ L concentration are 5pM is drawn with liquid-transfering gun, is added dropwise on the slide of coupling capture probe,
It is sucked after being incubated for 10min, and cleans slide twice with 1 × PBS of 100 μ L.
Fresh organic chicken (without object) that 5g is shredded is taken, the acetonitrile solution that 50mL contains 1 μM of object is added, fills
Divide and mixes 5min.Centrifuging and taking supernatant, by its with 2 × PBS be diluted to containing 5pM, 2pM, 1pM, 0.5pM, 0.2pM, 0.1pM,
The solution of 0.05pM and 0.03pM object.
Draw 2 × PBS solution of the above-mentioned Acetamiprid containing each concentration of 100 μ L or PCB-77 object respectively with liquid-transfering gun
And blank solution, it is added dropwise on slide, is sucked after 25 DEG C of incubation 60min, and clean slide twice with 1 × PBS of 100 μ L.
(3) single molecular fluorescence sensing is analyzed with data
100 μ L, which are drawn, with liquid-transfering gun contains the signal probe of 1nM, 2.5mM 3,4- methyl dihydroxy benzoate, 25nM original
Catechu adds oxygenase, and 4 × PBS of 1mM watermiscible vitamin E (anti-fluorescent dye photobleaching) is added dropwise on slide.
Slide is placed on total internal reflectance microscope objective table.
Signal probe excitation wavelength is 520nm, launch wavelength 593nm, the TIRF object that object lens are 100 times of amplification factor
Mirror.
Scintillation fluor point on shooting record slide, time for exposure 500ms record 10min.And with MATLAB, QuB software
The number of Analysis and Screening phosphor dot, for quantitative.
The unimolecule number (as Table 1 and Table 2 below) that fluorescence state change frequency in 10min is greater than 15 times is counted, with muscle
The concentration of object is abscissa in matrix, and the unimolecule number (fluorescence points) with fluorescence state change frequency greater than 15 times is vertical sits
Mark makes standard curve, as shown in Figure 5, it can be seen that for Acetamiprid experimental group, in the presence of 5pM Acetamiprid, finds really
The nucleic acid breath hybridization fluorescent recognized point 558 finds confirmation in the presence of the PCB-77 of 5pM for PCB-77 experimental group
Nucleic acid breath hybridization fluorescent point 364.
Fluorescence state change frequency when 1 various concentration Acetamiprid of table is greater than 15 unimolecule numbers
| Concentration (fM) | Unimolecule number |
| 30 | 3 |
| 50 | 5 |
| 100 | 9 |
| 200 | 25 |
| 500 | 71 |
| 1000 | 123 |
| 2000 | 230 |
| 5000 | 558 |
Fluorescence state change frequency when 2 various concentration PCB-77 of table is greater than 15 unimolecule numbers
| Concentration (fM) | Unimolecule number |
| 30 | 0 |
| 50 | 3 |
| 100 | 8 |
| 200 | 17 |
| 500 | 38 |
| 1000 | 66 |
| 2000 | 170 |
| 5000 | 364 |
The sequence of nucleic acid employed in the present embodiment is as shown in table 3.
Each nucleic acid sequence of table 3
Claims (1)
1. a kind of trace target object detecting method based on single molecular fluorescence sensing, includes the following steps:
(1) at least three kinds of standard solution containing object are prepared；
In the standard solution, the concentration of the object is 0 ~ 5000 fM；
(2) assist probes are fixed on slide, and aptamer is added, the assist probes and the aptamer are miscellaneous
It hands over；
Complementary base number is 13 ~ 15 between the aptamer and the assist probes；
The condition of the hybridization is as follows:
Temperature is 20 ~ 27 DEG C, and the time is 10 ~ 30 minutes；
It is connected between the assist probes and the slide by the interaction of biotin-avidin；
Biotin is marked on the assist probes；
Avidin is modified on the slide；
(3) it is added the standard solution on Xiang Suoshu slide, the object makes it from described in conjunction with the aptamer
It is detached from assist probes；
Condition of the object in conjunction with the aptamer is as follows:
Temperature is 20 ~ 27 DEG C, and the reaction time is 60 ~ 120min；
(4) signal probe is added on Xiang Suoshu slide, the assist probes being released in the signal probe and step (3) are sent out
Raw of short duration hybridization；
Complementary base number is 9 ~ 10 between the signal probe and the assist probes；
5 ' end label Cy3 fluorophors of the signal probe；
(5) slide is placed on the objective table of utilizing total internal reflection fluorescence microscope and is detected, record fluorescence state change frequency
Single molecular fluorescence points greater than 15 times, using the concentration of object described in the standard solution as abscissa, are become with fluorescence state
Changing single molecular fluorescence points of the number greater than 15 times is ordinate, makes standard curve；
The time of the detection is at least 10min；
(6) sample to be tested being repeated into step (3)-(5), single molecular fluorescence of the record fluorescence state change frequency greater than 15 times is counted,
The concentration of object described in the sample to be tested is arrived according to the standard curve；
Existing partial complementarity sequence is less than the assist probes and the core between the signal probe and the assist probes
Existing partial complementarity sequence between sour aptamers；
The sample to be tested is fresh and live agricultural product；
When the object is Acetamiprid, the sequence of the assist probes are as follows: 5 '-Biotin-
TTTTTTTTTTTCTTCATAATATGGTG-3 ', the sequence of the aptamer are as follows: 5 '-TGTAATTTGTCTGCAGCGGT
TCTTGATCGCTGACACCATATTATGAAGA-3 ', the sequence of the signal probe are as follows: 5 '-Cy3-ATTATGAAGA-3 '；
When the object is Polychlorinated biphenyls, the sequence of the assist probes are as follows: 5 '-Biotin-
TTTTTTTTTTCTTCGTAGCCCCGCC-3 ', the sequence of the aptamer are as follows: 5 '-GGCGGGGCTACGAAGTAGTGA
TTTTTTCCGATGGCCCGTG-3 ', the sequence of described probe are as follows: 5 '-Cy3-GGGCTACGAA-3 '.
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