CN109517880A - The method of gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction and DNA modification - Google Patents
The method of gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction and DNA modification Download PDFInfo
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- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
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Abstract
The method for the gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction (SDA) and DNA modification that the invention discloses a kind of.This method is in the presence of active Telomerase, and the extension product of substrate can cause probe Hairpin and Primer and SDA reaction occurs, and forming long, both ends has single-stranded double stranded DNA product.Then SDA product can hybridize with gold nano grain (DNA-AuNP) probe of DNA modification, and gold nano grain is caused to be assembled.The variation of gold nano grain dynamic light scattering signal (particle size) is measured, realizes the highly sensitive quantitative analysis to telomerase activation.This method is easy to operate, testing cost is low, required sample is few, high sensitivity and specificity it is good.Meanwhile this method is used for the detection of kinds cancer patient urine Telomerase Activity by we, the results showed that, the present invention has specificly-response to bladder cancer, provides new approach for the non-invasive diagnosis of bladder cancer.
Description
Technical field
The present invention relates to a kind of activity test method of telomerase, belong to cancer non-invasive diagnostic techniques field, and in particular to one
The method of gold nanoparticle detection urine Telomerase Activity of the kind based on chain replacement reaction and DNA modification.
Background technique
Bladder cancer is that the 4th common cancer, the diagnostic method of bladder cancer mainly include that cystoscopy and urine are thin in the world
Born of the same parents, which learn, to be checked.Cystoscopy is to diagnose the standard method of urinary system symptom, but its higher cost, and patient can be allowed to generate not
Fit or cause complication.Urinary cytology inspection is a kind of method of Noninvasive, its specificity with higher, but due to
Cast-off cells are less in urine, especially during rudimentary and infantile tumour, cause its sensitivity lower.Therefore, Yi Zhongwu
It creates, is simple, reliable Diagnosis of Bladder method is highly desirable.
The development of the neoformation analysis method of multi-crossed disciplines is that the diagnosis of improvement bladder cancer creates huge chance, is examined
The biomarker surveyed in urine is the effective way for obtaining bladder cancer important information, such as the detection of Telomerase.Telomere is position
In the nucleic acid for continuously repeating sequence (TTAGGG) of end of chromosome.Telomerase is a kind of ribonucleoprotein complex, it can be with
TTAGGG repetitive sequence is added in telomerase by its own intrinsic RNA template and reverse transcriptase and GAP-associated protein GAP and maintains end
Grain length.Telomerase activation is suppressed in most of normal human tissues, and telomere length successively decreases with the period division of cell,
Lead to the aging and death of cell.But it is activated and expresses in 85% or more malignant cell Telomerase Activity, make cancer
Cell can be constantly proliferated, therefore Telomerase is considered as the biomarker and therapy target of early-stage cancer diagnosis.1994,
A kind of telomeric repeat amplification scheme (TRAP) is developed, and for detecting the activity of Telomerase, and quantifies thin comprising a small amount of cancer
The Telomerase of the cell biopsy of born of the same parents.The TRAP of based on PCR is current most widely used Telomerase activity method, such as in real time
Fluorescence signal quantifies TRAP and is widely used in enzyme.Although having certain sensitivity, with strong background fluorescence signal
Non-specificity amplification is inevitable.
Therefore, developing a kind of simple, sensitive method come the detection for realizing telomerase activation is necessary and institute
Category field technical staff technical problem urgently to be solved.
Summary of the invention
Regarding to the issue above and demand, it is repaired the object of the present invention is to provide a kind of based on chain replacement (SDA) reaction and DNA
The method of the gold nanoparticle detection urine Telomerase Activity of decorations, passes through measurement UV-visible absorbance and gold nano grain
The highly sensitive quantitative analysis to telomerase activation is realized in the variation of dynamic light scattering signal (particle size).
In order to solve the above-mentioned technical problem, the technical solution adopted by the present invention is as follows.
The side of gold nanoparticle detection urine Telomerase Activity of the one kind based on chain replacement reaction (SDA) and DNA modification
Method mainly includes the following steps:
1) prepare reagent: gold nanoparticle (DNA-AuNPs) probe, Telomerase primer (TS to be measured including DNA modification
Primer), dNTPs, hairpin probe Hairpin, SDA primer (SDA Primer) and DNA extend enzyme；
2) extension: be added in containing Telomerase primer to be measured (TS primer), dNTPs and amplification buffer to
Telomere zyme extract is surveyed, constant-temperature incubation makes TS primer that extension occur, obtains extension reaction mixture；
3) SDA reacts: hairpin probe Hairpin and primer Primer being added in extension reaction mixture, is uniformly mixed
SDA reaction is carried out afterwards, and obtaining long, both ends is single-stranded double stranded DNA product；
4) DNA-AuNP probe is added in SDA product, assembles gold nano grain by DNA hybridization, used
Colorimetric and dynamic light scattering measure corresponding particle size variation, and solution colour changes, and average grain diameter becomes larger.
The method of above-mentioned detection urine Telomerase Activity, in step 1), the extension products of the TS primer can cause
SDA reaction, TS primer have nucleotide sequence (5`-AAT CCG TCG AGC AGA GTT-3 shown in SEQ ID NO:1
`), the sequence of extension products is 5`-AAT CCG TCG AGC AGA GTT AGGG (TTA GGG) n-3` (n=1~10).
The method of above-mentioned detection urine Telomerase Activity, in step 3), probe Hairpin includes two parts, a part
Participate in SDA reaction；Another part can hybridize with DNA-AuNP probe, and intermolecular arm is contained in the hairpin probe Hairpin
(such as Spacer Cn (n=1~10,000)), and by spacer CnIt is divided into two parts, a part causes SDA reaction；Another portion
Dividing can hybridize with DNA-AuNP probe.
The method of above-mentioned detection urine Telomerase Activity, in step 3), primer Primer includes two parts, a part ginseng
It is reacted with SDA；Another part can hybridize with DNA-AuNP probe, it is characterised in that: contain intermolecular arm in primer Primer
(such as Spacer Cn (n=1~10,000)).
The method of above-mentioned detection urine Telomerase Activity, preparation DNA-AuNP probe (have core shown in SEQ ID NO:4
Nucleotide sequence 5 '-SH-TTTTTTATCACATCA-3 '；With 5 '-SH- of nucleotide sequence shown in SEQ ID NO:5
TTTTTTAAGGAGTGT-3 '), DNA-AuNP probe is added in SDA product, gold nano grain is made by DNA hybridization
Aggregation.
The method of above-mentioned detection urine Telomerase Activity, in step 4), the DNA sequence dna of gold nano particle modification is according to spy
The complementary fragment of needle Hairpin and Primer are designed；It is dissipated according to the dynamic optical of UV, visible light absorbance and gold nano grain
It penetrates signal (particle size), measures the telomerase activation in sample.
The method of above-mentioned detection urine Telomerase Activity in step 4), measures UV, visible light absorbance and gold nano
The variation of grain dynamic light scattering signal (particle size) is realized to the analysis of telomerase activation and the diagnosis of bladder cancer.
The method of above-mentioned detection urine Telomerase Activity, Monitoring lower-cut are 3 cells；This method is particularly suitable for wing
The urine detection of Guang cancer patient.
The method of above-mentioned detection urine Telomerase Activity, the extraction process of urine telomerase are as follows:
Fresh urine sample is collected at 4 DEG C, is centrifuged 10 minutes under 850rpm, after being rinsed once with PBS again at 2300rpm, 4 DEG C
Precipitating, is then dissolved in 200 μ l lysis buffers by centrifugation 5 minutes, and is incubated for 30 minutes on ice, finally at 4 DEG C,
Be centrifuged 20 minutes under 12000rpm, supernatant shifted, be stored in -80 DEG C it is spare.
By above-mentioned technical proposal, the invention has the advantages that and advantageous effects:
1) in the presence of active Telomerase, method of the invention is to draw the extension product of its primer (TS Primer)
Send out SDA reaction, SDA product can hybridize with DNA-AuNP probe, cause gold nano grain to be assembled, by measurement is ultraviolet can
See the variation of absorbance and gold nano grain dynamic light scattering signal (particle size), realizes to the highly sensitive fixed of telomerase activation
Amount analysis.
2) method of the invention has many advantages, such as easy to operate, high sensitivity and specificity is good, at low cost, and this method
Suitable for kinds cancer patient, it is particularly suitable for the urine detections of bladder cancer patients.
3) the result shows that, the present invention to bladder cancer have specificly-response, for bladder cancer non-invasive diagnosis provide it is new
Approach.
Detailed description of the invention
Attached drawing is used to a further understanding of the present invention, and constitutes part of specification, with the embodiment of the present invention one
It rises and is used to explain the present invention, be not construed as limiting the invention.
Fig. 1 is to detect urine telomerase the present invention is based on the gold nanoparticle of chain replacement reaction (SDA) and DNA modification to live
The method schematic of property.
Fig. 2 is the suction that the present invention analyzes different samples (do not cause SDA and cause SDA) using ultraviolet-visible spectrophotometry
Luminosity figure.
The present invention is shown in Fig. 3 a and Fig. 3 b respectively (is not caused SDA and drawn using dynamic scattering analysis difference sample
Send out SDA) particle size distribution figure.
Fig. 4 is the particle size figure of present invention detection different number MCF-7 Cell Telomerase Activity, and illustration is 5 to 1000
The standard curve of a MCF-7 cell.
Fig. 5 is the particle size figure of present invention detection various cancers patient urine telomerase activation.
Specific embodiment
Chain replacement reaction (Strand displacement reaction, SDA) is used as a kind of isothermal nucleic acid amplifying technique
It is concerned, is to make hairpin probe and primer that cascade hybridization reaction occur under the induction for causing chain, forms long, both ends band list
The duplex DNA of chain.SDA can be in conjunction with a variety of probes, and gold nano grain (gold nanoparticles, AuNPs) is one
The most common nano material of kind is widely used in sensing due to its large specific surface area, optical property uniqueness, good biocompatibility
The building of platform.AuNPs also changes therewith with the change of dispersion/coherent condition, particle size.
Therefore, can be by AuNPs in conjunction with SDA, the dispersion/coherent condition and gold nano grain particle size of gold nano can
To be converted to the amount of SDA product.
The invention discloses a kind of gold nanoparticles based on chain replacement reaction (SDA) and DNA modification to detect urine middle-end
The method of telomerase activity.This method be in the presence of active Telomerase, the extension product of substrate can cause probe Hairpin and
SDA reaction occurs for Primer, and forming long, both ends has single-stranded double stranded DNA product.Then, SDA product can be with DNA modification
Gold nano grain (DNA-AuNP) probe hybridization, cause gold nano grain to be assembled.Gold nano grain dynamic optical is measured to dissipate
The variation of signal (particle size) is penetrated, realizes the highly sensitive quantitative analysis to telomerase activation.
As shown in Figure 1, for the present invention is based on the gold nanoparticles of DNA modification and SDA to detect urine Telomerase Activity
Method schematic.When not having Telomerase or Telomerase inactivates, gold nano grain keeps dispersity；And when active Telomerase is deposited
When, gold nano grain is assembled, and partial size significantly increases.
This method is in the presence of active Telomerase, and the extension product of substrate can cause probe Hairpin and primer
SDA reaction occurs for Primer, forms double stranded DNA product long, that both ends band is single-stranded.Then SDA product can be with DNA modification
The hybridization of gold nano grain (DNA-AuNP) probe, causes gold nano grain to be assembled.Measure UV, visible light absorbance and Jenner
The highly sensitive quantitative analysis to telomerase activation is realized in the variation of rice grain dynamic light scattering signal (particle size).
Method of the invention is easy to operate, testing cost is low, required sample is few, high sensitivity and specificity it is good.Meanwhile it should
The Monitoring lower-cut of method is 3 cells；This method is used for kinds cancer patient, especially bladder cancer cancer patient urine by us
The detection of Telomerase Activity.
Below by way of specific preferred embodiment combination attached drawing, invention is further described in detail, but the present invention and not only
It is limited to embodiment below.
As shown in Figure 1, the present invention relates to a kind of gold nanoparticles based on DNA modification and chain replacement reaction (SDA) detection
The method of urine Telomerase Activity.It is specific as follows:
1, the property of TTAGGG repetitive sequence can be added in the Telomerase primer end (TS primer) based on Telomerase:
The extension products of TS primer can cause SDA reaction, and TS primer has nucleotides sequence shown in SEQ ID NO:1
It arranges (5`-AAT CCG TCG AGC AGA GTT-3`), the sequence of extension products is 5`-AAT CCG TCG AGC AGA GTT
AGGG TTA GGG-3`；
2, SDA reacts:
Extension products and hairpin probe Hairpin (had into 5 '-ACACTCCTT of nucleotide sequence shown in SEQ ID NO:2
spacer18TCTTGGACTAACCCTAACCCTAAAACTTAGTCCAAGA-3 ') and Primer (have SEQ ID NO:3 shown in
5 '-TGA TGT GAT spacer18 TCT TGG AC-3 ' of nucleotide sequence) (dashed part is ginseng for mixing progress SDA reaction
With SDA reactive moieties), obtain SDA product long, that both ends band is single-stranded.Wherein, spacer18 is that DNA is connected with C18 polyethylene glycol
It connects, that is, 1 phosphoric acid of C18 polyethylene glycol, concrete structure formula are as follows:
3, hybridize:
Preparing DNA-AuNP probe (has 5 '-SH-TTTTTTATCACATCA- of nucleotide sequence shown in SEQ ID NO:4
3'；With nucleotide sequence 5 '-SH-TTTTTTAAGGAGTGT-3 ' shown in SEQ ID NO:5), DNA-AuNP probe is added
Into SDA product, assemble gold nano grain by DNA hybridization；
4, colorimetric and dynamic scattering analysis: measurement UV, visible light absorbance and gold nano grain dynamic light scattering signal
The variation of (particle size) is realized to the analysis of telomerase activation and the diagnosis of bladder cancer.
As shown in Fig. 2, Fig. 2 is that the present invention uses ultraviolet-visible spectrophotometry to analyze different sample absorbance distribution maps,
When not having Telomerase or Telomerase inactivates, SDA reaction, absorbance 0.7 or so cannot be caused；And when active Telomerase exists
When, the extension products of primer can cause SDA reaction, absorbance 0.5 or so.
As shown in figure 3, Fig. 3 is the particle size distribution figure that the present invention uses dynamic scattering analysis difference sample, when not having
When having Telomerase or Telomerase to inactivate (3a), SDA reaction cannot be caused, the partial size of gold nano grain is 41.3nm；And when activity
In the presence of Telomerase (3b), the extension products of primer can cause SDA reaction, and the partial size of gold nano grain increases to 435.5nm.
As shown in figure 4, showing the particle size of present invention detection different number MCF-7 Cell Telomerase Activity in Fig. 4
Figure, illustration are the standard curve of 5 to 1000 MCF-7 cells.
The cell number of MCF-7 and the partial size of gold nano grain are in good linear relationship, inspection within the scope of 10 to 1000
Rising limit is 3 cells.As shown in figure 4, showing the partial size of present invention detection various cancers patient urine telomerase activation in Fig. 4
Size figure.
The partial size of only bladder cancer's urine sample significantly increases, and the urine sample of normal person and other cancer patients
Partial size no significant change compared with the partial size of blank sample (Granzyme), show the present invention to bladder cancer have specificity
Response.
Embodiment 1
The side of gold nanoparticle detection urine Telomerase Activity of the one kind based on chain replacement reaction (SDA) and DNA modification
Method specifically comprises the following steps:
1) extraction of Telomerase
The extraction of cell telomerase:
By MCF-7 cell in the DMEM training containing 10% fetal calf serum, 100 μ g/ml penicillin and 100 μ g/ml streptomysins
It supports and is cultivated in base.After extracting cell with trypsase, 7 × 10 are had collected6A cell.Cell is dispersed in 1.5 milliliters of EP pipes,
It is centrifuged 5min at 1000rpm, and is washed twice with ice-cold PBS.Extracting solution is suspended in 200 μ L lysis buffers again,
After being incubated for 30 minutes on ice, it is centrifuged 20 minutes at 4 DEG C, 12000rpm.Supernatant is shifted, dilutes, is stored in standby at -80 DEG C
With.
The extraction of urine telomerase:
Fresh urine sample is collected at 4 DEG C, is centrifuged 10 minutes under 850rpm, after being rinsed once with PBS again at 2300rpm, 4 DEG C
Precipitating, is then dissolved in 200 μ l lysis buffers by centrifugation 5 minutes, and is incubated for 30 minutes on ice, finally at 4 DEG C,
Be centrifuged 20 minutes under 12000rpm, supernatant shifted, be stored in -80 DEG C it is spare.
2) the gold nano grain preparation of DNA modification
The preparation of AuNPs is and to have done adjustment appropriate according to classical reduction of sodium citrate method.By 2mL Fresh
38.8mM sodium citrate solution be added rapidly to the 1mM gold chloride HAuCl that 20mL boils4In solution, reaction solution is by yellowish discoloration
For black, then become purple, eventually becomes claret, continue to be heated to reflux and stir 10 minutes.Finally, reaction solution is allowed to be cooled to
Room temperature is simultaneously kept stirring, and is then filtered, is stored in spare in 4 DEG C of refrigerators with 0.45 micron of nylon leaching film.
AuNPs surface modification DNA:
Mercapto-modified DNA first uses tricresyl phosphate (2- chloroethyl) ester TCEP to handle, and is then added to by the molar ratio of 200:1
In previously prepared AuNPs solution, it is incubated at room temperature 16 hours.Then NaCl is added several times in next 44 hours
Salt aging is carried out, the ultimate density of NaCl is 0.1M.Finally, reaction solution is centrifuged 30 minutes with the revolving speed of 13800rpm to remove
Free DNA (being repeated 3 times), obtained oily sediment are dissolved in 10mM PBS buffer solution (pH 7.4,0.1M NaCl),
It is stored in spare in 4 DEG C of refrigerators.
3) progress of SDA reaction
Telomere zyme extract, 1.5 μM of TS primer, 1mM dNTPs, 0.2mg/mL BSA, 0.4U/mL will be contained
The solution of RNase inhibitor is incubated for 1.0h at 37 DEG C, and Hairpin is cooled to room temperature in 95 DEG C of heating 5min.Then will contain
The solution of Hairpin (1 μM), Primer (1 μM) and telomerase extended products are in 37 DEG C of reaction 1.5h.
The TS primer has nucleotide sequence (5`-AAT CCG TCG AGC AGA shown in SEQ ID NO:1
GTT-3`), the sequence of extension products be 5`-AAT CCG TCG AGC AGA GTT AGGG (TTA GGG) n-3` (n=1~
10)。
Contain intermolecular arm (such as Spacer C in probe Hairpinn(n=3~10,000)), and (such as by intermolecular arm
Spacer Cn(n=3~10,000)) it is divided into two parts, a part participates in SDA reaction；Another part can be visited with DNA-AuNP
Needle hybridization.
Contain intermolecular arm (such as Spacer C in primer Primern(n=3~10,000)), and by spacer CnIt is divided into
Two parts, a part cause SDA reaction；Another part can hybridize with DNA-AuNP probe.
4) SDA product is mixed with DNA-AuNP probe, and detects change of size.
The DNA sequence dna modified on gold nanoparticle is designed according to the complementary fragment of probe Hairpin and Primer, is made
DNA-AuNPs probe and SDA products thereof and the variation for causing color and dynamic light scattering signal (particle size), measure sample
In telomerase activation.
Appropriate DNA-AuNP probe is added into SDA product, assembles gold nano grain by DNA hybridization reaction,
Using colorimetric and dynamic light scattering measure corresponding particle size variation (solution colour becomes blue by red, average grain diameter by
It is small to become larger).
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, therefore
Without departing from the technical solutions of the present invention, to the above embodiments according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
Sequence table
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<120>method of the gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction and DNA modification
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Claims (9)
1. a kind of method of the gold nanoparticle detection urine Telomerase Activity based on chain replacement reaction and DNA modification, special
Sign is, comprising the following steps:
1) reagent prepares: gold nanoparticle (DNA-AuNPs) probe, Telomerase primer (TS to be measured including DNA modification
Primer), dNTPs, hairpin probe Hairpin, SDA primer (SDA Primer) and DNA extend enzyme；
2) end to be measured extension: is added in containing Telomerase primer to be measured (TS primer), dNTPs and amplification buffer
Granzyme extracting solution, constant-temperature incubation make TS primer that extension occur, obtain extension reaction mixture；
3) SDA reacts: hairpin probe Hairpin and primer Primer being added in extension reaction mixture, is uniformly mixed laggard
Row SDA reaction, obtaining long, both ends is single-stranded double stranded DNA product；
4) DNA-AuNP probe is added in SDA product, assembles gold nano grain by DNA hybridization, using colorimetric
Corresponding particle size variation is measured with dynamic light scattering.
2. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: described in step 1)
The extension products of TS primer can cause SDA reaction, and TS primer has nucleotide sequence (5`- shown in SEQ ID NO:1
AAT CCG TCG AGC AGA GTT-3`), the sequence of extension products is 5`-AAT CCG TCG AGC AGA GTT AGGG
(TTA GGG) n-3` (n=1~10).
3. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: in step 3), probe
Contain intermolecular arm (such as Spacer C in Hairpinn(n=3~10,000)), and by intermolecular arm (such as Spacer Cn(n=3
~10,000)) it is divided into two parts, a part participates in SDA reaction；Another part can hybridize with DNA-AuNP probe.
4. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: in step 3), primer
Contain intermolecular arm (such as Spacer C in Primern(n=3~10,000)), and by spacer CnIt is divided into two parts, a part
Cause SDA reaction；Another part can hybridize with DNA-AuNP probe.
5. the method for detection urine Telomerase Activity according to claim 4, it is characterised in that: preparation DNA-AuNP is visited
Needle (has nucleotide sequence 5 '-SH-TTTTTTATCACATCA-3 ' shown in SEQ ID NO:4；With shown in SEQ ID NO:5
Nucleotide sequence 5 '-SH-TTTTTTAAGGAGTGT-3 '), DNA-AuNP probe is added in SDA product, DNA hybridization is passed through
Assemble gold nano grain.
6. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: in step 4), Jenner
The DNA sequence dna modified on rice corpuscles is designed according to the complementary fragment of probe Hairpin and Primer, visits DNA-AuNPs
Needle and SDA products thereof and the variation for causing color and dynamic light scattering signal (particle size), measure the Telomerase in sample
Activity.
7. the method for detection urine Telomerase Activity according to claim 1, it is characterised in that: in step 4), measurement
The variation of UV, visible light absorbance and gold nano grain dynamic light scattering signal (particle size), realization divide telomerase activation
Analysis and the diagnosis of bladder cancer.
8. the method for detection urine Telomerase Activity according to claim 7, it is characterised in that:
The Monitoring lower-cut of this method is 3 cancer cells；This method is particularly suitable for the urine detection of bladder cancer patients.
9. the method for detection urine Telomerase Activity according to claim 7, it is characterised in that: urine telomerase
Extraction process is as follows:
Fresh urine sample is collected at 4 DEG C, is centrifuged 10 minutes under 850rpm, is centrifuged at 2300rpm, 4 DEG C again after being rinsed once with PBS
5 minutes, then precipitating is dissolved in 200 μ l lysis buffers, and is incubated on ice 30 minutes, finally at 4 DEG C, 12000rpm
It is lower centrifugation 20 minutes, supernatant is shifted, be stored in -80 DEG C it is spare.
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