CN108304932A - The structure of logic gate based on silver nanoclusters and its application in intelligent measurement - Google Patents
The structure of logic gate based on silver nanoclusters and its application in intelligent measurement Download PDFInfo
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Abstract
The invention belongs to the Molecular Logic Gates technical fields of analytical chemistry, are related to a kind of foundation of label-free, highly sensitive, highly selective miRNA intelligent detecting methods.The Molecular Logic Gates are built-up based on silver nanoclusters and graphene oxide, using the fluorescence intensity relative value of silver nanoclusters as basis for estimation, when relative intensity of fluorescence is more than 0.5, export as " 1 ", when relative intensity of fluorescence is less than 0.5, export as " 0 ".The Molecular Logic Gates include OR logic gates and INHIBIT logic gates, they can be used for examining whether two different miRNA (miR 21 and miR 141) in actual sample are respectively present.This logic detection method can identify the content of multiple target analytes in complex biological sample have potential application value in the early diagnosis of major disease, multivariate detection and clinical treatment etc..
Description
Technical field
The invention belongs to the Molecular Logic Gates technical fields of analytical chemistry, are related to a kind of label-free, highly sensitive, high selection
The foundation of the miRNA intelligent detecting methods of property.
Background technology
Molecular device is to put forward the 1980s, relates generally to two fields：Device based on molecular scale and
Device based on molecular material.Wherein, the device based on molecular scale is presently the most several hot spots neck active, that research is most wide
One of domain.Molecular device is most important for the development of the equipment miniaturization of analytical chemistry, molecular device itself have it is at low cost,
The features such as being swift in response is conducive to the development of portable instrument, to achieve the purpose that real-time, on-line checking.Molecular device is main
Including molecular rectifier, molecular memory, molecular motor, Molecular Logic Gates etc..This patent mainly using Molecular Logic Gates and
Biosensor carries out the intelligent processing of signal Analysis.
Due to the outstanding molecule distinguishabilities of DNA, the molecular logic device based on DNA is in medical diagnosis on disease, bio-sensing etc.
Field, which achieves, also achieves certain development.One considerable advantage of DNA molecular logic gate is exactly that it can be to low concentration
Object generates response signal, to complete to detect.However, label-free logical sensor of the foundation based on molecular level is seldom,
This is because logical sensor needs to combine multiple logic gates, caused by building process is more complicated.This logical sensor can
To realize the multivariate detection to the different target object in same sample, for pushing application of the Molecular Logic Gates in analytical chemistry
Have great importance.However up to now, only several molecular logic devices realize the multivariate detection to target molecule.
We utilize graphene oxide and silver nanoclusters, construct a multiple logic door based on OR and INHIBIT, realize for the first time
Multi-element intelligentization detection to two different miRNA (miR-21 and miR-141), compared with traditional sensor, it can be examined
It measures the presence of multiple analytes and carries out complicated logic analysis.
Invention content
The purpose of the present invention is intended to provide a kind of label-free multiple molecular based on silver nanoclusters and graphene oxide and patrols
Collect the application of door and the logical device in the detection of miRNA multi-element intelligentizations.The cascade multiple logics of this OR and INHIBIT
Door is for examining the combinational logic that whether two different miRNA (miR-21 and miR-141) are respectively present in actual sample
Circuit.This logic detection method can identify the content of multiple target analytes in complex biological sample.
The invention is realized in this way it is a kind of for identify in actual sample two different miRNA (miR-21 and
MiR-141) the combinational logic circuit whether being respectively present, specifically includes following steps：
The preparation for the silver nanoclusters that 1.DNA stablizes：The template Ag-DNA1 or Ag-DNA2 of silver nanoclusters are dissolved in phosphoric acid
In the buffer solution of sodium, 90 DEG C of annealing are heated to, are then cooled to room temperature.Solution is added in 1mM silver ions, concussion shakes up, puts
30min is set, NaBH is added4, silver ion reduction is acutely kept in dark place overnight after concussion 1min again.After the reaction was complete,
Obtain the silver nanoclusters solution of flaxen DNA protections.
2. the structure of the molecular logic platform based on silver nanoclusters and graphene oxide：By 100nM Ag-DNA1 and 100nM
The silver nanoclusters of Ag-DNA2 protections, are used as logical operation platform after being mixed 15 minutes with the graphene oxide of 15 μ g/mL.
The structure of 3.OR logic gates：Using molecular logic platform as substrate, miR-21 and miR-141 that various combination is added come
Build OR logic gates.When nucleic acid sequence (miR-21 and miR-141) is added, note input is " 1 ", is otherwise " 0 ".According to opposite
Whether fluorescence intensity level, which is more than 0.5, is used as basis for estimation, when relative intensity of fluorescence is more than 0.5, exports as " 1 ", when relatively glimmering
When luminous intensity is less than 0.5, export as " 0 ".MiR-21 and miR-141 can build an OR logic on molecular logic platform
Door.When inputting any one value in miR-21 or miR-141, output signal is " 1 ".
4. the structure of the INHIBIT logic gates based on miR-21 and P1：Using molecular logic platform as substrate, it is added different groups
The miR-21 and P1 of conjunction builds INHIBIT logic gates.MiR-21 and P1 can build one on molecular logic platform
INHIBIT logic gates.No matter when inputting P1, whether miR-21 is inputted simultaneously in system, output signal is " 0 ".Only body
When inputting miR-21 without inputting P1 in system, output signal is " 1 ".
5. the structure of the INHIBIT logic gates based on miR-141 and P2：Using molecular logic platform as substrate, it is added different
The miR-141 and P2 of combination builds INHIBIT logic gates.MiR-141 and P2 can build one on molecular logic platform
INHIBIT logic gates.No matter when inputting P2, whether miR-141 is inputted simultaneously in system, output signal is " 0 ".Only body
When inputting miR-141 without inputting P2 in system, output signal is " 1 ".
The multivariable of 6.miRNA detects：In order to judge whether be respectively present two different miRNA in sample
(miR-21 and miR-141), same sample is divided into four parts by us.First, four parts of same samples are separately added into GO/Ag-
DNA1/Ag-DNA2 molecular logic platforms are denoted as sample 1, sample 2, sample 3, sample 4.For first sample, do not add
Other inputs.For second sample, 1 μM of P1 is added.For third sample, 1 μM of P2 is added.For the 4th sample,
1 μM of P1 and 1 μM of P2 is added." 1 " and " 0 " is exported according to the relative intensity of fluorescence value of sample 1,2,3,4, judges sample with truth table
MiR-21 and miR-141 whether there is in product.
Description of the drawings
Fig. 1 (A) OR logic gates build schematic diagram.(B) fluorescence spectra of OR logic gates.(C) OR logic gates is relatively glimmering
Luminous intensity block diagram.(D) truth table of OR logic gates；
INHIBIT logic gates of the Fig. 2 (A) based on miR-21 and P1 builds schematic diagram.(B) based on miR-21's and P1
The fluorescence spectra of INHIBIT logic gates.(C) the relative intensity of fluorescence column of the INHIBIT logic gates based on miR-21 and P1
Figure.(D) truth table of INHIBIT logic gates；
INHIBIT logic gates of the Fig. 3 (A) based on miR-141 and P2 builds schematic diagram.(B) based on miR-141's and P2
The fluorescence spectra of INHIBIT logic gates.(C) the relative intensity of fluorescence column of the INHIBIT logic gates based on miR-141 and P2
Figure.(D) truth table of INHIBIT logic gates；
Fig. 4 (A) INHIBIT-OR cascaded logic doors are used for the schematic diagram of intelligent measurement miR-21 and miR-141.(B)
The truth table of INHIBIT-OR cascaded logic doors." " represent such case and can ignore；
Specific implementation mode
Below with reference to embodiment, the present invention will be further described, and the embodiment of the present invention is merely to illustrate the present invention's
Technical solution, and the non-limiting present invention.
Embodiment 1
The preparation of silver nanoclusters：5 μM of Ag-DNA1 or Ag-DNA2 are dissolved in the buffer solution (10mM of sodium phosphate
Na2HPO4/NaH2PO4,100mM CH3COONa,5mM Mg(CH3COO)2, pH 7.5) in, 10 points are then heated at 90 DEG C
Then clock is slowly cooled to room temperature.Then by 30 μM of AgNO3It is added in 5 μM of Ag-DNA1 or Ag-DNA2 solution, mixing
Afterwards, mixed solution is being protected from light lower placement 30 minutes, is making Ag+Ion is fully acted on C bases.Finally, again into buffer system
The secondary NaBH for being added 30 μM4Solution acutely shakes 1 minute.It is protected from light at 4 DEG C overnight, fluorescence silver nanoclusters is made.
Embodiment 2
The structure of OR logic gates based on silver nanoclusters and graphene oxide：Using molecular logic platform as substrate, it is added not
OR logic gates are built with the miR-21 and miR-141 of combination.When nucleic acid sequence (miR-21 and miR-141) is added, note is defeated
Enter for " 1 ", is otherwise " 0 ".It is used as basis for estimation according to whether relative intensity of fluorescence value is more than 0.5, when relative intensity of fluorescence is big
When 0.5, exports and exported as " 0 " for " 1 " when relative intensity of fluorescence is less than 0.5.When no any input, silver nanoclusters
Fluorescence quenched by graphene oxide, relative intensity of fluorescence be less than 0.5, be denoted as output be " 0 ".Due to miR-21 and Ag-
The Sequence of DNA1 is complementary, so when miR-21 is added, Ag-DNA1 can hybridize to form DNA/RNA double-strand knots with miR-21
Structure, the duplex structure can be desorbed from surface of graphene oxide.Therefore, the silver nanoclusters of Ag-DNA1 protections can equally leave oxidation
Graphene surface, fluorescence intensity enhancing, relative intensity of fluorescence are more than 0.5, and it is " 1 " to be denoted as output.Similarly, due to miR-141 and
The Sequence of Ag-DNA2 is complementary, so when miR-141 is added, Ag-DNA2 can hybridize that form DNA/RNA bis- with miR-141
Chain structure, the duplex structure can be desorbed from surface of graphene oxide.Therefore, the silver nanoclusters of Ag-DNA2 protections can equally leave
Surface of graphene oxide, fluorescence intensity enhancing, relative intensity of fluorescence are more than 0.5, and it is " 1 " to be denoted as output.Certainly, the two is deposited simultaneously
When, Ag-DNA1 protection silver nanoclusters and Ag-DNA2 protection silver nanoclusters can be all desorbed from surface of graphene oxide, phase
0.5 is more than to fluorescence intensity, it is " 1 " to be denoted as output.It can be seen that miR-21 and miR-141 can be on molecular logic platform
Build an OR logic gate.When platform inputs any one value in miR-21 or miR-141, output signal is " 1 ".
Embodiment 3
The structure of INHIBIT logic gates based on miR-21 and P1：Using molecular logic platform as substrate, various combination is added
MiR-21 and P1 build INHIBIT logic gates.When nucleic acid sequence (miR-21 and P1) is added, note input is " 1 ", otherwise
For " 0 ".It is used as basis for estimation according to whether relative intensity of fluorescence value is more than 0.5, when relative intensity of fluorescence is more than 0.5, output
It is exported as " 0 " for " 1 " when relative intensity of fluorescence is less than 0.5.When no any input, the fluorescence of silver nanoclusters is aoxidized
Graphene is quenched, and relative intensity of fluorescence is less than 0.5, and it is " 0 " to be denoted as output.Due to the Sequence of miR-21 and Ag-DNA1
Complementation, so when miR-21 is added, Ag-DNA1 can hybridize to form DNA/RNA duplex structures with miR-21, the duplex structure meeting
It is desorbed from surface of graphene oxide.Therefore, the silver nanoclusters of Ag-DNA1 protections can equally leave surface of graphene oxide, fluorescence
Intensity enhancing, relative intensity of fluorescence are more than 0.5, and it is " 1 " to be denoted as output.And P1 and Ag-DNA1, Ag-DNA2 sequence be not enough
Complementary base, so when P1 is added, hybridization reaction will not occur for P1 and other DNA, and such silver nanoclusters can not leave oxygen
Graphite alkene surface, fluorescence signal is constant, and relative intensity of fluorescence is less than 0.5, and it is " 0 " to be denoted as output.Certainly, miR-21 and P1 are same
When in the presence of, since miR-21 and P1 is complete complementary, so miR-21 and P1 can preferentially be hybridized, such Ag-DNA1
It can not just hybridize with miR-21.Such silver nanoclusters can not equally leave surface of graphene oxide, and fluorescence signal is constant, phase
0.5 is less than to fluorescence intensity, it is " 0 " to be denoted as output.It can be seen that miR-21 and P1 can build one on molecular logic platform
A INHIBIT logic gates.
Embodiment 4
The structure of INHIBIT logic gates based on miR-141 and P2：Using molecular logic platform as substrate, it is added different groups
The miR-141 and P2 of conjunction builds INHIBIT logic gates.When nucleic acid sequence (miR-141 and P2) is added, note input is " 1 ",
Otherwise it is " 0 ".It is used as basis for estimation according to whether relative intensity of fluorescence value is more than 0.5, when relative intensity of fluorescence is more than 0.5,
Output is " 1 ", when relative intensity of fluorescence is less than 0.5, is exported as " 0 ".When no any input, the fluorescence quilt of silver nanoclusters
Graphene oxide is quenched, and relative intensity of fluorescence is less than 0.5, and it is " 0 " to be denoted as output.Due to the sequence of miR-141 and Ag-DNA2
Partial complementarity, so when miR-141 is added, Ag-DNA2 can hybridize to form DNA/RNA duplex structures with miR-141, the double-strand
Structure can be desorbed from surface of graphene oxide.Therefore, the silver nanoclusters of Ag-DNA2 protections can equally leave graphene oxide table
Face, fluorescence intensity enhancing, relative intensity of fluorescence are more than 0.5, and it is " 1 " to be denoted as output.And P2 and Ag-DNA1, Ag-DNA2 sequence do not have
There are enough complementary bases, so when P2 is added, hybridization reaction will not occur for P2 and other DNA, and such silver nanoclusters can not
Surface of graphene oxide is left, fluorescence signal is constant, and relative intensity of fluorescence is less than 0.5, and it is " 0 " to be denoted as output.Certainly, miR-
When 141 and P2 is existed simultaneously, since miR-141 and P2 is complete complementary, so miR-141 and P2 can preferentially be hybridized,
Ag-DNA2 in this way can not just hybridize with miR-141.Such silver nanoclusters can not equally leave surface of graphene oxide, glimmering
Optical signal is constant, and relative intensity of fluorescence is less than 0.5, and it is " 0 " to be denoted as output.It can be seen that miR-141 and P2 can be patrolled in molecule
It collects and builds an INHIBIT logic gate on platform.
OR the and INHIBIT logic gates built in embodiment are applied to the intelligence inspection of multiple miRNA in same sample
It surveys, concrete operation method and the following application example of result：
Application example 1
In order to judge whether to be respectively present two different miRNA (miR-21 and miR-141) in sample, we
It needs to export to complete signal using INHIBIT-OR cascaded logics door.First, same sample (raw sample) is divided into four by us
Part.Then, four parts of same samples (raw sample) will be separately added into GO/Ag-DNA1/Ag-DNA2 molecular logic platforms, and is denoted as sample
Product 1, sample 2, sample 3, sample 4.For first sample, other inputs are not added.For second sample, it is added 1 μM
P1.For third sample, 1 μM of P2 is added.For the 4th sample, 1 μM of P1 and 1 μM of P2 is added.When first sample does not have
When having apparent fluorescence signal, one can consider that miR-21 and miR-141 is not present in raw sample, test terminates.If the
When one sample produces fluorescence signal, illustrate in raw sample at least containing one in miR-21 and miR-141.In this way we
Fluorometric investigation is carried out to second sample.If when second no generation fluorescence signal, illustrate to contain miR-21 in raw sample,
This is because second sample and first sample are compared and are added into P1 so that the miR-21 in raw sample is inhibited by, can not
Hybridization generates fluorescence signal.If second when still having fluorescence signal, illustrates at least to contain miR-141 in raw sample.We
It needs further to test whether raw sample contains only miR-141.This just needs to carry out fluorometric investigation to third sample, if the
When three samples do not generate fluorescence signal, illustrate that raw sample contains only miR-141.This is because third sample is than first
Sample adds P2 more, so that the miR-141 in raw sample is inhibited by P2, can not hybridize generation fluorescence signal.If third sample
Product still have fluorescence signal, illustrate that raw sample contains two kinds of miRNA of miR-21 and miR-141 simultaneously.It ought only add simultaneously in this way
When entering P1 and P2, fluorescence signal can be just quenched (see the 4th sample).Specific truth table is shown in attached drawing 4B.This combination
Good conceptual model is provided in terms of intellectualized detection of the detection method of logic for analytical chemistry.
Claims (4)
1. a kind of structure of logic gate of silver nanoclusters and its application in intelligent measurement, which is characterized in that the molecule
Logic gate is that fluorescent quenching occurs after being contacted based on silver nanoclusters and graphene oxide, and hybridization forms the original of fluorescence recovery after double-strand
Manage it is built-up, using the fluorescence intensity relative value of silver nanoclusters as basis for estimation, when relative intensity of fluorescence be more than 0.5 when,
Output is " 1 ", when relative intensity of fluorescence is less than 0.5, is exported as " 0 ".The Molecular Logic Gates include OR logic gates and
Cascaded logic door that INHIBIT logic gates and the two are connected in series simultaneously is applied to logic detection.
2. the construction method and logic detection application of OR logic gates as described in claim 1 and INHIBIT logic gates, special
Sign is as follows：
(1) OR logic gates：By the silver nanoclusters of 100nM Ag-DNA1 and 100nM Ag-DNA2 protections, stone is aoxidized with 15 μ g/mL
Black alkene is used as logical operation platform after mixing 15 minutes.Believe using RNA chain miR-21 and miR-141 as input according to shown in Fig. 1
Number, the fluorescence intensity of silver nanoclusters builds OR logic gates as output signal.
(2) the INHIBIT logic gates based on miR-21 and P1：It is molten with silver nanoclusters used in (1) and graphene oxide mixing
Liquid as molecular logic platform, according to shown in Fig. 2 using RNA/DNA chain miR-21 and P1 as input signal, fluorescence intensity conduct
Output signal builds INHIBIT logic gates.
(3) the INHIBIT logic gates based on miR-141 and P2：It is molten with silver nanoclusters used in (1) and graphene oxide mixing
Liquid as molecular logic platform, according to shown in Fig. 3 using RNA/DNA chain miR-141 and P2 as input signal, fluorescence intensity conduct
Output signal builds INHIBIT logic gates.
(4) the multivariable detection of miRNA：Judge two kinds whether are respectively present in sample using INHIBIT-OR cascaded logic doors
Different miRNA (miR-21 and miR-141) exports " 1 " and " 0 " according to the relative intensity of fluorescence value of sample, is judged with truth table
MiR-21 and miR-141 whether there is in sample.
(5) in the structure of the logic gate as described in claim 1 and 2, required DNA sequence dna is as follows：
Ag-DNA1:TGAACATCAGACTGATAACCTAATCCCCCCCCCCCC
Ag-DNA2:CGATCTTTACCTGACAGTCTTA ATCCCCCCCCCCCC
P1:TCAACATCAGTCTGATAAGCTA
P2:CCATCTTTACCAGACAGTGTTA
miR-21:UAGCUUAUCAGACUGAUGUUGA
miR-141:UAACACUGUCUGGUAAAGAUGG。
3. in the structure of logic gate as described in claim 1, input oligonucleotide chain (miR-21, miR-141, P1 and P2)
Concentration and Ag-DNA1/Ag-DNA2 concentration ratios are 10:1.
4. the logic detection work(to miRNA may be implemented in the molecular logic device based on silver nanoclusters as described in claim 1
Energy.
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| CN110113044A (en) * | 2019-05-22 | 2019-08-09 | 燕山大学 | A kind of upper conversion logic door construction method and system |
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| CN111218497A (en) * | 2020-01-16 | 2020-06-02 | 台州学院 | Construction of polymer nano-pore and detection of miRNA (micro ribonucleic acid) by using same |
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