Fluorescence quantum/nano-metal particle conjugate and preparation and application
Technical field
The present invention relates to nano material, especially a kind of fluorescence quantum/nano-metal particle conjugate, its preparation method and its application in detecting metallic ion.
Background technology
In environment (especially in the fresh water), the pollution of metallic ion such as mercury, lead, copper, manganese, cadmium, chromium etc. has great harm to human beings'health and economic sustainable development.Therefore, the detection of metallic ion (carrying out real-time and highly sensitive metal ion detection especially at the scene) becomes more and more important.Yet; Traditional metal ion inspection; Need expensive instrument and tediously long sample preparation, analytical test and data processing step like chromatography, atomic absorption spectrography (AAS) etc., be difficult to accomplish that sample (like water body, food, beverage etc.) is carried out real-time scene to be detected.
(Quantum Dot QD), is called nanocrystalline or artificial atom again to quantum dot, is a kind of nano particle of the accurate zero dimension of being made up of II-VI family or III-V family element (quasi-zero-dimensional).Usually, quantum dot in the size on three dimensions all below 100 nanometers (nm).Because quantum confined effect (quantum confinement effect), electronics in the quantum dot and hole have the discrete energy levels structure, can emitting fluorescence after being excited.Through shape, structure and the size of control quantum dot, can regulate the size of its energy gap width, exciton bind energy and the electronic states such as energy blue shift of exciton easily.Along with reducing gradually of quantum dot size, the blue shift phenomenon appears in the optical absorption spectra of quantum dot.Size is more little, and it is also remarkable more then to compose the blue shift phenomenon, promptly so-called quantum size effect.Because quantum dot can absorb the photon that all are higher than its band-gap energy, and the optical wavelength (being color) of emission has size-dependent, so the different similar quantum dot of useful size forms the different a series of labels of emission wavelength (being color).
Quantum dot can be divided into box-shaped quantum dot, spherical quantum dot, tetrahedron quantum dot, Cylindrical Quantum Dots, cube quantum dot, dish type quantum dot and outfield (electric field and magnetic field) and induce quantum dot by its geometric configuration; By the quantum sealing process in its electronics and hole, quantum dot can be divided into 1 type quantum dot and 2 type quantum dots; Form by its material, quantum dot can be divided into the elemental semiconductor quantum dot again, compound semiconductor quantum dot and heterojunction quantum dot.In addition, atom and molecular cluster, ultramicron and how empty silicon etc. also all belong to the quantum dot category.
(Nanometer Particle NP) can be used as a kind of general fluorescence and buries in oblivion group metal nanoparticle.People such as Maxwell (Dustin J.Maxwell; Jason R.Taylor; And Shuming Nie.Self-Assembled Nanoparticle Probes for Recognition and Detection of Biomolecules.Journal of the American Chemical Society.2002; 124,9606-9612) at the two ends of oligonucleotide probe molecule difference marking nano gold grain (AuNP) and fluorescence excitation group, probe is because base complementrity forms " hair fastener " structure; Fluorescence excitation group and nanogold particle near, cause that fluorescence excitation buries in oblivion; And when probe with after the specificity target DNA combines, its conformation changes, nanogold particle separates with the fluorescence excitation group, thereby inspires fluorescence.The real-time fluorescence that this principle can be used for nucleic acid detects, and the detection of single base mutation polymorphism etc.
One Chinese patent application CN101482508A discloses a kind of method of highly sensitive detection trace metal ion; Comprise: the precious metal that will modify with ssDNA probe and the quantum dot of modifying with ssDNA probe are connected to form conjugate through the single stranded DNA as the substrate of the DNA enzyme that the metal target ion is had specific activity; Said conjugate is contacted with sample in the presence of said DNA enzyme, and when in the sample during driftlessness metallic ion, thereby the fluorescence of precious metal cancellation fluorescence quantum detects more weak fluorescence intensity; When containing the metal target ion in the sample; The DNA enzyme is activated and cuts off substrate DNA, and precious metal is separated with fluorescence quantum, and precious metal disappears to the quenching effect of fluorescence quantum fluorescence; Thereby detect the fluorescence intensity of enhancing, realize detection thus metallic ion.
Yet, still need various fluorescence quantums/nano-metal particle conjugate to satisfy different demands, particularly detect metallic ion with higher sensitivity.
Summary of the invention
One aspect of the present invention provides a kind of fluorescence quantum/nano-metal particle conjugate, comprising:
-the fluorescence quantum modified by first ssDNA probe;
-the nano-metal particle modified by second ssDNA probe; With
The 3rd single stranded DNA of said nano-metal particle of-coupling and said fluorescence quantum; Wherein said the 3rd single stranded DNA comprises and complementary first sequence that combines of the partial sequence at least of first ssDNA probe, second sequence that combines with the complementation of partial sequence at least of second ssDNA probe and the 3rd sequence that between first sequence and second sequence, exists with single stranded form
Distance between wherein said nano-metal particle and the said fluorescence quantum makes the fluorescence that said nano-metal particle can the said fluorescence quantum of cancellation sends; Make said nano-metal particle leave the no longer fluorescence that sends of the said fluorescence quantum of cancellation of said fluorescence quantum thereby said the 3rd sequence can exist and activate when this object had the DNA enzyme of specific activity to be cut off by said DNA enzyme at object, and the mol ratio of said fluorescence quantum and said nano-metal particle is about 1: 10 to about 20: 1.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, wherein said fluorescence quantum are selected from CdSe quantum dot, CdS quantum dot, CdTe quantum dot, CdSe/ZnS quantum dot, CdTe/CdS/ZnS quantum dot, CdSe/CdS quantum dot and its combination in any.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, wherein said nano-metal particle are selected from gold nano grain, silver nano-grain and its combination in any.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, wherein said fluorescence quantum are of a size of 1 nanometer to 50 nanometer, are preferably 2 nanometer to 10 nanometers.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, wherein said nano-metal particle are of a size of 2 nanometer to 100 nanometers, are preferably 5 nanometer to 30 nanometers.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, the substrate chain that wherein said the 3rd sequence is said DNA enzyme.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate; Wherein said object is selected from natural and synthetic organic and mineral compound; Be preferably selected from and said nano-metal particle different metallic and ion and compound, more preferably be selected from mercury, lead, copper, manganese, cadmium, chromium, uranium and ion thereof and oxide.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate; The mol ratio of wherein said fluorescence quantum and said nano-metal particle is preferably about 1: 10, about 1: 5, about 1: 2, about 1: 1, about 2: 1, about 5: 1, about 10: 1 or about 15: 1, more preferably about 3.2: 1.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate; Wherein said the 3rd single stranded DNA also comprises in the 4th sequence between first sequence and the 3rd sequence and/or the 5th sequence between second sequence and the 3rd sequence, said the 4th sequence and/or the 5th sequence can with said DNA enzyme complementary pairing.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, wherein said fluorescence quantum/nano-metal particle conjugate also comprise and said the 4th sequence and/or the complementary said DNA enzyme that combines of the 5th sequence.
Some embodiments of the fluorescence quantum according to the present invention/nano-metal particle conjugate, wherein the particle diameter of nano-metal particle is the 5-100 nanometer, preferred 5-30 nanometer, more preferably from about 13 nanometers; The mol ratio of first ssDNA probe and fluorescence quantum is 10-200: 1, and preferred 50: 1; The concentration of the fluorescence quantum of being modified by first ssDNA probe is 10-200nM, preferred 50-150nM, more preferably 80nM; The mol ratio of second ssDNA probe and nano metal ion is 10-200: 1, and preferred 100: 1; The concentration of the nano metal ion of being modified by second ssDNA probe is 5-100nM, preferred 25-50nM, more preferably 25nM; Concentration as the 3rd single stranded DNA of substrate DNA is 50-500nM, preferred 100-400nM, more preferably 200nM.
Another aspect of the present invention provides a kind of method for preparing aforementioned according to fluorescence quantum of the present invention/nano-metal particle conjugate, comprising:
-fluorescence quantum of being modified by first ssDNA probe is provided;
-nano-metal particle of being modified by second ssDNA probe is provided;
-the 3rd single stranded DNA is provided; Comprise can with complementary first sequence that combine of the partial sequence at least of first ssDNA probe, can with complementary second sequence that combine of the partial sequence at least of second ssDNA probe and the 3rd sequence that between first sequence and second sequence, exists with single stranded form, said the 3rd sequence can object exist and activation when this object is had the DNA enzyme of specific activity by said DNA enzyme cut-out;
-the said fluorescence quantum of being modified by first ssDNA probe, the said nano-metal particle of being modified by second ssDNA probe and said the 3rd single stranded DNA are mixed obtaining said fluorescence quantum/nano-metal particle conjugate,
The wherein said fluorescence quantum of being modified by first ssDNA probe is about 1: 10 to about 20: 1 with the mol ratio of said nano-metal particle by the modification of second ssDNA probe; Be preferably about 1: 10, about 1: 5, about 1: 2, about 1: 1, about 2: 1, about 5: 1, about 10: 1 or about 15: 1, more preferably about 3.2: 1.
The aforementioned method of preparation according to the present invention according to fluorescence quantum of the present invention/nano-metal particle conjugate; Wherein said fluorescence quantum and said nano-metal particle are being mixed with the time through said nano-metal particle of the 3rd single stranded DNA coupling and said fluorescence quantum; The annealing curve of used DNA hybridization is to keep 5 minutes down at 80 degrees centigrade, then cool to room temperature.
Of the present inventionly provide a kind of more on the other hand and aforementionedly be used to detect the purposes of object according to fluorescence quantum of the present invention/nano-metal particle conjugate.
Of the present invention a kind of composition is provided on the other hand again, contains aforementioned according to fluorescence quantum of the present invention/nano-metal particle conjugate.When needed, said composition also contain can when object exists, be activated and cut off the 3rd sequence this object is had the DNA enzyme of specific activity.
Of the present invention a kind of composition is provided on the other hand again, contains aforementionedly according to fluorescence quantum of the present invention/nano-metal particle conjugate, said conjugate comprises and said the 4th sequence and/or the complementary said DNA enzyme that combines of the 5th sequence.
In some cases, above-mentioned composition also comprises sodium chloride, and its concentration is 0.01-0.3mM, preferred 0.15M.
Of the present invention a kind of kit is provided on the other hand again, contain with good grounds fluorescence quantum of the present invention/nano-metal particle conjugate with can when object exists, be activated and cut off the 3rd sequence this object is had the DNA enzyme of specific activity.
Of the present invention a kind of kit is provided on the other hand again; Contain with good grounds fluorescence quantum of the present invention/nano-metal particle conjugate; Said conjugate comprises and said the 4th sequence and/or the complementary DNA enzyme that combines of the 5th sequence, when this object exists, is activated to cut off the 3rd sequence thereby wherein said DNA enzyme has specific activity to object.
Of the present invention a kind of kit is provided on the other hand again, contains:
-the fluorescence quantum modified by first ssDNA probe;
-the nano-metal particle modified by second ssDNA probe; With
-Di three single stranded DNAs; Comprise can with complementary first sequence that combine of the partial sequence at least of first ssDNA probe, can with complementary second sequence that combine of the partial sequence at least of second ssDNA probe and the 3rd sequence that between first sequence and second sequence, exists with single stranded form, said the 3rd sequence can object exist and activation when this object is had the DNA enzyme of specific activity by said DNA enzyme cut-out; With
-with said the 3rd sequence DNA enzyme that is substrate,, wherein said DNA enzyme when this object exists, is activated cutting off the 3rd sequence thereby having specific activity to object,
The wherein said fluorescence quantum of being modified by first ssDNA probe is about 1: 10 to about 20: 1 with the mol ratio of said nano-metal particle by the modification of second ssDNA probe; Be preferably about 1: 10, about 1: 5, about 1: 2, about 1: 1, about 2: 1, about 5: 1, about 10: 1 or about 15: 1, more preferably about 3.2: 1.
The method that object in a kind of test sample is provided on the other hand again of the present invention comprises:
-provide according to above-mentioned fluorescence quantum of the present invention/nano-metal particle conjugate or its composition;
-provide can when said object exists, be activated and cut off the 3rd sequence this object is had the DNA enzyme of specific activity;
-said fluorescence quantum/nano-metal particle conjugate is contacted with said sample with said DNA enzyme;
Fluorescence intensity before and after the-measurement contact;
Confirm there is object in the said sample during-fluorescence intensity before the fluorescence intensity after the contact is greater than contact and/or according to the typical curve calculation sample in the concentration of object.
The method that object in a kind of test sample is provided on the other hand again of the present invention comprises:
-above-mentioned fluorescence quantum/nano-metal particle conjugate or its composition according to the DNA of containing enzyme of the present invention are provided, wherein said DNA enzyme has specific activity and when said object exists, can be activated and cut off the 3rd sequence said object;
-said fluorescence quantum/nano-metal particle conjugate is contacted with said sample;
Fluorescence intensity before and after the-measurement contact;
Confirm there is object in the said sample during-fluorescence intensity before the fluorescence intensity after the contact is greater than contact and/or according to the typical curve calculation sample in the concentration of object.
Be surprised to find that; According to fluorescence quantum of the present invention/nano-metal particle conjugate; When the mol ratio of wherein fluorescence quantum and nano-metal particle is in specific scope, for example about 1: 10 to about 20: 1, be preferably about 1: 10, about 1: 5, about 1: 2, about 1: 1, about 2: 1, about 5: 1, about 10: 1 or about 15: 1; More preferably about 3.2: 1 o'clock; Can reduce the fluorescence intensity of (before promptly contacting) before detecting, and improve the recovery that detects back (promptly contacting the back) fluorescence intensity, improve the sensitivity that detects thus significantly with sample with sample.
Embodiment
Below in conjunction with embodiment embodiments more of the present invention are further introduced, but be not intended to limit protection scope of the present invention.
Below be the conventional method that example explanation preparation has specific fluorescence quantum (QD) and fluorescence quantum/nano-metal particle (QD-NP) conjugate of nano-metal particle (NP) mol ratio with golden nanometer particle (AuNP).
The structure of QD-AuNP conjugate forms through single stranded DNA complementary pairing, hybridization, may further comprise the steps:
-obtain fluorescence quantum (QD-DNA) through the carboxylic group and the terminal amino reaction formation amido link of first ssDNA probe on QD surface by the modification of first ssDNA probe;
-effect acquisition through the strong coordination bonding in terminal sulfydryl of second ssDNA probe and AuNP surface is by the second ssDNA probe gold nano-particles modified (Au-DNA);
-adding the 3rd single stranded DNA, the complementary pairing through the 3rd single stranded DNA and first ssDNA probe and second ssDNA probe obtains the QD-AuNP conjugate.
When preparation QD-AuNP conjugate, also can add corresponding D NA enzyme.In some cases, said DNA endonuclease capable combines with the partial sequence of the 3rd single stranded DNA is complementary.
In the process of preparation QD-DNA and Au-DNA, can in reaction, add excessive DNA, the dna probe number on QD and AuNP surface is calculated through the DNA concentration that measurement is free in solution in the reaction back.When forming the QD-AuNP conjugate; Can the mol ratio (for example mol ratio) of QD and NP be adjusted to: for example 20: 1,15: 1,10: 1,5: 1,3.2: 1,2: 1,1: 1,1: 2,1: 5,1: 10,1: 15,1: 20 etc., then with being connected as the 3rd single stranded DNA of substrate and optional DNA enzyme.The formation of QD-AuNP conjugate can be confirmed through measuring fluorescence intensity.
Embodiment 1: preparation ssDNA probe gold nano-particles modified
Golden nanometer particle synthetic can referring to method of the prior art (J.J.Storhoff, R.Elghanian, R.C.Mucic, C.A.Mirkin, R.L.Letsinger.J.Am.Chem.Soc., 1998,120,1959-1964).98 ml waters are added in the two-mouth bottle.The HAuCl that adds 2 milliliters of 50mM
4HAuCl in the feasible bottle of solution
4Concentration be 1mM, under agitation be heated to backflow, add the sodium citrate solution of 10 milliliters of 38.8mM then rapidly, color in 1 minute from pale yellow become dark red.Continue to reflux 20 minutes, stop heating and under agitation be cooled to room temperature (23-25 degree centigrade).The particle diameter of gained nano particle is 13 nanometers, and concentration is 10nM.Transmission electron microscope (TEM) shows that the gained nano particle is for spherical.
Because the sodium citrate gold nano-particles modified is under high salt concn unstable, further uses BSPP (C
6H
5P (C
6H
4SO
3K)
2) handle golden nanometer particle the surface with enhanced stability.Particularly, in new synthetic 100mL nano-Au solution, add the 0.1gBSPP powder, gentle shaken overnight under the room temperature.Subsequently, (5 '-TTTTTTTTTACGCGTACTAGC, concentration is 25nM, DNA: AuNP=100: 1) gold nano-particles modified with the ssDNA probe of the stronger sulfhydrylation of compatibility through aglucon exchange.It is 13 nanometers that the dynamic laser scatterometer records particle diameter.Adopt similar approach also to prepare the for example golden nanometer particle of 16 nanometers, 25 nanometers and 35 nanometers of other particle diameter.The size distribution of all these golden nanometer particles is all less than 10%.
Embodiment 2: the fluorescence quantum that the preparation ssDNA probe is modified
Through the single stage method prepared in reaction fluorescence quantum (referring to Q.B.Wang, D.K.Seo.JMater Sci., 2009,44,816; Q.B.Wang, Y.Liu, Y.Ke, H.Yan.Angew.Chem., Int.Ed., 2008,47,316; Q.B.Wang, N.Iancu, D.K.Seo.Chem.Mater., 2005,17:4762; And Q.B.Wang, Y.Xu, X.Zhao, Y.Chang, Y.Liu, L.Jiang, J.Sharma, D.K.Seo, H.Yan.J.Am.Chem.Soc., 2007,129,6380).
Particularly, be prepared as follows the CdSe/ZnS core/shell type quantum dot that MPA modifies.With 0.1 mM P
2S
5, 0.5 gram 3-mercaptopropionic acid (MPA) and 0.3 milliliter of butylamine mix also heating for dissolving at 10 milliliters of N-methyl-2-in than pyrrolidone (NMP).Behind the cool to room temperature, add the CdSe nuclear particle (Bioisystech Co., Ltd provides by thing source, BeiJing ZhongKe, and fluorescence peak is 525nm) that 0.01 gram oleyl amine is modified, then with 0.05 gram ZnCl
2Solution mix.Mixed solution heats the CdSe/ZnS core/shell type quantum dot that obtained the MPA modification in 1 hour down at 70 degrees centigrade.It is in 7.5 the phosphate buffer that gained CdSe/ZnS core/shell type quantum dot is scattered in pH.
The fluorescence quantum (QD-DNA) that single stranded DNA is modified can obtain through the carboxyl and the terminal amino reaction of DNA on QD surface; Wherein 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimine (EDC) and N-hydroxy-succinamide (NHS) as coupling agent (referring to Q.B.Wang; H.Wang; C.Lin, J.Sharma, S.Zou; Y.Liu.Chem.Commum., 2010,46,240).
Particularly; N-hydroxysulphosuccinimide (Sulfo-NHS) solution of the EDC solution of 50 microlitre 1M and 50 microlitre 1M is added in the damping fluid of the CdSe/ZnS core/shell type quantum dot that the above-mentioned MPA of 200 microlitres modifies; The DNA (5 '-TCA CAG ATGAGT-amine-3 ', the DNA: QD=50: 1) that add 80nM then.Under agitation incubated at room is spent the night.The CdSe/ZnS core/shell type quantum dot (QD-DNA) of gained dna modification stores down at 4 degrees centigrade after filtering purifying.
Embodiment 3: preparation fluorescence quantum/nano-metal particle conjugate
In QD concentration is in the QD-DNA solution from embodiment 2 of 200nM; The concentration that adds Au is that the particle diameter from embodiment 1 of 8nM is 13 nanometer Au-DNA solution; Wherein keep QD in the QD-Au structure: Au=5: 1, i.e. QD-DNA solution 2 microlitres, Au-DNA solution 10 microlitres; And then adding has specific DNA enzyme (40 μ M to object in system; 0.1 microlitre) and corresponding substrate DNA (40 μ M, 0.1 microlitre) and sodium chloride solution to make the concentration of sodium chloride be 0.15mM, spending the night annealing hybridization.The annealing curve of DNA hybridization is to keep 5 minutes down at 80 degrees centigrade, slow then cool to room temperature.
When object was lead, said DNA enzyme was 5 '-CAT CTC TTC TCC GAG CCGGTC GAA ATA GTG AGT-3 ', and said substrate DNA is 3 '-GTG CTC AAC TGTGTA GAG AAG GrA TAT CAC TCA AGT GTC TAC TCA-5 '.
When object is copper; Said DNA enzyme is 5 '-GGT AAG CCT GGG CCT CTTTCT TTT TAA GAA AGA AC-3 ', and said substrate DNA is 3 '-GTG CTC AACTGT CCA TTC GGC ATA ATC TTT CTT CGA AGT GTC TAC TCA-5 '.
Above sequence is all available from IDT (Integrated DNA Technologies) company.
Embodiment 4: detect metallic ion with fluorescence quantum/nano-metal particle conjugate
According to embodiment 3, use to plumbous DNA enzyme and substrate DNA to have prepared the QD-AuNP conjugate, add lead ion then with 0.2 μ M, wherein measured before the adding lead ion and fluorescence intensity afterwards.In addition, as emphasical with the fluorescence of the potpourri of the Au-DNA of DNA enzyme and QD-DNA with reference to also having measured the fluorescence intensity of QD-DNA and not contained substrate DNA.The concentration of QD all is consistent in all samples.
The result shows, QD-DNA is about 500 as the fluorescence intensity of free QD, and the fluorescence intensity of the potpourri of Au-DNA and QD-DNA is about 480, and whether this explanation contains Au-DNA to the fluorescence intensity influence of QD not quite; The initial fluorescent intensity of QD-AuNP be about 100 (be about free QD fluorescence intensity 20%), the fluorescence intensity of phase specific ionization QD has remarkable decline, explains that the fluorescence that the AuNP that is coupled at together sends QD has the cancellation effect; After the lead ion of 0.2 μ M adds; Thereby lead ion has activated the DNA enzyme and has cut off substrate DNA; Cause separating between the QD and AuNP among the QD-AuNP, AuNP descends to the cancellation effect of the fluorescence that QD sends, make fluorescence intensity return to free QD fluorescence intensity 85%.
To also having carried out similar experiment to the DNA enzyme of copper and the QD-AuNP conjugate of substrate DNA preparation according to embodiment 3 usefulness.The result shows, the initial fluorescent intensity of QD-AuNP is about 50 (is about free QD 10%), and same explanation is coupled at the fluorescence that AuNP together sends QD has the cancellation effect; Behind the copper ion that adds 0.2 μ M, fluorescence intensity return to free QD fluorescence intensity 56%.
Embodiment 5: the ratio of fluorescence quantum and nano-metal particle is to the influence of fluorescence intensity
Adopt the same procedure of embodiment 3 and to plumbous DNA enzyme and substrate DNA, through changing the usage ratio of QD-DNA solution and Au-DNA solution, prepared a series of QD-AuNP conjugates, wherein the value of QD:AuNP was respectively 1: 10,3.2: 1 and 25: 1.Measure its initial fluorescent intensity respectively and add 1 μ M Pb
2+After fluorescence intensity.The result shows as QD: AuNP=25: in the time of 1, initial fluorescent intensity is about 300, adds 1 μ M Pb
2+Back fluorescence intensity is increased to about 350; As QD: AuNP=3.2: in the time of 1, initial fluorescent intensity is about 50, adds 0.2 μ M Pb
2+Back fluorescence intensity is increased to about 450; As QD: AuNP=1: in the time of 10, initial fluorescent intensity is about 100, adds 1 μ M Pb
2+Back fluorescence intensity increases to about 150.Above result shows, as QD: AuNP=3.2: in the time of 1, conjugate has shown best sensitivity in metal ion detection, and QD: AuNP is too high or too low the sensitivity of detection is descended.
Do not receive any theory constraint, think that the Au-DNA that in QD-DNA, adds different proportion can produce different cancellation effects.If the QD/NP ratio is too low, make NP and QD realize separating even add metallic ion, the fluorescence that the too much NP that is dispersed in the solution also can cancellation QD makes that the detection signal of reading is not obvious, sensitivity decline.If the QD/NP ratio is too high, even NP has realized being connected with the DNA enzyme through DNA substrate chain with QD,, can not carry out cancellation effectively to the fluorescence of QD because the number of NP is very little, be equivalent to improve the background noise that detects, sensitivity is descended.Therefore, the ratio of suitably selecting QD: NP is about 1: 10 to about 20: 1, for example about 3.2: 1 o'clock, can make the fluorescence intensity of QD-NP conjugate before detection lower, and the degree that fluorescence intensity is recovered after detection is bigger, thereby has improved the sensitivity that detects.
Below the mode of explanation has been described the present invention by way of example.But, should be appreciated that the present invention never only is limited to these embodiments.Those of ordinary skill can carry out various modifications or change to the present invention, and these modifications and change all belong to protection scope of the present invention.