CN109852613A - Functional gold nanoparticles highly sensitive nucleic acid detection method based on nucleic acid probe modification - Google Patents
Functional gold nanoparticles highly sensitive nucleic acid detection method based on nucleic acid probe modification Download PDFInfo
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Abstract
The invention belongs to DNA detection technique fields, disclose a kind of functional gold nanoparticles highly sensitive nucleic acid detection method based on nucleic acid probe modification；Surface modification probe is complete complementary probe and half complementary probe；The probe tails of the preparation method of the surface modification probe carry out sulfydryl modification, pass through nanogold outer layer PEG molecule, directly with nanometer gold surface by sulfydryl in conjunction with；Obtain three kinds of detection reaction systems: ccpDNA-PEG-AuNPs, hcpDNA1-PEG-AuNPs, hcpDNA2-PEG-AuNPs.Present invention detection is quick: 30min completes reaction；It is easy to operate；System is simple, does not need accurate temperature control, does not need enzyme and participates in reaction；It is of less demanding to detection device, it is only necessary to UV-Visible spectral detection；High sensitivity: femtomole rank.
Description
Technical field
The invention belongs to DNA detection technique field more particularly to a kind of functional gold nanoparticles based on nucleic acid probe modification
Highly sensitive nucleic acid detection method.
Background technique
Currently, the prior art commonly used in the trade is such that PCR is most widely used DNA detection technique, it is particularly suitable for
The detection of trace amount DNA.However PCR needs the primer for different drone design complexity, and needs precise control of temperature
Circulation detects (point-of-care test, POCT) so being difficult to use in by bed.Isothermal duplication is not due to needing accurate temperature
Loop control, one temperature condition of entire reaction needs are spent, therefore be expected to substitution PCR to become more effective DNA detection means.
However PCR and isothermal amplification require to use different enzymes, and cryo-conservation is needed before use, participate in needing when reaction most
Suitable reaction temperature, therefore reaction reagent storage and reaction condition are required stringent.Have benefited from the development of chemistry with nanotechnology,
Many new detection methods are introduced in detection of nucleic acids, such as electrochemistry, light spectrum image-forming, chromogenic reaction etc..Wherein utilize nanometer
The change of surface plasma resonance (localized surface plasmon resonance, LSPR) signal of gold is as core
The method of sour inspection policies is very suitable for POCT since its fast high-sensitive operates detection.The DNA of fast high-sensitive degree is examined
It surveys for disease early diagnosis and accurate differentiation important role.Most widely used DNA detection PCR needs at least 1 at present
Small the reaction time, while design of primers is complicated, entire amplification procedure needs accurate temperature control.Nanogold is unique
Matter is widely used and quickly detects with large biological molecule, at present in field of nucleic acid detection, the various inspection policies based on nanogold
It receives significant attention, such as chromogenic reaction, Surface enhanced Raman spectroscopy and electrochemistry.However these detection methods need syncaryon
The technologies such as acid amplification or the amplification of enzyme-linked grade, according to signal reading method, there are two types of the detection of nucleic acids strategies based on nanogold LSPR.
The first is since dielectric change of the nanogold to its ambient enviroment or surface adsorbate is very sensitive.The addition of determined nucleic acid or
The interaction that person's gold surface occurs can all change the dielectric constant or refraction coefficient on its surface, and accordingly cause LSPR red shift.
Second is to guide between nanogold mutually to assemble by nucleic acid, causes the suction in the high wavelength region of ultraviolet-visible spectrum
Light value increases or color change occurs for reaction solution.Two kinds of detection of nucleic acids strategies based on nanogold LSPR all do not need enzyme
It participates in, reaction temperature is not strict with, can be completed under room temperature environment, entire reaction process is within 30min, detection
Ultraviolet specrophotometer is infrastructure device together, therefore is very suitable for POCT.Unsuitable POCT complicated for operation other quickly inspections
It surveys.Detection without amplified signal is not able to satisfy low abundance DNA detection demand in clinical sample only in nanomole rank.Therefore urgently
A kind of quick, simple and highly sensitive DNA detection reagent need to be developed.
In conclusion problem of the existing technology is: needing that various amplifications etc. is combined to put at present in nucleic acid detection method
Big technology, unsuitable POCT complicated for operation other quickly detection.
It solves the difficulty and meaning of above-mentioned technical problem: nanogold LSPR method being used alone at present, detects the spirit of nucleic acid
Sensitivity is inadequate, needs to combine the amplification methods such as other amplifications, but will increase the complexity of operation in this way, reduces extensive POCT and answer
Potential.
Field quick detection nucleic acid meaning (POCT meaning): POCT be health care workers medical field application it is small-sized,
Instrument that is portable, can obtaining inspection result immediately is detected, or the arbitrary site except hospital consulting room implements
A variety of detection projects clinical examination.The significance of POCT is mainly reflected in, and is obtained in the time earlier related to disease
Information realization early stage disorder in screening and diagnosis, such as doctor can be helped to identify patient earlier the detection of myocardial injury markers
Myocardial damage situation, to suspicious crowd carry out tumor-marker analyte detection be expected to realize tumour early screening and discovery.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of functional gold nanoparticles based on nucleic acid probe modification
Highly sensitive nucleic acid detection method.
The invention is realized in this way a kind of surface modification probe, the surface modification probe be complete complementary probe and
Half complementary probe；
The sequence of the complete complementary probe are as follows: SEQ ID NO:2；The sequence of half complementary probe is SEQ ID NO:
3 and SEQ ID NO:4.
Further, the substrate of the surface modification probe: the nanogold of low-molecular-weight polyethylene glycol modification.
Another object of the present invention is to provide a kind of preparation method of surface modification probe, the surface modification is visited
The probe tails of the preparation method of needle carry out sulfydryl modification, pass through nanogold outer layer PEG molecule, directly pass through with nanometer gold surface
Sulfydryl combines；Obtain three kinds of detection reaction systems: ccpDNA-PEG-AuNPs, hcpDNA1-PEG-AuNPs, hcpDNA2-PEG-
AuNPs。
Another object of the present invention is to provide a kind of functional gold nanoparticles using the surface modification probe are highly sensitive
Nucleic acid detection method is spent, the functional gold nanoparticles highly sensitive nucleic acid detection method includes:
Target: single-stranded target DNA；
- CCP method: in ccpDNA-PEG-AuNPs solution, due to mutual between PEG molecule and surface DNA probe
Effect, nano particle have moiety aggregation, and UV detection spectrum upper surface is that high wavelength period acromion occurs.When be added tDNA with
Ccp probe complete complementary on ccpDNA-PEG-AuNPs, breaks the interaction between PEG molecule and surface DNA probe,
Nano particle depolymerization, UV detect the peak value that spectrum upper surface is high wavelength period acromion and reduce.
Another object of the present invention is to provide a kind of functional gold nanoparticles using the surface modification probe are highly sensitive
Spend nucleic acid detection method, the functional gold nanoparticles highly sensitive nucleic acid detection method include: hcpDNA1-PEG-AuNPs and
In the solution that hcpDNA2-PEG-AuNPs is mixed in equal volume, nano particle has moiety aggregation, and it is Gao Bo that UV, which detects spectrum upper surface,
There is acromion in long section；After tDNA is added, tDNA with hcp1 the and hcp2 probes complementary in nanogold, forms sandwich knot respectively
Structure breaks the interaction between PEG molecule and surface DNA probe, nano particle depolymerization, and it is height that UV, which detects spectrum upper surface,
The peak value of wavelength period acromion reduces.
In conclusion advantages of the present invention and good effect are as follows: detection is quick: 30min completes reaction；It is easy to operate；Body
System is simple, does not need accurate temperature control, does not need enzyme and participates in reaction；It is of less demanding to detection device, it is only necessary to UV-
Visible spectral detection；High sensitivity: femtomole rank.
Detailed description of the invention
Fig. 1 is the functional gold nanoparticles highly sensitive nucleic acid detection provided in an embodiment of the present invention based on nucleic acid probe modification
Method flow diagram.
Fig. 2 is the functional gold nanoparticles highly sensitive nucleic acid detection provided in an embodiment of the present invention based on nucleic acid probe modification
Method implementation flow chart.
Fig. 3 is Electronic Speculum phenogram and absorption spectrum characterization schematic diagram provided in an embodiment of the present invention；
In figure: (a) the Electronic Speculum phenogram of PEG-AuNP；(b) UV-Visble absorption spectrum characterizes: PEG-AuNP (black),
HcpDNA1-PEG-AuNP (red), the hcpDNA2-PEG-AuNP (blue), the ccpDNA-PEG-AuNP (green).
Fig. 4 is UV-Visble absorption spectrum schematic diagram provided in an embodiment of the present invention；
In figure: various concentration tDNA uses (a) CCP method and (b) MIX method to detect respectively；
Fig. 5 be (a) provided in an embodiment of the present invention with the increase of tDNA concentration at 580nm absorbance value (A580)
Variation, black are CCP method, and red is MIX method；(b) in CCP method 10-15To 10-12The tDNA and absorbance of M concentration
Value carries out linear fit；(c) in MIX method 10-15To 10-13The tDNA and absorbance value of M concentration carry out linear fit.
Fig. 6 is the A580 that the two methods of mispairing single stranded DNA provided in an embodiment of the present invention relative to target dna detect
It is worth schematic diagram.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
Surface modification probe provided in an embodiment of the present invention is complete complementary probe and half complementary probe；
The sequence of complete complementary probe are as follows: SEQ ID NO:2；The sequence of half complementary probe is SEQ ID NO:3 and SEQ
ID NO:4。
As shown in Figure 1, the functional gold nanoparticles high sensitivity core provided in an embodiment of the present invention based on nucleic acid probe modification
Detection method includes the following steps for acid:
In S101:ccpDNA-PEG-AuNPs solution, due to the interaction between PEG molecule and surface DNA probe,
Nano particle has moiety aggregation, and UV detection spectrum upper surface is that high wavelength period acromion occurs；
S102: when the ccp probe complete complementary being added on tDNA and ccpDNA-PEG-AuNPs, break PEG molecule and
Interaction between the DNA probe of surface, nano particle depolymerization, UV detect the peak value that spectrum upper surface is high wavelength period acromion and drop
It is low.
Application principle of the invention is further described with reference to the accompanying drawing.
1. substrate: the nanogold (gold of low-molecular-weight polyethylene glycol (Poly Ethylen Glycol, PEG) modification
Nanoparticles, AuNP) i.e. PEG-AuNPs；
2. surface modification probe
Complete complementary probe (complete complementary capture probe, ccpDNA)；
- half complementary probe (halfcomplementary capture probe, hcpDNA)
3. prepared by detection probe
Probe tails carry out sulfydryl modification, pass through nanogold outer layer PEG molecule, directly pass through sulfydryl knot with nanometer gold surface
It closes.Obtain three kinds of detection reaction systems: ccpDNA-PEG-AuNPs, hcpDNA1-PEG-AuNPs, hcpDNA2-PEG-AuNPs；
4. the corresponding two kinds of detection methods of two kinds of probes
Target: single-stranded target DNA (tDNA)；
- CCP method: in ccpDNA-PEG-AuNPs solution, due to mutual between PEG molecule and surface DNA probe
Effect, nano particle have moiety aggregation, and UV detection spectrum upper surface is that high wavelength period acromion occurs.When be added tDNA with
Ccp probe complete complementary on ccpDNA-PEG-AuNPs, breaks the interaction between PEG molecule and surface DNA probe,
Nano particle depolymerization, UV detect the peak value that spectrum upper surface is high wavelength period acromion and reduce；
MIX method: in the solution that hcpDNA1-PEG-AuNPs and hcpDNA2-PEG-AuNPs are mixed in equal volume, due to
Interaction between PEG molecule and surface DNA probe, nano particle have moiety aggregation, and it is height that UV, which detects spectrum upper surface,
There is acromion in wavelength period.After tDNA is added, tDNA with hcp1 the and hcp2 probes complementary in nanogold, is formed sandwich respectively
Structure, breaks the interaction between PEG molecule and surface DNA probe, nano particle depolymerization, and UV detection spectrum upper surface is
The peak value of high wavelength period acromion reduces.
1 determined nucleic acid of table and probe sequence design
Wherein tDNA is target DNA to be measured, and ccpDNA is the probe with tDNA complete complementary, is used for CCP method；hcpDNA1
It is that half complementary probe, modification are mixed for the side MIX after on PEG-AuNP in equal volume respectively with the both ends tDNA with hcpDNA2
Method.
M4T is the single stranded DNA for having 4 base mispairings relative to tDNA, and underline position is mispairing point.NC be relative to
The single stranded DNA of the complete mispairing of tDNA, i.e. random sequence.
Application effect of the invention is explained in detail below with reference to experiment.
The two is used for the Evaluation on specificity to method
Electronic Speculum phenogram (b) the UV-Visble absorption spectrum of Fig. 3 (a) PEG-AuNP characterizes: PEG-AuNP (black),
HcpDNA1-PEG-AuNP (red), the hcpDNA2-PEG-AuNP (blue), the ccpDNA-PEG-AuNP (green).
PEG-AuNP good dispersion is shown under electron microscope, it is uniform in size.UV figure illustrates the successful modification of nucleic acid sequence, phase
There is similar red shift for the LSPR of PEG-AuNP, hcpDNA1-PEG-AuNP and hcpDNA2-PEG-AuNP, is moved on to by 521
527nm, and since ccpDNA nucleic acid sequence is longer, the LSPR of ccpDNA-PEG-AuNP has bigger red shift, is moved on to by 521
529nm。
Fig. 4 UV-Visble absorption spectrum: various concentration tDNA uses (a) CCP method and (b) MIX method to detect respectively, arrow
Head direction is shown, with the increase of tDNA concentration, the absorbance value (i.e. A580) at wavelength 580nm is reduced；Various concentration
TDNA with (c) hcpDNA1-PEG-AuNP and (d) hcpDNA2-PEG-AuNP solution reaction respectively, there is no absorbance value intensity
Change, tDNA and two kinds of probes illustrate that variation caused by tDNA is effective in MIX method there is no reacting.
For Fig. 5 (a) with the variation of the increase of tDNA concentration absorbance value (A580) at 580nm, black is CCP method,
Red is MIX method；(b) in CCP method 10-15To 10-12The tDNA and absorbance value of M concentration carry out linear fit；(c) right
10 in MIX method-15To 10-13The tDNA and absorbance value of M concentration carry out linear fit.
LOD (limit ofdetection) is carried out plus the method for 3 times of standard errors by blank signal to be calculated, CCP
LOD with MIX is respectively 124aM and 2.54fM, the technology that the remote super existing LSPR based on AuNP of sensitivity changes.
The A580 value that Fig. 6 is detected relative to the mispairing single stranded DNA of target dna with two methods, there are ability by only tDNA
Cause effective A580 to change, illustrates that reaction system specificity is good.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>the first affiliated hospital, army medical university, ground force, the Chinese People's Liberation Army
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Claims (5)
1. a kind of surface modification probe, which is characterized in that the surface modification probe is complete complementary probe and half complementary probe；
The sequence of the complete complementary probe are as follows: SEQ ID NO:2；The sequence of half complementary probe be SEQ ID NO:3 and
SEQ ID NO:4。
2. surface modification probe as described in claim 1, which is characterized in that the substrate of the surface modification probe: low molecule
Polyethyleneglycol modified nanogold.
3. a kind of preparation method of surface modification probe as described in claim 1, which is characterized in that the surface modification probe
The probe tails of preparation method carry out sulfydryl modification, pass through nanogold outer layer PEG molecule, directly pass through sulfydryl with nanometer gold surface
In conjunction with；Obtain three kinds of detection reaction systems: ccpDNA-PEG-AuNPs, hcpDNA1-PEG-AuNPs, hcpDNA2-PEG-
AuNPs。
4. a kind of functional gold nanoparticles highly sensitive nucleic acid detection method using surface modification probe described in claim 1,
It is characterized in that, the functional gold nanoparticles highly sensitive nucleic acid detection method includes:
Target: single-stranded target DNA；
- CCP method: in ccpDNA-PEG-AuNPs solution, due to the interaction between PEG molecule and surface DNA probe,
Nano particle has moiety aggregation, and UV detection spectrum upper surface is that high wavelength period acromion occurs；As addition tDNA and ccpDNA-PEG-
Ccp probe complete complementary on AuNPs, breaks the interaction between PEG molecule and surface DNA probe, nano particle solution
Poly-, UV detects the peak value that spectrum upper surface is high wavelength period acromion and reduces.
5. a kind of functional gold nanoparticles highly sensitive nucleic acid detection method using surface modification probe described in claim 1,
It is characterized in that, the functional gold nanoparticles highly sensitive nucleic acid detection method includes: hcpDNA1-PEG-AuNPs and hcpDNA2-
In the solution that PEG-AuNPs is mixed in equal volume, nano particle has moiety aggregation, and UV detects spectrum upper surface as the appearance of high wavelength period
Acromion；After tDNA is added, tDNA with hcp1 the and hcp2 probes complementary in nanogold, forms interlayer structure, breaks respectively
Interaction between PEG molecule and surface DNA probe, nano particle depolymerization, it is high wavelength period that UV, which detects spectrum upper surface,
The peak value of acromion reduces.
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Cited By (1)
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