CN105866079A - A molecular logic gate method building based on interactions among a fluorochrome NMM, G-quadruplex DNA, crown ether and metal ions - Google Patents
A molecular logic gate method building based on interactions among a fluorochrome NMM, G-quadruplex DNA, crown ether and metal ions Download PDFInfo
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Abstract
The invention relates to a molecular logic gate method building based on interactions among a fluorochrome NMM, G-quadruplex DNA, crown ether and metal ions. The molecular logic gates are built based on principles that the fluorescence intensity is increased when the fluorochrome NMM is in contact with the G-quadruplex DNA or the crown ether and that fluorescence is quenched when the fluorochrome NMM is in contact with the metal ions. The fluorescence intensity of the fluorochrome NMM is adopted as a determining criterion, when the fluorescence intensity is less than 75, 0 is outputted and when the fluorescence intensity is higher than 75, 1 is outputted. The molecular logic gates comprise an INHIBIT type logic gate, an IMPLY1 type logic gate and an IMPLY2 type logic gate. Compared with the prior art, the method is efficient, sensitive, simple, rapid and good in selectivity, and can be used for anti-tumor researching, metal ion detection, amino acid detection, and other fields.
Description
Technical field
The present invention relates to molecular computer technical field, be specifically related to a kind of based on fluorescent dye NMM, G-tetra-chain
The Molecular Logic Gates construction method that body DNA, crown ether and metal ion interact.
Background technology
Electronic computer changes the life style of the mankind to a great extent, becomes human society and enters postindustrial
One of the key technology progress in change epoch.Gate realizes logical operations rule in binary system, and constitutes
The arithmetic core of conditional electronic computer.Simple gate is made up of transistor, and these combinations can make to represent two
The high and low level of road input signal produces corresponding high and low Level output signal after by them.High,
Low level represents " 1 " " 0 " in binary system or "true" "false" in logic respectively, thus realizes logic fortune
Calculate.Arieh Aviram in 1988 proposes a tremendous and breakthrough imagination and builds the most on a molecular scale
Stand and perform logical operation, indicating the birth of Molecular Logic Gates.Binary system phenomenon on molecular level is gradually by people
Institute cognitive.Since de Silva reports first molecule-type AND logic gate, grinding of Molecular Logic Gates
Study carefully and achieve huge progress.Relative to conventional semiconductors, the advantage that construction logic operation is maximum on a molecular scale
It is that yardstick is little, reconfigurability good.The change that will be able to be activated by external condition such as light, pH, electricity or chemistry input
Credit or biomolecule have been obtained for extensive concern as function element, the research building Molecular Logic Gates.
G-tetra-stranded structure is the special nucleic acid secondary structure of discovered in recent years, and it is the most easily formed, distribution
Quite varied and there is important biological function.G-tetra-serobila of respective regions participates in lengthening of telomeres, DNA
Replicate, transcribe, meiosis, the important life process such as gene recombinaton, play antitumor, antiviral, suppression
Angiogenesiss etc. act on.Researchers detect the existence of G-tetra-serobila the most in vitro and parse its crystal knot
Structure, the method for various these structures of detection such as specificity fluorescent probe, antibody etc. are the most constantly found or synthesize.Porphyrin
Organic micromolecule has the binding ability of high specific to G-tetra-serobila DNA, utilizes potassium, the lead ion etc. can
Inducing the DNA sequence rich in G base to form G-tetra-stranded structure, porphyrin molecule NMM inserts G-tetra-chain
Body structure produces fluorescence signal, and therefore NMM is the fluorescent dye of a kind of energy specific recognition G-tetra-stranded structure.
Crown ether as a kind of macro ring ethers organic compound, be mainly used in the efficient phase transfer catalyst of Minute Organic Synthesis,
Chelating agent, noble metal and rare earths separation extraction extractant etc., ion is had selection to make by the void structure of crown ether
With, such as, 12-crown-4 and lithium ion complexation and not with sodium, potassium ion complexation；18-crown-6 not only with potassium ion network
Close, also can with diazol complexation, but not with lithium or sodium ion complexation.Willner seminar is based on 18 hat 6 ethers pair
The selection of potassium ion, and the regulating and controlling effect that potassium ion is to G-tetra-stranded structure, devise multiple configuration
DNA enzymatic, therefore can also use it for the design of Molecular Logic Gates.Heavy metal ion is as the important dirt of environment
Dye thing, there is the biggest impact in healthy on human body.Such as, mercury ion is highly stable bivalence gold in water body
Belonging to ion, the brain of people can by the approach such as drinking water and food by human intake, and be produced permanent by it
Damage.In recent years, DNA molecular has obtained paying close attention to widely with the effect of metal ion, this is because metal from
Son can form stable complex with specific DNA base.Such as: mercury ion (Hg2+), silver ion (Ag+)
Can make to be formed between thymus pyrimidine (T) and cytosine (C) T-Hg respectively2+-T and C-Ag+The similar base such as-C is joined
To structure.Studies have found that again, some metal ions, such as copper ion, iron ion, mercury ion etc. contaminates with fluorescence
Between material NMM, chelation can occur, these ions can the fluorescence of cancellation NMM, therefore based on metal from
Son and fluorescent dye and nucleic acid between contacting, these specific functions have great application prospect, such as metal from
The detection of son, the detection etc. of DNA structure, and it is many that the effect between these materials and contact can be utilized to construct
Planting different Molecular Logic Gates, therefore build efficient and sensible, simply, quickly, the System of Logic that selectivity is good has
Important meaning.
Summary of the invention
The purpose of the present invention be contemplated to provide a kind of efficient and sensible, simple, quickly, selectivity good based on fluorescence
The Molecular Logic Gates construction method that dyestuff NMM, G-tetra-serobila DNA, crown ether and metal ion interact.
The purpose of the present invention can be achieved through the following technical solutions: a kind of based on fluorescent dye NMM, G-
The Molecular Logic Gates construction method that four serobila DNA, crown ether and metal ion interact, described molecular logic
Door is to contact fluorescence intensity based on fluorescent dye NMM with G-tetra-serobila DNA or crown ether to strengthen, fluorescent dye
The principle that NMM contacts fluorescent quenching with metal ion is built-up, with the fluorescence intensity of fluorescent dye NMM
For basis for estimation, when fluorescence intensity is less than 75, it is output as 0, when fluorescence intensity is more than 75, is output as 1；
Described Molecular Logic Gates includes that INHIBIT type gate, IMPLY1 type gate and IMPLY2 type are patrolled
Collect door.
Described INHIBIT type gate, IMPLY1 type gate and IMPLY2 type gate construction method divide
As follows:
(1) taking fluorescent dye NMM solution is template, with the one in metal ion solution or crown ether solution or
Two kinds is input signal, and crown ether and NMM occur to interact and the fluorescence intensity of NMM is strengthened, metal from
Son and NMM occur chelation to make the fluorescent quenching of NMM, with the fluorescence intensity of NMM for output signal structure
Build INHIBIT type gate；
(2) mixed solution taking fluorescent dye NMM and crown ether is template, with Fe3+Solution or EDTA solution
In one or both be input signal, Fe3+Chelation is occurred to make the fluorescent quenching of NMM with NMM,
EDTA Yu NMM and crown ether are without effect, EDTA and Fe3+Chelation is occurred to make the fluorescence of NMM recover,
IMPLY1 type gate is built for output signal with the fluorescence intensity of NMM and crown ether effect；
(3) mixed solution taking fluorescent dye NMM and G-tetra-serobila DNA is template, with Hg2+Solution or
One or both in cysteine solution are input signal, Hg2+Chelation is occurred to make NMM's with NMM
Fluorescent quenching, cysteine and NMM and G-tetra-serobila DNA are without effect, cysteine and Hg2+In conjunction with making
The fluorescence of NMM and G-tetra-serobila DNA recovers, with the fluorescence intensity of NMM and G-tetra-serobila DNA effect
IMPLY2 type gate is built for output signal.
Between heretofore described NMM and crown ether, the mechanism of effect is mainly due to NMM itself in aqueous
Fluorescence the lowest, but its fluorescence in organic solvent is very strong, and this is owing to water reduces the energy level of NMM, makes
It is susceptible to nonradiative transition under light excites, and energy level in organic solvent is higher, is easier to spoke
Penetrating transition, simultaneously because NMM and crown ether are all macrocyclic compounds, and the radius of both rings is close, so adds
After entering 18 hat 6 ethers, there is pi-pi accumulation, so that NMM is in a kind of anhydrous cavity in easy and NMM
In, block the water cancellation to NMM fluorescence, when there being light to excite, NMM is easier to radiation transistion.
NMM is as the probe of a kind of good identification G-tetra-serobila DNA simultaneously, and it is inserted into G-tetra-serobila
In structure so that it is fluorescence intensity is obviously enhanced, and the effect of metal ion and NMM is mainly by chelation,
EDTA also has chelation to iron ion, with NMM, Competition can occur by the amount controlling EDTA,
Thus capture out by iron ion.Containing sulfydryl in cysteine, it can be good at being combined with mercury ion, so
Addition cysteine is also with the Competition between it and NMM, then captures from NMM by mercury ion
Come.In the present invention, raw materials is cheap, and preparation is simple, easily stores, and the method utilizing fluorescence, operation
Simple and sensitive, it is possible to realize the detection to metal ion, aminoacid and DNA structure well.
Metal ion described in step (1) includes Fe3+And Hg2+, described crown ether includes 18 hat 6 ethers.
The molar concentration of described NMM solution is 0.8～1.2 μM；
In step (1), NMM and 18 hat 6 ethers concentration ratio be 1:(25000～70000), NMM with
The concentration ratio of mercury ion is 1:(80～120), NMM is 1:(200～400 with the concentration ratio of iron ion)；
In step (2), NMM and 18 hat 6 ethers concentration ratio be 1:(25000～70000), NMM with
The concentration ratio of mercury ion is 1:(80～120), iron ion is 1:(3～5 with the concentration ratio of EDTA)；
In step (3), the concentration ratio of NMM Yu G4 is 1:(1～3), the concentration ratio of NMM and mercury ion
Example is 1:(80～120), mercury ion is 1:(3～7 with the concentration ratio of cysteine).
Wherein, the concentration of 18 hat 6 ethers can not be the highest or the lowest, the highest if metal ion dense that add below
Degree also can increase accordingly, and for all increasing accordingly with the EDTA of NMM competition or the concentration of cysteine
Adding, so concentration is moderate, the metal ion being simultaneously introduced can not be the highest or too for the concentration of cancellation NMM
Low, the highest if quenching effects the best, but the concentration adding the material of chelated metal ions is also required to the highest,
Could recover the fluorescence of NMM, the effect that otherwise NMM fluorescence rises is the most bad, therefore, used in the present invention
The concentration of material is the best results summed up through many experiments and the higher concentration of discrimination.
When step (1) described metal ion is Fe3+, when described crown ether is 18 hat 6 ether, with Fe3+Solution or
One or both in 18 hat 6 ethereal solutions are input signal, and 18 hat 6 ethers occur interaction to make with NMM
The fluorescence intensity of NMM strengthens, Fe3+Chelation is occurred to make the fluorescent quenching of NMM, with NMM with NMM
Fluorescence intensity be output signal build INHIBIT1 type gate；
When step (1) described metal ion is Hg2+, when described crown ether is 18 hat 6 ether, with Hg2+Solution or
One or both in 18 hat 6 ethereal solutions are input signal, and 18 hat 6 ethers occur interaction to make with NMM
The fluorescence intensity of NMM strengthens, Hg2+Chelation is occurred to make the fluorescent quenching of NMM, with NMM with NMM
Fluorescence intensity be output signal build INHIBIT2 type gate.
Although above-mentioned two gate broadly falls into INHIBIT type, but its output result is different, mainly by
In in design we by Hg in two doors2+(the 3rd) and Fe3+(the second) addition sequence is designed as difference,
Causing its output result also to differ, four kinds of output results of INHIBIT1 type are (0,1,0,0), INHIBIT2
Four kinds of type output result is (0,0,1,0).It practice, when the input of gate is two kinds, that input condition is just
Have four kinds, output situation altogether just have 16 kinds (because each corresponding two kinds of outputs of input, i.e. 0 and 1,
The output result that those four kinds of input conditions are corresponding just has 2 × 2 × 2 × 2 that is 16 kind).So, INHIBIT1 and
INHIBIT2 is exactly two kinds therein, in the present invention design other two kinds of gates be IMPLY1 type and
IMPLY2 type.
The NMM solution of described 0.8～1.2 μM is to store liquid dilution by the NMM of 100 μMs to obtain.
The NMM storage liquid of described 100 μMs is through the following steps that prepare: NMM is dissolved in DMSO solvent
In, mix homogeneously obtains the NMM that concentration is 5mM and stores liquid, and-20 DEG C keep in Dark Place, standby；Again from this
Taking out part storage liquid in the NMM storage liquid of 5mM, add triple distillation water, being configured to concentration is 100 μMs
NMM store liquid, 4 DEG C keep in Dark Place, standby.
Described Tris-HCl buffer contains Tris and HCl, and wherein, the concentration of Tris is 8～12mM,
The pH of Tris-HCl buffer is 7.0～7.5.
The concentration of described G-tetra-serobila DNA is 100 μMs, is prepared by following steps: will be containing rich G sequence
Add Tris-KCl buffer in the DNA of row, form the solution that concentration is 100 μMs of DNA, mix homogeneously
Rear sealing, stands 8min in 90 DEG C of thermostatic water bath, is then cooled to room temperature, preserve at a temperature of 4 DEG C, standby
With.
Described rich G sequence is 5 '-AGGGTTAGGGTTAGGGTTAGGG-3 '.
Containing Tris and KCl in described Tris-KCl buffer, wherein, the concentration of Tris is 8～12mM,
The concentration of KCl is 30～60mM, and the pH of Tris-KCl buffer is 7.0～7.5.
Compared with prior art, beneficial effects of the present invention is embodied in following several respects:
(1) present invention utilizes porphyrin fluorescent dye NMM and macro ring ethers Organic substance 18 to be preced with the work of 6 ethers first
By mechanism, construct INHIBIT1 type gate and INHIBIT2 type gate, can efficient and sensible, simple,
The metal ion being quickly detected from solution；
(2) utilize IMPLY1 type gate and the IMPLY2 type gate of the present invention, can quickly detect amino
Acid, and antineoplastic research is had good directive function.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of INHIBIT1 type gate；
Fig. 2 is INHIBIT1 type gate fluorescence intensity figure of NMM when varying input signal；
Fig. 3 is the logic figure of INHIBIT1 type gate；
Fig. 4 is the fluorescence spectrum figure that NMM and 18C6 acts in different solutions
Fig. 5 is the schematic diagram of INHIBIT2 type gate；
Fig. 6 is INHIBIT2 type gate fluorescence intensity figure of NMM when varying input signal；
Fig. 7 is the logic figure of INHIBIT2 type gate；
Fig. 8 is the schematic diagram of IMPLY1 type gate；
Fig. 9 is IMPLY1 type gate fluorescence intensity figure of NMM when varying input signal；
Figure 10 is the logic figure of IMPLY1 type gate；
Figure 11 is the schematic diagram of IMPLY2 type gate；
Figure 12 is IMPLY2 type gate fluorescence intensity figure of NMM when varying input signal；
Figure 13 is the logic figure of IMPLY2 type gate.
Detailed description of the invention
Elaborating embodiments of the invention below, the present embodiment enters under premised on technical solution of the present invention
Row is implemented, and gives detailed embodiment and concrete operating process, but under protection scope of the present invention is not limited to
The embodiment stated.
Embodiment 1
The preparation of Tris-HCl buffer (Tris:10mM, pH=7.4): really weigh Tris 0.6057g in 500
In mL beaker, it is completely dissolved with 400mL triple distillation water, is regulated pH to 7.4 with dilute hydrochloric acid solution,
Move liquid to 500mL volumetric flask.Beaker triple distillation water washs three times, and rinse liquid is transferred in volumetric flask, fixed
Holding, mix homogeneously is standby.
The preparation of NMM storage liquid (2mL, 100 μMs): accurately weigh NMM 5mg, adds 1.72mL
DMSO is dissolved, mix homogeneously, and-20 DEG C keep in Dark Place, standby, and concentration is 5mM, more molten from this
Liquid takes 40 μ L, adds 1960 μ L triple distillation water, be i.e. configured to the storage liquid that concentration is 100 μMs, keep away for 4 DEG C
Light preserves, standby.
The preparation of 18 hats 6 ether storage liquid (10mL, 1M): accurately weigh 18 hat 6 ether 2.6432g, use 10mL
Triple distillation water dissolution, mix homogeneously is standby.
The preparation of mercury ion storage liquid (10mL, 10mM): accurately weigh solid chlorine hydrargyrum 0.0272g, use 10mL
Triple distillation water dissolution, mix homogeneously is standby.
The structure of INHIBIT1 type gate, its principle is as it is shown in figure 1, with mercury ion (0.1mM) and 18C6
(50mM) being input signal, with the fluorescence intensity of NMM (1 μM) as output signal, fluorescence intensity is more than
75, corresponding true value is 1, and less than 75, corresponding true value is 0.When input is (0,0), from NMM
Storage liquid takes 10 μ L, adds 990 μ L Tris-HCl buffer, under excitation wavelength is 399nm, survey it glimmering
Light intensity, owing to NMM autofluorescence intensity in aqueous is the lowest, so output valve is 0；When merely entering hydrargyrum
During ion (1,0), adding 10 μ L mercury ion storage liquid, ambient temperatare is put 10 minutes, owing to mercury ion can
The fluorescence of cancellation NMM, so output valve is 0；When merely entering 18C6 (0,1), add 50 μ L18C6
Storage liquid, owing to NMM and 18 hat 6 ethers are all macrocyclic compounds, the addition of 18 hat 6 ethers, may heap
Amass on NMM surface, hinder the water quenching effect to NMM, cause the fluorescence intensity of NMM to raise, because of
This output valve is 1；When being simultaneously entered mercury ion and 18 hat 6 ethers (1,1), it is separately added into 10 μ L mercury ions
Storage liquid and 50 μ L18C6 store liquid, and ambient temperatare puts 10 minutes, and mercury ion still can cancellation NMM glimmering
Light, hinders the effect of NMM and 18 hat 6 ethers, and therefore output valve is 0.
Under varying input signal, the fluorescence intensity of fluorescent dye NMM detects as in figure 2 it is shown, return the true of its correspondence
Value table is as shown in table 1.
Table 1INHIBIT1 type gate truth table
| Input 1 (Hg2+) | Input 2 (18C6) | Output (NMM) |
| 0 | 0 | 0 |
| 0 | 1 | 1 |
| 1 | 0 | 0 |
| 1 | 1 | 0 |
According to this truth table, it can be deduced that the logic figure of INHIBIT1 type gate is as shown in Figure 3.
The mechanism that NMM and 18 hat 6 ether interacts is probed into as shown in Figure 4, and wherein a line represents 1 μM
NMM fluorescence intensity in acetonitrile solvent；B line represents that the NMM of 1 μM is in Tris-HCl buffer
Fluorescence intensity；C line represents the 18C6 of 50mM fluorescence intensity in acetonitrile solvent；D line represents 50mM's
18C6 fluorescence intensity in Tris-HCl buffer；E line represents the 18C6 of NMM and 50mM of 1 μM
Fluorescence intensity in Tris-HCl buffer.From this figure, it can be seen that the fluorescence that NMM itself is in aqueous
The lowest, but its fluorescence in organic solvent is very strong, crown ether all unstressed configuration in aqueous solution or organic solvent,
But 18C6 but can promote the fluorescence intensity of NMM to occur significantly raised in aqueous, it can therefore be concluded that by
It is all macrocyclic compounds in NMM and crown ether, and the radius of both rings is close, after 18 hat 6 ethers add,
There is pi-pi accumulation in easy and NMM, so that NMM is in a kind of anhydrous cavity, has blocked water to NMM
The cancellation of fluorescence, when there being light to excite, NMM is easier to radiation transistion occur, so the rise of fluorescence can be caused.
Embodiment 2
The preparation of iron ion storage liquid (10mL, 10mM): accurately weigh Iron(III) chloride hexahydrate 0.0270g,
Being dissolved in 10mL triple distillation water, mix homogeneously is standby.
Meanwhile, Tris-HCl buffer, NMM storage liquid and 18 hat 6 ethers same as in Example 1 are used to deposit
Liquid storage.
The structure of INHIBIT2 type gate, its principle is as it is shown in figure 5, with 18C6 (50mM) and iron ion
(0.3mM) as input signal, with the fluorescence intensity of NMM (1 μM) as output signal, fluorescence intensity is big
In 75, corresponding true value is 1, and less than 75, corresponding true value is 0.When input is (0,0), from
NMM storage liquid takes 10 μ L, adds 990 μ L Tris-HCl buffer, under excitation wavelength is 399nm
Survey its fluorescence intensity, owing to NMM autofluorescence intensity in aqueous is the lowest, so output valve is 0；When only
During input 18C6 (0,1), add 50 μ L18C6 and store liquid, owing to NMM can be deposited between 18 hat 6 ethers
Surface, hinders the water quenching effect to NMM, causes the fluorescence intensity of NMM to raise, and therefore output valve is
1；When merely entering iron ion (1,0), adding 30 μ L iron ion storage liquid, ambient temperatare is put 10 minutes, by
In iron ion can the fluorescence of cancellation NMM, so output valve is 0；When be simultaneously entered 18 hat 6 ethers and ferrum from
During son (1,1), being separately added into 50 μ L18C6 storage liquid and 30 μ L iron ions storage liquid, ambient temperatare puts 10
Minute, iron ion still can the fluorescence of cancellation NMM, hinder NMM and 18 effects being preced with 6 ethers, the most defeated
Going out value is 0.
Under varying input signal, the fluorescence intensity of fluorescent dye NMM detects as shown in Figure 6, returns the true of its correspondence
Value table is as shown in table 2.
Table 2INHIBIT2 type gate truth table
| Input 1 (18C6) | Input 2 (Fe3+) | Output (NMM) |
| 0 | 0 | 0 |
| 0 | 1 | 0 |
| 1 | 0 | 1 |
| 1 | 1 | 0 |
According to this truth table, it can be deduced that the logic figure of INHIBIT2 type gate is as shown in Figure 7.
Embodiment 3
The preparation of EDTA storage liquid (10mL, 50mM): accurately weigh EDTA0.1461g, solution 10mL
In triple distillation water, mix homogeneously is standby.
Use Fe same as in Example 2 simultaneously3+Storage liquid, Tris-HCl buffer, NMM store liquid and 18
It is preced with 6 ether storage liquid
The structure of IMPLY1 type gate, its principle as shown in Figure 8, with EDTA (1mM) and iron ion
(0.3mM) as input signal, make with the fluorescence intensity that NMM (1 μM) and 18C6 (50mM) acts on
For output signal, fluorescence intensity is more than 75, and corresponding true value is 1, and less than 75, corresponding true value is 0.
When input is (0,0), from NMM storage liquid, takes 10 μ L, add 50 μ L18C6 and store liquid, add
940 μ L Tris-HCl buffer, survey its fluorescence intensity under excitation wavelength is 399nm, only exist NMM in system
And 18C6, both interact, make the fluorescence intensity of NMM raise, in this, as initial fluorescence value, the most defeated
Going out value is 1；When merely entering object iron ion (0,1), add 30 μ L iron ion storage liquid, under room temperature
Place 10 minutes, due to the effect between iron ion and NMM, cause the fluorescent quenching of NMM, the most defeated
Going out value is 0；When merely entering EDTA, add 20 μ LEDTA and store liquid, due to EDTA Yu NMM and
Without interacting between 18C6, so fluorescence intensity is almost unchanged, output valve is still 1；When be simultaneously entered ferrum from
When son and EDTA (1,1), it is simultaneously introduced 30 μ L iron ion storage liquid and 20 μ LEDTA store liquid, under room temperature
Placing 10 minutes, due to EDTA energy chelates ferric ions, and both binding abilities are better than iron ion and NMM
Between action intensity, so NMM still can be more or less the same with initial strength with 18C6 effect, fluorescence intensity,
Therefore, output valve is still 1.
Under varying input signal, the fluorescence intensity of fluorescent dye NMM detects as it is shown in figure 9, return the true of its correspondence
Value table is as shown in table 3.
Table 3IMPLY1 type gate truth table
| Input 1 (EDTA) | Input 2 (Fe3+) | Output (NMM+18C6) |
| 0 | 0 | 1 |
| 0 | 1 | 0 |
| 1 | 0 | 1 |
| 1 | 1 | 1 |
According to this truth table, it can be deduced that the logic figure of IMPLY1 type gate is as shown in Figure 10.
Embodiment 4
The preparation of Tris-KCl buffer (Tris:10mM, KCl:50mM, pH=7.4): really weigh Tris
0.6057g, KCl 1.8638g, in 500mL beaker, is completely dissolved with 400mL triple distillation water,
Regulate pH to 7.4 with dilute hydrochloric acid solution, move liquid to 500mL volumetric flask.Beaker triple distillation water washs three
Secondary, rinse liquid is transferred in volumetric flask, constant volume, and mix homogeneously is standby.
The formation of G-tetra-stranded structure: the Tris-KCl of respective volume will be added in the DNA containing rich G sequence
Buffer, makes its mix homogeneously by liquid-transfering gun compressing, seals, and stands 8min, then in 90 DEG C of thermostatic water bath
It is slowly cooled to room temperature, puts in 4 DEG C of refrigerators and save backup.The concentration of the DNA solution of preparation is 100 μMs.
DNA sequence is 22AG:5 '-AGGGTTAGGGTTAGGGTTAGGG-3 '.
The preparation of cysteine (Cys) storage liquid (10mL, 10mM): accurately weigh cysteine 0.01212g,
With 10mL triple distillation water dissolution, mix homogeneously is standby.
Meanwhile, Hg same as in Example 1 is used2+Storage liquid, Tris-HCl buffer and NMM store liquid.
The structure of IMPLY2 type gate, its principle as shown in figure 11, with mercury ion (0.1mM) and half Guang
Propylhomoserin (0.5mM) is input signal, is defeated with the fluorescence intensity that NMM (1 μM) and G4 (1 μM) act on
Going out signal, fluorescence intensity is more than 75, and corresponding true value is 1, and less than 75, corresponding true value is 0.When
When input signal is (0,0), adding 10 μ L NMM and store liquid, 10 μ L G4 store liquid, and room temperature places 30
Minute, system only exists NMM and G4, owing to NMM can be inserted in G-tetra-stranded structure so that it is glimmering
Light is obviously enhanced, and therefore using this fluorescence intensity as Initial output signal, i.e. output valve is 1；When merely entering half Guang
During propylhomoserin (0,1), add cysteine storage liquid 50 μ L, due to cysteine and NMM, equal nothing between G4
Interact, and on the effect of NMM and G4 without impact, fluorescence intensity is basically unchanged, and therefore output valve is still
1；When merely entering mercury ion (1,0), adding 10 μ L mercury ion storage liquid, room temperature is placed 10 minutes, due to
Mercury ion can be combined with NMM, hinders NMM to be inserted in G-tetra-stranded structure, makes the fluorescence of NMM
Intensity significantly reduces, and therefore output valve is 0；When both are simultaneously entered, add 10 μ L mercury ions storage liquid and
50 μ L cysteine storage liquid, room temperature is placed 30 minutes, owing to containing the combination of sulfydryl and mercury ion on cysteine
Ability is relatively strong, and its action intensity is more than the mercury ion cancellation intensity to NMM, the therefore fluorescence intensity of NMM
Change is little, and output valve is still 1.
Under varying input signal, the fluorescence intensity of fluorescent dye NMM detects as shown in figure 12, returns its correspondence
Truth table is as shown in table 4.
Table 4IMPLY2 type gate truth table
| Input 1 (Hg2+) | Input 2 (Cys) | Output (NMM+G4) |
| 0 | 0 | 1 |
| 0 | 1 | 1 |
| 1 | 0 | 0 |
| 1 | 1 | 1 |
According to this truth table, it can be deduced that the logic figure of IMPLY2 type gate is as shown in figure 13.
Embodiment 5
Using and the identical construction method of embodiment 1～4, difference is that each solution concentration used is different, tool
Body difference is:
(1) concentration of NMM solution used by is 0.8 μM；
(2) used by, the concentration of 18 hat 6 ethereal solutions is 20mM；
(3) Hg used by2+The concentration of solution is 64 μMs；
(4) Fe used by3+The concentration of solution is 0.16mM；
(5) concentration of EDTA solution used by is 0.48mM；
(6) used by, the concentration of G-tetra-serobila DNA solution is 0.8 μM；
(7) concentration of cysteine solution used by is 0.192mM；
(8) in the Tris-HCl buffer used by, the concentration of Tris be 8mM, pH be 7.0；
(9) in the Tris-KCl buffer used by, the concentration of Tris be the concentration of 8mM, KCl be 30mM,
PH is 7.0.
Experiments verify that, the INHIBIT1 type gate identical with embodiment 1～4, INHIBIT2 type can be set up
Gate, IMPLY1 type gate and IMPLY2 type gate.
Embodiment 6
Using and the identical construction method of embodiment 1～4, difference is that each solution concentration used is different, tool
Body difference is:
(1) concentration of NMM solution used by is 1.2 μMs；
(2) used by, the concentration of 18 hat 6 ethereal solutions is 84mM；
(3) Hg used by2+The concentration of solution is 144 μMs；
(4) Fe used by3+The concentration of solution is 0.48mM；
(5) concentration of EDTA solution used by is 2.4mM；
(6) used by, the concentration of G-tetra-serobila DNA solution is 3.6 μMs；
(7) concentration of cysteine solution used by is 1.008mM；
(8) in the Tris-HCl buffer used by, the concentration of Tris be 12mM, pH be 7.5；
(9) in the Tris-KCl buffer used by, the concentration of Tris be the concentration of 12mM, KCl be 60mM,
PH is 7.5.
Experiments verify that, the INHIBIT1 type gate identical with embodiment 1～4, INHIBIT2 type can be set up
Gate, IMPLY1 type gate and IMPLY2 type gate.
Claims (10)
1. one kind based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and metal ion interact point
Sub-gate construction method, it is characterised in that described Molecular Logic Gates is based on fluorescent dye NMM and G-
Four serobila DNA or crown ether contact fluorescence intensity strengthen, and fluorescent dye NMM contacts fluorescent quenching with metal ion
Principle built-up, with the fluorescence intensity of fluorescent dye NMM as basis for estimation, when fluorescence intensity is less than
When 75, it is output as 0, when fluorescence intensity is more than 75, is output as 1；
Described Molecular Logic Gates includes that INHIBIT type gate, IMPLY1 type gate and IMPLY2 type are patrolled
Collect door.
One the most according to claim 1 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that described INHIBIT type logic
Door, IMPLY1 type gate and IMPLY2 type gate construction method are as follows:
(1) taking fluorescent dye NMM solution is template, with the one in metal ion solution or crown ether solution or
Two kinds is input signal, and crown ether and NMM occur to interact and the fluorescence intensity of NMM is strengthened, metal from
Son and NMM occur chelation to make the fluorescent quenching of NMM, with the fluorescence intensity of NMM for output signal structure
Build INHIBIT type gate；
(2) mixed solution taking fluorescent dye NMM and crown ether is template, with Fe3+Solution or EDTA solution
In one or both be input signal, Fe3+Chelation is occurred to make the fluorescent quenching of NMM with NMM,
EDTA Yu NMM and crown ether are without effect, EDTA and Fe3+Chelation is occurred to make the fluorescence of NMM recover,
IMPLY1 type gate is built for output signal with the fluorescence intensity of NMM and crown ether effect；
(3) mixed solution taking fluorescent dye NMM and G-tetra-serobila DNA is template, with Hg2+Solution or
One or both in cysteine solution are input signal, Hg2+Chelation is occurred to make NMM's with NMM
Fluorescent quenching, cysteine and NMM and G-tetra-serobila DNA are without effect, cysteine and Hg2+In conjunction with making
The fluorescence of NMM and G-tetra-serobila DNA recovers, with the fluorescence intensity of NMM and G-tetra-serobila DNA effect
IMPLY2 type gate is built for output signal.
One the most according to claim 1 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
Metal ion interact Molecular Logic Gates construction method, it is characterised in that the metal described in step (1) from
Attached bag includes Fe3+And Hg2+, described crown ether includes 18 hat 6 ethers.
One the most according to claim 2 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that described NMM solution mole dense
Degree is 0.8～1.2 μM；
In step (1), the molar concentration ratio of NMM and 18 hat 6 ethers is 1:(25000～70000), NMM
It is 1:(80～120 with the molar concentration ratio of mercury ion), NMM is 1 with the molar concentration ratio of iron ion:
(200～400)；
In step (2), the molar concentration ratio of NMM and 18 hat 6 ethers is 1:(25000～70000), NMM
It is 1:(200～400 with the molar concentration ratio of iron ion), iron ion is 1 with the molar concentration ratio of EDTA:
(3～5)；
In step (3), the molar concentration ratio of NMM Yu G4 is 1:(1～3), NMM rubs with mercury ion
Your concentration ratio is 1:(80～120), mercury ion is 1:(3～7 with the molar concentration ratio of cysteine).
One the most according to claim 4 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that described 0.8～1.2 μM
NMM solution is to store liquid dilution by the NMM of 100 μMs to obtain；The NMM of described 100 μMs deposits
Liquid storage is through the following steps that prepare: being dissolved in by NMM in DMSO solvent, mix homogeneously obtains concentration and is
The NMM of 5mM stores liquid, and-20 DEG C keep in Dark Place, standby；Store liquid from the NMM of this 5mM again and take
Go out part storage liquid, add triple distillation water, be configured to the NMM that concentration is 100 μMs and store liquid, 4 DEG C of lucifuges
Preserve, standby.
One the most according to claim 5 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that described Tris-HCl buffer
Containing Tris and HCl, wherein, the concentration of Tris is 8～12mM, and the pH of Tris-HCl buffer is 7.0～7.5.
One the most according to claim 3 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that when described metal ion is Fe3+,
When described crown ether is 18 hat 6 ether, with Fe3+One or both in solution or 18 hat 6 ethereal solutions are input signal,
18 hat 6 ethers occur to interact with NMM and the fluorescence intensity of NMM are strengthened, Fe3+Chela is there is with NMM
The cooperation fluorescent quenching making NMM, builds INHIBIT1 type with the fluorescence intensity of NMM for output signal and patrols
Collect door；
When described metal ion is Hg2+, when described crown ether is 18 hat 6 ether, with Hg2+Solution or 18 hat 6 ethers
One or both in solution are input signal, and 18 hat 6 ethers occur to interact with NMM and make NMM's
Fluorescence intensity strengthens, Hg2+Chelation is occurred to make the fluorescent quenching of NMM, with the fluorescence of NMM with NMM
Intensity is that output signal builds INHIBIT2 type gate.
One the most according to claim 2 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that described G-tetra-serobila DNA leads to
Cross following steps to prepare: add Tris-KCl buffer by the DNA containing rich G sequence, close after mix homogeneously
Envelope, stands 8min in 90 DEG C of thermostatic water bath, is then cooled to room temperature, preserve at a temperature of 4 DEG C, standby.
One the most according to claim 8 based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether and
The Molecular Logic Gates construction method that metal ion interacts, it is characterised in that described rich G sequence is
5’-AGGGTTAGGGTTAGGGTTAGGG-3’。
One the most according to claim 8 is based on fluorescent dye NMM, G-tetra-serobila DNA, crown ether
The Molecular Logic Gates construction method interacted with metal ion, it is characterised in that described Tris-KCl buffering
Containing Tris and KCl in liquid, wherein, the concentration of Tris is 8～12mM, and the concentration of KCl is 30～60mM,
The pH of Tris-KCl buffer is 7.0～7.5.
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