CN108315400A - A kind of visualization of heavy metal ion quantifies new detecting method - Google Patents
A kind of visualization of heavy metal ion quantifies new detecting method Download PDFInfo
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Abstract
The present invention provides a kind of colorimetric sensing new methods based on supper-fast PCR, are used for Hg2+Visualization super sensitivity detection.The new method is according to mercury ion thymidine mispairing, design supper-fast PCR (Polymerase Chain Reaction, PCR amplimer) and template, simultaneously by the primer in combination with enzyme-linked nucleic acid self assembly colour developing module, a kind of the functional nucleic acid new detecting method and biosensor of the emerging mercury ion target based on mispairing are established in integration.The detection method and biosensor that the present invention is established, it is more faster than conventional method, sensitiveer, has the advantages that high specificity, high sensitivity, reliable testing result, it can be with Simplified analysis detecting step, shorten analysis time, it is often more important that make it possible on-line real-time measuremen, easy to carry and field work, in heavy metal field of fast detection, there is extraordinary application prospect.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of method and biosensor of detection heavy metal.
Background technology
Mercury seldom exists with the state of simple metal in nature, with mercuric sulphide, chlorine sulphur mercury ore, sulphur antimony mercury usually in mineral reserve
The states such as mine exist.Mercury is a kind of heavy metal element having huge poison, with the continuous acceleration of Progress in industrialization, more and more mercury
Gathered by food chain, it is more serious to the harm of human body.It is soaked by number of ways such as water, air, food, drug, cosmetics
Enter human body, human body is damaged.Especially pregnant woman will pay special attention to avoid contact with mercury, because either organic mercury (methylates
Mercury) and inorganic mercury the health of fetus can all be had an impact.
Traditional Hg2+Detection method includes mainly atomic absorption spectrography (AAS) (AAS), atomic fluorescence spectrometry (AFS), inductance
Coupled plasma mass, electrochemical analysis method, atomic absorption spectrophotometry etc., the detection of these methods is sensitive, accurate,
But generally require longer detection cycle and professional's operation.Due to organic dyestuff, the chemical sensor of albumen, DNA, film
The advantages of, so that it is rapidly developed in terms of mercury detection and analysis, but there is also water solubility, sensitivity, stability, selectivity etc. are each
The deficiency of aspect.It is applied to the continuous development of colorimetric method along with novel nano-material, largely compensates for tradition side
The deficiency of method.Since colorimetric method is easy to operate, low to instrument requirements, obtained as a kind of method quickly, effectively detecting mercury
Broad development.But traditional colorimetric detection mercury method still has certain limitation, such as complex for operation step, sensitivity and choosing
The organic solvent that selecting property is bad, need to use easily pollutes the environment, therefore free of contamination, easy to be fast there is an urgent need to develop
Speed, highly sensitive and high specific method meet the needs of trace meter mercury detection.
Invention content
The detection method and biosensor that the present invention establishes, overcome the deficiency of existing detection technique, realize counterweight
That metal carries out is accurate, quickly, the detection and analysis that is simple and efficient, be a kind of cheap, quick, sensitive, high specificity, lowest detection
1.3pM is limited far below Hg in tap water as defined in the World Health Organization (WHO)2+Limited Doses 30nmol/L mercury ion detection
New method.
It is an object of the present invention to provide a kind of detection methods of metal, and the method includes beyond body nucleic acids to expand skill
At least one of art, the reaction system of the isothermal DNA amplification include following 1) -2):
1) sense primer, the sense primer include:Complementary series A, linking arm, complementary series B, can specific amplification mould
The nucleotide sequence of plate and the nucleotide that can be combined with metal to be measured;The linking arm is located at the complementary series A and complementary sequence
Between arranging B, it is described can specific amplification template nucleotide sequence and the nucleotide that can be combined with metal to be measured on described
Swim the 5 ' ends or 3 ' ends of primer;The complementation of the nucleotide sequence of the complementary series A and the complementary series B and/or reverse complemental;
The linking arm includes the structure that can inhibit polymerase combination and/or the knot that can inhibit new chain extension in beyond body nucleic acid amplification procedure
Structure;
2) template, the template include the nucleotide of the nucleotide phase complementation combined with metal to be measured;
Specifically, the reaction system of the isothermal DNA amplification further includes downstream primer;The downstream primer includes
Can specific amplification template nucleotide sequence.
Specifically, the template further includes complementary series H and sequence I;Wherein, the complementary series H draws with the upstream
In object can specific amplification template nucleotide sequence is complementary and/or reverse complemental;The nucleotide sequence of the sequence I and institute
State in downstream primer can specific amplification template nucleotide sequence it is identical.
It is described complementary including complementary or reverse complemental defined in the prior art or common knowledge, and/or according to existing skill
Principle of complementarity defined in art or common knowledge carries out complementary or reverse complemental.
The polymerase includes the polymerase that can be used for beyond body nucleic acid amplification.
It is described can the nucleotide sequence of specific amplification template specifically include drawing designed by the characteristic sequence according to template
Object sequence;The characteristic sequence includes characteristic sequence defined in the prior art or common knowledge;The design includes existing skill
Design method recorded in art or common knowledge.
Described A, B, H, I are served only for distinguishing different complementary series or sequence, are not used in sequence.
At least one of specifically, the method further includes following 1) -3):
1) isothermal DNA amplification includes supper-fast PCR reactions, the reaction process packet of the supper-fast PCR reactions
It includes:90-98 DEG C, 2-6s;50-60 DEG C, 2-8s;Total 20-40 cycle;
2) linking arm includes the compound for having backbone;
3) nucleotide that can be combined with metal to be measured specifically includes the nucleotide containing thymidine or cytimidine.
Specifically, the reaction process of the supper-fast PCR reactions includes:95 DEG C, 2s;58 DEG C, 3s;Totally 30 cycles;
Again specifically, the reaction system middle and upper reaches primer of supper-fast PCR reactions and downstream primer it is a concentration of common
10 times or more of PCR concentration;Also specifically, being 20 times;Further include DNA polymerizations in the reaction system of the supper-fast PCR reactions
Enzyme, 10 times or more of a concentration of regular-PCR concentration of the archaeal dna polymerase, also specifically, being 60 times;)
At least one of specifically, the method further includes following 1) -4):
1) when the reaction system of the isothermal DNA amplification includes the sense primer, the sense primer packet
It includes:By SEQ ID № in sequence table:1 and SEQ ID №:What nucleotide sequence shown in 2 obtained after being connected by linking arm draws
Object;
2) when the reaction system of the isothermal DNA amplification includes the sense primer, the sense primer packet
It includes:By SEQ ID № in sequence table:1 and/or SEQ ID №:Nucleotide sequence shown in 2 is by one or several nucleotide
Replace and/or lack and or add and with SEQ ID № in sequence table:1 and/or SEQ ID №:Nucleotide sequence shown in 2 has
The primer obtained after thering is the nucleotide sequence of identical function to be connected by linking arm;
3) when the reaction system of the isothermal DNA amplification includes the template, the template includes:Sequence table
Middle SEQ ID №:Nucleotide sequence shown in 4;
4) when the reaction system of the isothermal DNA amplification includes the template, the template includes:By sequence
SEQ ID № in table:Nucleotide sequence shown in 4 by one or several nucleotide substitution and/or lack and or add and with
SEQ ID № in sequence table:The nucleotide sequence with the same function of nucleotide sequence shown in 4.
Specifically, when the reaction system of the isothermal DNA amplification includes the downstream primer, the downstream is drawn
Object includes:SEQ ID № in sequence table:Nucleotide sequence shown in 3, and/or by SEQ ID № in sequence table:Nucleosides shown in 3
Acid sequence by one or several nucleotide substitution and/or lack and or add and with SEQ ID № in sequence table:Shown in 3
Nucleotide sequence nucleotide sequence with the same function;
The function includes that the amplification of the template can be achieved.
Specifically, the linking arm is the chemistry knot of oxyethyleneglycol bridge, oxyethyleneglycol
Structure is:
Specifically, the sense primer includes:AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol
bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT
Specifically, the downstream primer further include marked in the downstream primer it is immune labeled;The immune labeled packet
Include biotin;The biotin labeling is on 5 ' first nucleotide in end of the downstream primer;The labeling method belongs to existing
There is technology;Again specifically, the downstream primer is Biotin-TCATCGCACCGTCAAAGGAACC;
Specifically, the method further includes nucleic acid self assembly chromogenic reaction, the reaction of the nucleic acid self assembly chromogenic reaction
System includes hair fastener sequence, and the hair fastener sequence includes hair fastener sequence one and/or hair fastener sequence two, and the hair fastener sequence one is wrapped
It includes:The arbitrary nucleotide of complementary series C, complementary series D and 3 or more, described 3 or more arbitrary nucleotide are located at the hair
5 ' the ends and/or 3 ' ends of card sequence;The hair fastener sequence two includes complementary series E and complementary series F;The complementary series D with
Complementary series F complementations and/or reverse complemental;The complementary series C and the complementary series A and/or complementary series B complementations and/
Or reverse complemental, the complementary series C and complementary series E complementations and/or reverse complemental.
Described one and two, for distinguishing different hair fastener sequences, are not used in sequence;It is different that described C, D, E, F are served only for difference
Complementary series, be not used in sequence.
At least one of specifically, the hair fastener sequence includes following 1) -4):
1) the hair fastener sequence one includes SEQ ID № in sequence table:Nucleotide sequence shown in 5;
2) the hair fastener sequence two includes SEQ ID № in sequence table:Nucleotide sequence shown in 6;
3) the hair fastener sequence one includes by SEQ ID № in sequence table:Nucleotide sequence shown in 5 passes through one or several
The substitution of a nucleotide and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 5 has identical
The nucleotide sequence of function;
4) the hair fastener sequence two includes by SEQ ID № in sequence table:Nucleotide sequence shown in 6 passes through one or several
The substitution of a nucleotide and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 6 has identical
The nucleotide sequence of function.
The function includes:The sense primer can open the hairpin structure of the hair fastener sequence one by base complementrity,
And the hair fastener sequence one can realize the extension of itself nucleotide chain under the action of TDT enzymes;The hair fastener sequence two can lead to
Cross the hairpin structure that base complementrity opens the hair fastener sequence one;The hair fastener sequence one and hair fastener sequence two can be mutual by base
The mutual hairpin structure of mutual partially unrolling is mended, and is interconnected with one another;What the hair fastener sequence one and hair fastener sequence two were formed
In connector, hair fastener sequence one can also realize the extension of itself nucleotide chain under the action of TDT enzymes simultaneously.
Specifically, the nucleic acid self assembly chromogenic reaction further includes following 1) -2) described at least one:
1) it is described in reaction system and claim 4 and/or 5 claim 1,2 and/or 3 the methods to be prepared
Hair fastener sequence mixing in method, 20min is incubated in 37 DEG C;TdT enzyme reaction systems are added, after 37 DEG C are reacted 20min, in 75
DEG C heating 10min;Then hemin and the buffer-induced liquid of tetra- serobilas of G- are added and is incubated 20min in 37 DEG C;Finally plus
Enter ABTS2-Chromogenic reaction is carried out with hydrogen peroxide;
2) in the reaction system of the nucleic acid self assembly chromogenic reaction, the end of the hair fastener sequence one or hair fastener sequence two is dense
Degree is 2 μM.
Further include purification step specifically, before the addition TdT enzyme reaction systems;Specifically, the purification step packet
It includes by connecting biotin in the downstream primer, life is carried with the method purifying that close enzyme chain nucleic is combined using biotin
The target product of object element.
At least one of specifically, the method further includes following 1) -4):
1) judge whether contain object to be measured in determinand by the color change of end reaction system;
2) object to be measured in determinand is calculated to make the method for standard curve by the color of end reaction system
Concentration;
3) dual or Multiple detection is realized by increasing the type of sense primer and hairpin one, hair fastener sequence two.
4) further include being purified to beyond body nucleic acid amplified production before nucleic acid self assembly chromogenic reaction.
Specifically, when the color of reaction system changes, judge to contain object to be measured in determinand;Again specifically,
When the color of reaction system is blue-green, judge to contain object to be measured in determinand;
The type of the hairpin one includes:Complementary series C in the hairpin one and the same upstream
The hairpin of complementary series A and/or B complementation or reverse complemental in primer is otherwise same kind of hairpin one is
Different types of hairpin one;The identical sense primer nucleotide sequence is the same sense primer.
Specifically, the purifying includes kits or magnetic beads for purifying method;The purifying includes removal reaction system
In other impurities in addition to beyond body nucleic acid amplified production and hair fastener sequence link product.
It is described it is a further object to provide a kind of kit and/or biosensor for detecting metal
At least one of kit and/or biosensor include following 1) -2) described:
1) sense primer, the sense primer include:Complementary series A, linking arm, complementary series B, can specific amplification mould
The nucleotide sequence of plate and the nucleotide that can be combined with metal to be measured;The linking arm is located at the complementary series A and complementary sequence
Between arranging B, it is described can specific amplification template nucleotide sequence and the nucleotide that can be combined with metal to be measured on described
Swim the 5 ' ends or 3 ' ends of primer;The complementation of the nucleotide sequence of the complementary series A and the complementary series B and/or reverse complemental;
The linking arm includes the structure that can inhibit polymerase combination and/or the knot that can inhibit new chain extension in beyond body nucleic acid amplification procedure
Structure;
2) template, the template include the nucleotide of the nucleotide phase complementation combined with metal to be measured.
It is described complementary including complementary or reverse complemental defined in the prior art or common knowledge, and/or according to existing skill
Principle of complementarity defined in art or common knowledge carries out complementary or reverse complemental.
The polymerase includes the polymerase that can be used for beyond body nucleic acid amplification.
It is described can the nucleotide sequence of specific amplification template specifically include drawing designed by the characteristic sequence according to template
Object sequence;The characteristic sequence includes characteristic sequence defined in the prior art or common knowledge;The design includes existing skill
Design method recorded in art or common knowledge.
Described A, B are served only for distinguishing different complementary series or sequence, are not used in sequence.
The nucleotide that can be combined with metal to be measured specifically includes the nucleotide containing thymidine or cytimidine.
At least one of specifically, the kit and/or biosensor further include following 1) -5) described:
1) hair fastener sequence, the hair fastener sequence include hair fastener sequence one and/or hair fastener sequence two, and the hair fastener sequence one is wrapped
It includes:The arbitrary nucleotide of complementary series C, complementary series D and 3 or more, described 3 or more arbitrary nucleotide are located at the hair
5 ' the ends and/or 3 ' ends of card sequence;The hair fastener sequence two includes complementary series E and complementary series F;The complementary series D with
Complementary series F complementations and/or reverse complemental;The complementary series C and the complementary series A and/or complementary series B complementations and/
Or reverse complemental, the complementary series C and complementary series E complementations and/or reverse complemental.
Described one and two, for distinguishing different hair fastener sequences, are not used in sequence;It is different that described C, D, E, F are served only for difference
Complementary series, be not used in sequence.
2) downstream primer, the downstream primer include can specific amplification template nucleotide sequence;
Specifically, when the reaction system of the isothermal DNA amplification includes the downstream primer, the downstream is drawn
Object includes:SEQ ID № in sequence table:Nucleotide sequence shown in 3, and/or by SEQ ID № in sequence table:Nucleosides shown in 3
Acid sequence by one or several nucleotide substitution and/or lack and or add and with SEQ ID № in sequence table:Shown in 3
Nucleotide sequence nucleotide sequence with the same function;
The function includes that the amplification of the template can be achieved;
Specifically, the downstream primer further include marked in the downstream primer it is immune labeled;The immune labeled packet
Include biotin;The biotin labeling is on 5 ' first nucleotide in end of the downstream primer;The labeling method belongs to existing
There is technology;)
3) when the kit and/or biosensor include the sense primer, the sense primer includes:By sequence
SEQ ID № in list:1 and SEQ ID №:The primer that nucleotide sequence shown in 2 obtains after being connected by linking arm;
4) when the kit and/or biosensor include the sense primer, the sense primer includes:By sequence
SEQ ID № in list:1 and/or SEQ ID №:Nucleotide sequence shown in 2 by one or several nucleotide substitution and/
Or lack and or add and with SEQ ID № in sequence table:1 and/or SEQ ID №:Nucleotide sequence shown in 2 has identical
The primer that the nucleotide sequence of function obtains after being connected by linking arm;
The function includes that the amplification of the template can be achieved;
The linking arm includes the compound for having backbone;
Specifically, the linking arm is the chemistry knot of oxyethyleneglycol bridge, oxyethyleneglycol
Structure is:
5) when the kit and/or biosensor include the template, the template packet further includes complementary series H
With sequence I;Wherein, in the complementary series H and sense primer can specific amplification template nucleotide sequence it is complementary and/or
Reverse complemental;The nucleotide sequence of the sequence I in downstream primer can the nucleotide sequence of specific amplification template it is identical.
It is described complementary including complementary or reverse complemental defined in the prior art or common knowledge, and/or according to existing skill
Principle of complementarity defined in art or common knowledge carries out complementary or reverse complemental.
It is described can the nucleotide sequence of specific amplification template specifically include drawing designed by the characteristic sequence according to template
Object sequence;The characteristic sequence includes characteristic sequence defined in the prior art or common knowledge;The design includes existing skill
Design method recorded in art or common knowledge.
Described H, I are served only for distinguishing different complementary series or sequence, are not used in sequence.
Specifically, the template includes:SEQ ID № in sequence table:Nucleotide sequence shown in 4;And/or by sequence table
Middle SEQ ID №:Nucleotide sequence shown in 4 by one or several nucleotide substitution and/or lack and or add and and sequence
SEQ ID № in list:The nucleotide sequence with the same function of nucleotide sequence shown in 4.
The function includes that the amplification of the template can be achieved;
At least one of specifically, the hair fastener sequence includes following 1) -4):
1) the hair fastener sequence one includes SEQ ID № in sequence table:Nucleotide sequence shown in 5;
2) the hair fastener sequence two includes SEQ ID № in sequence table:Nucleotide sequence shown in 6;
3) the hair fastener sequence one includes by SEQ ID № in sequence table:Nucleotide sequence shown in 5 passes through one or several
The substitution of a nucleotide and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 5 has identical
The nucleotide sequence of function;
4) the hair fastener sequence two includes by SEQ ID № in sequence table:Nucleotide sequence shown in 6 passes through one or several
The substitution of a nucleotide and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 6 has identical
The nucleotide sequence of function.
The function includes:The sense primer can open the hairpin structure of the hair fastener sequence one by base complementrity,
And the hair fastener sequence one can realize the extension of itself nucleotide chain under the action of TDT enzymes;The hair fastener sequence two can lead to
Cross the hairpin structure that base complementrity opens the hair fastener sequence one;The hair fastener sequence one and hair fastener sequence two can be mutual by base
The mutual hairpin structure of mutual partially unrolling is mended, and is interconnected with one another;What the hair fastener sequence one and hair fastener sequence two were formed
In connector, hair fastener sequence one can also realize the extension of itself nucleotide chain under the action of TDT enzymes simultaneously.
Specifically, the kit and/or biosensor further include following 1) -5):
1)AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-
CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT;
2)Biotin-TCATCGCACCGTCAAAGGAACC;
3) SEQ ID № in sequence table:Nucleotide sequence shown in 4;
4) SEQ ID № in sequence table:Nucleotide sequence shown in 5;
5) SEQ ID № in sequence table:Nucleotide sequence shown in 6;
Wherein, the chemical constitution of oxyethyleneglycol is:
Specifically, the kit and/or biosensor further include TdT reaction buffers, dATP, dGTP, TdT enzyme,
Hemin, the buffer-induced liquid of tetra- serobilas of G-, ABTS2-, hydrogen peroxide, home-made XP beads, surface modification
At least one of the magnetic bead of one layer of close enzyme chain nucleic.
It is also another object of the present invention to provide any the method for the present invention, any kit of the present invention and/or
The application of biosensor.
Specifically, the application includes following 1) -4) at least one of application:
1) metal is detected;
2) product for preparing detection metal and/or the application in Related product;
3) dual or multi-metal detection;
4) application in the product and/or Related product detected for dual or multi-metal is prepared.
Specifically, the metal includes Hg2+。
Optionally, any application does not include the diagnosing and treating side of the disease described in patent law of china Article 25
Method.
A kind of colorimetric sensing new method based on supper-fast PCR that the present invention establishes:
(1) this method establishes supper-fast PCR reaction systems, and the normal PCR process for taking 3 hours or so is reduced to
2.5 minutes, significantly reduce the used time of PCR reactions;
(2) supper-fast PCR reaction systems are carried enzyme-linked nucleic acid self assembly to develop the color module, solving that normal PCR is difficult to can
Depending on changing the problem quantitatively detected;
(3) supper-fast PCR reaction systems are carried into enzyme-linked nucleic acid self assembly colour developing module, realizes the ultrafast of heavy metal
Speed, quantitative, Visual retrieval.
The specific embodiment of the present invention provides a kind of colorimetric sensing new method based on supper-fast PCR, is used for Hg2+
Visualization super sensitivity detection.The new method designs supper-fast PCR according to mercury ion thymidine mispairing
The amplimer of (Polymerase Chain Reaction, PCR), while by the primer in combination with enzyme-linked nucleic acid self assembly
Develop the color module, and a kind of functional nucleic acid new detecting method of the emerging mercury ion target based on mispairing is established in integration.
The present invention has following advantageous effects:
1) detection method and biosensor that the present invention is established, it is more faster than conventional method, sensitiveer, have special
Property strong, high sensitivity, reliable testing result the advantages that, analysis time can be shortened with Simplified analysis detecting step, it is often more important that
Make it possible that on-line real-time measuremen, easy to carry and field work have extraordinary answer in heavy metal field of fast detection
Use foreground.
2) detection method and biosensor that the present invention is established can be achieved at the same time to Hg2+Specific detection, detection
Specific good, high sensitivity, testing result is reliable, can visually distinguish, detection process is efficient and convenient, in daily monitoring or city
Field screening etc. is of great significance.Specifically, sensitivity experiment the result shows that, the detection method established of the present invention and life
Object sensor is to Hg2+Detection be limited to 1.3pM, the high sensitivity of detection;Specific test the result shows that, the present invention is established
Detection method and biosensor are to Cu2+No cross reaction is, it can be achieved that Hg2+Specific detection;Mark-on reclaims test experience knot
Fruit shows the detection method that the present invention is established and biosensor to Hg2+Detected value close to practical mark-on value, detection knot
Fruit is reliable, accuracy is high.
3) detection method and biosensor that the present invention is established, solve normal PCR and are difficult to visualize quantitative detection
Problem, realize heavy metal Hg2+Supper-fast, quantitative, Visual retrieval.
Description of the drawings
Fig. 1 is the structure chart of supper-fast PCR device.
Fig. 2 is the expanding effect verification result figure of supper-fast PCR reactions;Wherein, swimming lane 1 is DNA molecular amount standard;Swimming lane
2 (do not add Hg for negative control in reaction system2+) result;Swimming lane 3, swimming lane 4 and swimming lane 5 are to add Hg in reaction system2+
Result.
Fig. 3 is Hg2+Canonical plotting.
Fig. 4 is specificity experiments result figure, wherein 1 be micropore 1,2 be micropore 2,3 be micropore 3,4 is micropore 4.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiments《Molecular Cloning:A Laboratory guide》
Listed specific method carries out in one book of (third edition) J. Pehanorm Brookers, or is carried out according to kit and product description.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, one kind are for detecting heavy metal Hg2+Supper-fast PCR colorimetric sensing new method foundation
(1) experiment material
The nucleotide sequence of primer designed by the present embodiment is shown in Table 1 and sequence table.
Table 1
In table 1, the nucleotide on the left of the linking arm (oxyethyleneglycol bridge) of sense primer Primer 1
Sequence is SEQ ID № in sequence table:Nucleotide sequence shown in 1, the nucleotides sequence on the right side of linking arm are classified as SEQ in sequence table
ID №:The chemical constitution of nucleotide sequence shown in 2, linking arm is:
In table 1, downstream primer Primer 2 is by SEQ ID № in sequence table:First core of the nucleotide shown in 35 '
Thuja acid, which is marked, to be obtained after biotin.
In table 1, the nucleotides sequence of template Template is classified as SEQ ID № in sequence table:Nucleotide sequence shown in 4.
In table 1, hair fastener sequence 1-2 (Hairpin 1, Hairpin 2) is followed successively by SEQ ID № in sequence table respectively:5、
SEQ ID №:Nucleotide sequence shown in 6.
Listed sequence is artificial synthesized in table 1.
SYBR Gold nucleic acid dyes, terminal deoxy transferase (TdT), 10 × TdT buffer, deoxyadenosine triphosphate
(dATP), triphosphoric acid deoxy-guanine (dGTP), dNTP, Ex Taq archaeal dna polymerases, 10 × Taq buffer, chlorine high ferro are blood red
Element, mercury chloride, magnetic bead Sera-mag SpeedBeads, magnetic beadMyOneTMStreptavidin T1, nucleic acid
Molecular weight standard ultra-low range DNA ladder are purchased from silent winged science and technology (the Thermo Scientific Life of match
Technologies).Experimental water is all from Milli-Q pure water systems.
(2) supper-fast PCR device is built
The primary structure of supper-fast PCR device is as shown in Figure 1, its specific structure, connection type and operation principle, work
Process includes:
Supper-fast PCR device uses the capillary (20uL, 04 929 292 001, Roche) of Light Cycler models
As the sample rooms PCR, by way of rapid centrifugation, sample can gather each capillary one end, band after the completion of centrifugation respectively
There is the capillary of sample to be fixed on plastic stent.Plastic stent is connected to stepper motor (42JSF630AS-1000, Just
Motioin Control) on, high temperature of the capillary sample room at 95 DEG C being fixed on plastic stent is driven by the stepper motor
Between water-bath and 58 DEG C of medium temperature water-bath recycle conversion, realize in supper-fast PCR reaction process reaction temperature variation and
Control.The stepper motor is powered by Switching Power Supply (S-100-24, Elecall), using DC servo motor driver
(YZ-ACSD60, Moving) and Labview (version 2014) realize stepper motor above-mentioned cycle conversion frequency or
The control of time.Temperature measurement is realized using the thermocouple being encapsulated in capillary.The amplification and linearisation of thermocouple signal
Processing procedure then using temperature transmitter (SBWR-2260, K, Yuancheng) transmit and using Arduino UNO v1.0
Chip is handled.The analog signal of the temperature received is converted into digital signal by Arduino UNO chips, then by
Arduino IDE (version 1.8.1) module executes operation.
(3) supper-fast PCR reactions
1) supper-fast PCR reaction systems are prepared, are specifically shown in Table 2:
Table 2
4 groups of reactions are carried out, wherein do not add Hg in the reaction system of negative control group2+;In the reaction system of experimental group one
Add Hg2+;Experimental group two and two repetitions that experimental group three is experimental group one are tested, and Hg is added in reaction system2+.In table 2
Primer Primer-1, Primer-2 be followed successively by primer Primer 1, Primer 2 described in above-mentioned table 1 respectively.
2) supper-fast PCR reaction process:
According to table 2,10 microlitres of reaction systems are prepared on ice, are immediately placed in the supper-fast PCR reactions that step (2) is built
It is controlled into trip temperature in device, temperature control and recurring number are shown in Table 3:
Table 3
After the completion of supper-fast PCR reactions, the magnetic bead " home-made XP beads " synthesized using this laboratory is according to production
The size of object segment purifies reaction system, obtains the product for only having metal ion to combine amplification.
This laboratory synthesis magnetic bead " home-made XP beads " be according to " magnetic bead Sera-mag SpeedBeads " into
The processing of row PEG-8000, NaCl, Tris-HCl, EDTA obtain, and specific preparation method is shown in document:Tian,Jingjing,et
al."Visual Single Cell Detection of Dual-Pathogens based on Multiplex Super
PCR(MS-PCR)and Asymmetric Tailing HCR(AT-HCR)."Sensors and Actuators B:
Chemical(2018).Available online 3 January 2018。
3) the expanding effect verification of supper-fast PCR reactions:
After completing above-mentioned supper-fast PCR reaction process, verified using the prestained agarose gel electrophoresis of 2% ethidium bromide
The expanding effect of supper-fast PCR reaction systems, deposition condition:120V for 25min, camera system:Molecular Imager
Gel Doc XR(Bio-Rad)。
The expanding effect verification result of supper-fast PCR reaction is shown in Fig. 2, Fig. 2 the result shows that:In Hg2+In the presence of, the present invention is built
Vertical supper-fast PCR reaction systems and reaction process realize the amplification of template, while magnetic bead plays and goes to clean well
The effect of matter.
(4) foundation of enzyme-linked nucleic acid self assembly colour developing module and Hg2+Visual retrieval
1) sensitivity experiment
Hg2+Standard curve drafting:
By 2 hairpin probes (HairpinProbe) listed by above-mentioned table 1:Hairpin 1, Hairpin 2 is respectively with super
Pure water is dissolved to 100 μM, in 95 DEG C heat 5min, after be slowly dropped to room temperature;
According to the supper-fast PCR reaction systems and reaction process (including magnetic beads for purifying step) described in above-mentioned steps (three)
Reaction is completed, unlike:Multigroup reaction system is established, i.e., according to reaction system listed by table 2, is separately added into through gradient dilution
Hg2+So that Hg in differential responses system2+Final concentration be followed successively by 10pM, 100pM, 200pM, 300pM, 500pM, 800pM,
1000pM。
By the above-mentioned Hg added with various concentration2+Multiple reaction systems complete reaction after, in each reaction system respectively
The ultra-pure water solution of the Hairpin 1 and Hairpin 2 of above-mentioned preparation is added, is then added in each reaction system respectively again
Self assembly buffer solution (8mM Na2HPO4,2.5mM NaH2PO4,0.15M NaCl,2mM MgCl2, pH 7.4) so that it is each anti-
It is 50 μ L to answer the total volume of system, and makes the final concentration of of each hairpin probe (HairpinProbe) in each reaction system
2μM;All reaction systems of gained in 37 DEG C be incubated 20min, obtain HCR (hybridization chain reaction, HCR,
Cross chain reaction) product;Then the magnetic bead of the close enzyme chain nucleic of one layer of surface modification is reused
MyOneTMStreptavidin T1 go the HCR double-stranded products of junction belt biotin labeling, removal to be not attached to HCR double-stranded products
On the impurity such as hairpin probe, remove magnetic bead finally by high eluting salt, obtain HCR product after purification.
The functionalization nucleic acid self-assembly system of TdT enzymatics is established, system includes:(match is silent to fly 1 × TdT reaction buffers
Technological product), 0.4mMdATP, 0.6mMdGTP, 20U/ μ LTdT enzymes and 50 μ L HCR product after purification react 20min in 37 DEG C
Afterwards, 10min termination enzymatic reactions are heated in 75 DEG C forms rich G sequence;10 μ L enzymatic reaction products are taken, 1 μ L chlorine siderosis is added
Red pigment (hemin) liquid storage (10 μM), 32 μ L G-, the tetra- buffer-induced liquid of serobila (100mM 2- (4-morpholine)
Ethanesulfonic acid (MES), 40mMKCl, with 0.05%Triton X-100, pH 5.5), 23 μ L ultra-pure waters;In 37
DEG C be incubated 20min formed tetra- serobilas of G;8 μ L ABTS are added2-Liquid storage (20mM) and 8 μ L hydrogen peroxide (H2O2) liquid storage (20mM) in
Room temperature, which is protected from light, is incubated 5min.
After the completion of reaction Hg is drawn in the OD values of 415nm using spectrophotometer detection reaction solution2+Standard curve, paint
The results are shown in Figure 3 for system.
The determination of minimum detection limit:
According to the standard curve of drafting and 3 σ principles, Hg is determined2+Detection limit be respectively 1.3pM, illustrate the present invention built
Vertical new detecting method high sensitivity.
2) accuracy is tested
Mark-on reclaims test experience:
According to described in above-mentioned steps (three) supper-fast PCR reaction systems and reaction process complete reaction, unlike:
Multigroup reaction system is established, i.e., according to reaction system listed by table 2, is separately added into the Hg of final concentration of 100,400,500pM2+, instead
It develops the color according to step described in above-mentioned sensitivity experiment after answering, after the completion, according to made standard curve, this is calculated
The Hg that the established method of invention detects2+Concentration, testing result is shown in Table 4.
The detected value (mark-on reclaims value) for the new method that the present invention establishes in table 4 illustrates that the present invention is built close to mark-on amount
Vertical new detecting method testing result is reliable, and accuracy is high.
Table 4
3) specificity experiments
4 reaction systems are established, i.e., will be added final concentration of 500pM's in the supper-fast reaction systems of PCR listed by table 2
Hg2+As experimental group one, while final concentration is added respectively it is the Hg of 500pM2+、Cu2+As experimental group two, final concentration is added
For the Cu of 500pM2+It is both not added with Hg as experimental group three, negative control2+Also it is not added with Cu2+Conduct experimental group four, according to above-mentioned
Described in step (3) supper-fast PCR reactions (in addition to the type and concentration of metal ion in system do corresponding replace, Qi Tajun
Unanimously) reacted.
By 2 hairpin probes (Hairpin Probe) listed by above-mentioned table 1:Hairpin 1, Hairpin 2 is respectively with super
Pure water is dissolved to 100 μM, and 5min are heated in 95 DEG C, after to be slowly dropped to room temperature spare.
4 reaction systems (10ul systems) for completing supper-fast PCR reactions are added separately to coloring reaction system i.e. 4
In micropore (ultra-pure water solution of Hairpin 1 and Hairpin 2 is dissolved in advance in 4 micropores).The reactant of experimental group one
System is added in the micropore 1 that number is 1, and the reaction system of experimental group two is added in the micropore 2 that number is 2, experimental group three
Reaction system is added in the micropore 3 that number is 3, and the reaction system of experimental group four is added in the micropore 4 that number is 4;Divide again
Self assembly buffer solution (8mM Na are added not in each micropore2HPO4,2.5mM NaH2PO4,0.15M NaCl,2mM MgCl2,
PH 7.4), and make final concentration of 2 μM of each hairpin probe (Hairpin Probe), each micropore is 50 microlitres, institute
There is micropore to be incubated 20min in 37 DEG C, HCR purified products are obtained after purification respectively through magnetic bead.
Above-mentioned HCR purified products are added separately to containing 1 × TdT reaction buffers (the silent winged technological product of match),
In the micropore of 0.4mMdATP, 0.6mMdGTP, 20U/ μ LTdT enzymes, after 37 DEG C are reacted 20min, heats 10min in 75 DEG C and terminate
Enzymatic reaction forms rich G sequence;1 μ L hemin liquid storages (10 μM), the induction of 32 μ L G-, tetra- serobilas are added in enzymatic reaction product
Buffer solution (100mM 2- (4-morpholine) ethanesulfonic acid (MES), 40mMKCl, with 0.05%Triton
X-100, pH 5.5), 23 μ L ultra-pure waters;It is incubated 20min in 37 DEG C and forms tetra- serobilas of G;8 μ L ABTS are added2-Liquid storage (20mM) with
8 μ L hydrogen peroxide (H2O2) liquid storage (20mM) in room temperature be protected from light be incubated 5min.
Experimental result as shown in figure 4, Fig. 4 the result shows that the detection method established of the present invention and biosensor to Cu2+
No cross reaction is, it can be achieved that Hg2+Specific detection.
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore it is interpreted as the limitation to the scope of the claims of the present invention, as long as skill obtained in the form of equivalent substitutions or equivalent transformations
Art scheme should all be fallen within the scope and spirit of the invention.
Sequence table
<110>China Agricultural University
<120>A kind of visualization of heavy metal ion quantifies new detecting method
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agagagagag agggaaagag agag 24
<210> 2
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ctctctcttt ccctctctct ctctgtgaaa ttatcgccac gttcggtttt 50
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tcatcgcacc gtcaaaggaa cc 22
<210> 4
<211> 102
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tcatcgcacc gtcaaaggaa cctcagtatc agtgctatac gtcgatcagt acccctgtta 60
tgtgcaaaaa aaaaaatttt ccgaacgtgg cgataatttc ac 102
<210> 5
<211> 53
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctctctcttt ccctctctct ctctcggggc agagagagag agggaaagtc tct 53
<210> 6
<211> 48
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
agagagagag agggaaagag agagctttcc ctctctctct ctgccccg 48
Claims (10)
1. a kind of detection method of metal, which is characterized in that the method includes isothermal DNA amplification, the beyond body nucleic acid
At least one of the reaction system of amplification technique includes following 1) -2):
1) sense primer, the sense primer include:Complementary series A, linking arm, complementary series B, can specific amplification template
Nucleotide sequence and the nucleotide that can be combined with metal to be measured;The linking arm be located at the complementary series A and complementary series B it
Between, it is described can specific amplification template nucleotide sequence and the nucleotide that can be combined with metal to be measured be located at the sense primer
5 ' end or 3 ' end;The complementation of the nucleotide sequence of the complementary series A and the complementary series B and/or reverse complemental;The company
It includes the structure that can inhibit polymerase combination and/or the structure that can inhibit new chain extension in beyond body nucleic acid amplification procedure to connect arm;
2) template, the template include the nucleotide of the nucleotide phase complementation combined with metal to be measured.
At least one of 2. according to the method described in claim 1, it is characterized in that, the method further includes following 1) -3):
1) isothermal DNA amplification includes supper-fast PCR reactions, and the reaction process of the supper-fast PCR reactions includes:
90-98 DEG C, 2-6s;50-60 DEG C, 2-8s;Total 20-40 cycle;
2) linking arm includes the compound for having backbone;
3) nucleotide that can be combined with metal to be measured specifically includes the nucleotide containing thymidine or cytimidine.
3. according to the method described in claim 1 and/or 2, which is characterized in that the method further includes following 1) -4) in extremely
Few one kind:
1) when the reaction system of the isothermal DNA amplification includes the sense primer, the sense primer includes:It will
SEQ ID № in sequence table:1 and SEQ ID №:The primer that nucleotide sequence shown in 2 obtains after being connected by linking arm;
2) when the reaction system of the isothermal DNA amplification includes the sense primer, the sense primer includes:It will
SEQ ID № in sequence table:1 and/or SEQ ID №:Nucleotide sequence shown in 2 passes through the substitution of one or several nucleotide
And/or lack and or add and with SEQ ID № in sequence table:1 and/or SEQ ID №:Nucleotide sequence shown in 2 has phase
The primer that the nucleotide sequence of congenerous obtains after being connected by linking arm;
3) when the reaction system of the isothermal DNA amplification includes the template, the template includes:SEQ in sequence table
ID №:Nucleotide sequence shown in 4;
4) when the reaction system of the isothermal DNA amplification includes the template, the template includes:It will be in sequence table
SEQ ID №:Nucleotide sequence shown in 4 by one or several nucleotide substitution and/or lack and or add and and sequence
SEQ ID № in table:The nucleotide sequence with the same function of nucleotide sequence shown in 4;
Specifically, when the reaction system of the isothermal DNA amplification includes the downstream primer, the downstream primer packet
It includes:SEQ ID № in sequence table:Nucleotide sequence shown in 3, and/or by SEQ ID № in sequence table:Nucleotides sequence shown in 3
Row by one or several nucleotide substitution and/or lack and or add and with SEQ ID № in sequence table:Nucleosides shown in 3
Acid sequence nucleotide sequence with the same function;
Specifically, the linking arm is the chemical constitution of oxyethyleneglycol bridge, oxyethyleneglycol
For:
Specifically, the sense primer includes:AGAGAGAGAGAGGGAAAGAGAGAG-oxyethylenegl ycol
bridge-CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT;
Specifically, the downstream primer further include marked in the downstream primer it is immune labeled;It is described immune labeled including life
Object element;The biotin labeling is on 5 ' first nucleotide in end of the downstream primer;Again specifically, the downstream primer is
Biotin-TCATCGCACCGTCAAAGGAACC。
4. according to the method described in claim 1,2 and/or 3, which is characterized in that the method further includes nucleic acid self assembly colour developing
Reaction, the reaction system of the nucleic acid self assembly chromogenic reaction includes hair fastener sequence, and the hair fastener sequence includes hair fastener sequence one
And/or hair fastener sequence two, the hair fastener sequence one include:The arbitrary nucleotide of complementary series C, complementary series D and 3 or more,
Described 3 or more arbitrary nucleotide is located at the 5 ' ends and/or 3 ' ends of the hair fastener sequence;The hair fastener sequence two includes complementation
Sequence E and complementary series F;The complementary series D and complementary series F complementations and/or reverse complemental;The complementary series C and institute
It states complementary series A and/or complementary series B complementations and/or reverse complemental, the complementary series C and complementary series E is complementary
And/or reverse complemental.
5. according to the method described in claim 4, it is characterized in that, the hair fastener sequence includes following 1) -4) at least one
Kind:
1) the hair fastener sequence one includes SEQ ID № in sequence table:Nucleotide sequence shown in 5;
2) the hair fastener sequence two includes SEQ ID № in sequence table:Nucleotide sequence shown in 6;
3) the hair fastener sequence one includes by SEQ ID № in sequence table:Nucleotide sequence shown in 5 passes through one or several cores
The substitution of thuja acid and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 5 has identical function
Nucleotide sequence;
4) the hair fastener sequence two includes by SEQ ID № in sequence table:Nucleotide sequence shown in 6 passes through one or several cores
The substitution of thuja acid and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 6 has identical function
Nucleotide sequence.
6. according to the method described in claim 4 and/or 5, which is characterized in that under the nucleic acid self assembly chromogenic reaction further includes
State 1) -2) described at least one:
1) it is reaction system and claim 4 and/or 5 the methods claim 1,2 and/or 3 the methods to be prepared
In hair fastener sequence mixing, in 37 DEG C be incubated 20min;TdT enzyme reaction systems are added, after 37 DEG C are reacted 20min, are added in 75 DEG C
Hot 10min;Then hemin and the buffer-induced liquid of tetra- serobilas of G- are added and is incubated 20min in 37 DEG C;It is eventually adding
ABTS2-Chromogenic reaction is carried out with hydrogen peroxide;
2) in the reaction system of the nucleic acid self assembly chromogenic reaction, the final concentration of the hair fastener sequence one or hair fastener sequence two is equal
It is 2 μM.
7. a kind of kit and/or biosensor for detecting metal, which is characterized in that the kit and/or biology
At least one of sensor includes following 1) -2) described:
1) sense primer, the sense primer include:Complementary series A, linking arm, complementary series B, can specific amplification template
Nucleotide sequence and the nucleotide that can be combined with metal to be measured;The linking arm be located at the complementary series A and complementary series B it
Between, it is described can specific amplification template nucleotide sequence and the nucleotide that can be combined with metal to be measured be located at the sense primer
5 ' end or 3 ' end;The complementation of the nucleotide sequence of the complementary series A and the complementary series B and/or reverse complemental;The company
It includes the structure that can inhibit polymerase combination and/or the structure that can inhibit new chain extension in beyond body nucleic acid amplification procedure to connect arm;
2) template, the template include the nucleotide of the nucleotide phase complementation combined with metal to be measured.
8. kit according to claim 7 and/or biosensor, which is characterized in that the kit and/or biology
At least one of sensor further includes following 1) -5) described:
1) hair fastener sequence, the hair fastener sequence include hair fastener sequence one and/or hair fastener sequence two, and the hair fastener sequence one includes:
The arbitrary nucleotide of complementary series C, complementary series D and 3 or more, described 3 or more arbitrary nucleotide are located at the hair fastener
5 ' the ends and/or 3 ' ends of sequence;The hair fastener sequence two includes complementary series E and complementary series F;The complementary series D with mutually
Complementary series F complementations and/or reverse complemental;The complementary series C and the complementary series A and/or complementary series B it is complementary and/or
Reverse complemental, the complementary series C and complementary series E complementations and/or reverse complemental;
2) downstream primer, the downstream primer include can specific amplification template nucleotide sequence;
3) when the kit and/or biosensor include the sense primer, the sense primer includes:By sequence table
Middle SEQ ID №:1 and SEQ ID №:The primer that nucleotide sequence shown in 2 obtains after being connected by linking arm;
4) when the kit and/or biosensor include the sense primer, the sense primer includes:By sequence table
Middle SEQ ID №:1 and/or SEQ ID №:Nucleotide sequence shown in 2 passes through the substitution of one or several nucleotide and/or lacks
Lose and/or addition and with SEQ ID № in sequence table:1 and/or SEQ ID №:Nucleotide sequence shown in 2 has identical function
Nucleotide sequence connected by linking arm after obtained primer;
5) when the kit and/or biosensor include the template, the template packet further includes complementary series H and sequence
Arrange I;Wherein, in the complementary series H and sense primer can specific amplification template nucleotide sequence it is complementary and/or reversed
It is complementary;The nucleotide sequence of the sequence I in downstream primer can the nucleotide sequence of specific amplification template it is identical;
Specifically, the template includes:SEQ ID № in sequence table:Nucleotide sequence shown in 4;And/or it will be in sequence table
SEQ ID №:Nucleotide sequence shown in 4 by one or several nucleotide substitution and/or lack and or add and and sequence
SEQ ID № in table:The nucleotide sequence with the same function of nucleotide sequence shown in 4;
At least one of specifically, the hair fastener sequence includes following 1) -4):
1) the hair fastener sequence one includes SEQ ID № in sequence table:Nucleotide sequence shown in 5;
2) the hair fastener sequence two includes SEQ ID № in sequence table:Nucleotide sequence shown in 6;
3) the hair fastener sequence one includes by SEQ ID № in sequence table:Nucleotide sequence shown in 5 passes through one or several cores
The substitution of thuja acid and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 5 has identical function
Nucleotide sequence;
4) the hair fastener sequence two includes by SEQ ID № in sequence table:Nucleotide sequence shown in 6 passes through one or several cores
The substitution of thuja acid and/or lack and or add and with SEQ ID № in sequence table:Nucleotide sequence shown in 6 has identical function
Nucleotide sequence;
Specifically, the downstream primer includes:SEQ ID № in sequence table:Nucleotide sequence shown in 3, and/or by sequence table
Middle SEQ ID №:Nucleotide sequence shown in 3 by one or several nucleotide substitution and/or lack and or add and and sequence
SEQ ID № in list:The nucleotide sequence with the same function of nucleotide sequence shown in 3;
Specifically, the linking arm is the chemical constitution of oxyethyleneglycol bridge, oxyethyleneglycol
For:
Specifically, the downstream primer further include marked in the downstream primer it is immune labeled.
9. kit according to claim 8 and/or biosensor, which is characterized in that the kit and/or biology
Sensor further includes following 1) -5):
1)AGAGAGAGAGAGGGAAAGAGAGAG-oxyethyleneglycol bridge-
CTCTCTCTTTCCCTCTCTCTCTCTGTGAAATTATCGCCACGTTCGGTTTT;
2)Biotin-TCATCGCACCGTCAAAGGAACC;
3) SEQ ID № in sequence table:Nucleotide sequence shown in 4;
4) SEQ ID № in sequence table:Nucleotide sequence shown in 5;
5) SEQ ID № in sequence table:Nucleotide sequence shown in 6;
Wherein, the chemical constitution of oxyethyleneglycol is:
10. claim 1,2,3,4,5 and/or 6 the methods, claim 7,8 and/or 9 the kit and/or biology pass
The application of sensor.
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