CN106198662A - A kind of electrochemical method simultaneously detecting two kinds of tumor markerses - Google Patents
A kind of electrochemical method simultaneously detecting two kinds of tumor markerses Download PDFInfo
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- CN106198662A CN106198662A CN201610769134.9A CN201610769134A CN106198662A CN 106198662 A CN106198662 A CN 106198662A CN 201610769134 A CN201610769134 A CN 201610769134A CN 106198662 A CN106198662 A CN 106198662A
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Abstract
This patent discloses a kind of electrochemical method simultaneously detecting two kinds of tumor markerses, relate to technical field of electrochemical detection.Utilize object and the specific binding active force of aptamers more than this feature of active force between aptamers and its DNA complementary series, make two kinds of objects respectively with the respective aptamers of capture probe competition binding on electrode, thus reach object specific recognition and detection object convert purpose；By two metal ion species tracers are incorporated into combined with electrochemical detection method in detection system, it is achieved the difference output of two kinds of object signals.This method fast response time, detection sensitivity are high.
Description
Technical field
The present invention relates to technical field of electrochemical detection, a kind of with the capture spy on object and electrode
Pin competition binding aptamers is identification step, and metal ion complex carries out electro chemical analysis as tracer and detects two simultaneously
The method planting tumor markers.
Background technology
In recent years, the sickness rate of cancer and mortality rate present the most high trend, and this trend is also continuing increasing
Greatly.After human body produces cancerous tumor cell, the metabolism of cancerous cell and normal cell have the biggest difference, and cancerous cell is also simultaneously
Can produce and be different from Normocellular small-molecule substance, such as enzyme, hormone, antigen, little peptide and microRNA etc..These little molecules
Material is exactly so-called tumor markers.The contents level of related neoplasms mark is the important of cancer early warning and diagnosis
Index.It is not single for it should be noted that the contents level of tumor markers carrys out qualitative assessment cancer, that is cell occurs certain
After a kind of canceration, the tumor markers of its correspondence may have two kinds or two or more, under multiple tumor markerses identify,
Can Precise Diagnosis cancer.Therefore, a kind of analysis method that can simultaneously detect Diagnostic Value of Several Serum Tumor Markers is developed, cancer early stage
Early warning and diagnosis aspect have and important meaning.
Aptamers, English name is aptamer, be a bit of can the nucleotides sequence of specific binding target molecule
Row, generally comprise 20 ~ 50 length of nucleotides.Compared with traditional immune combination, aptamers and the knot of target molecule
Closing intensity suitable with the bond strength of antigen-antibody, the even affinity between the former is greater than the latter, and aptamers can
Synthesize in large quantity, and its prepared method is simpler.In recent years, there are some researches show, when the target molecule of aptamers is egg
When white matter or little peptide, its binding ability is better than the intensity of base pair complementarity between DNA and DNA.Therefore, divide when target
In the presence of son, the base pair complementarity effect that aptamers is complementary between sequence will be destroyed, and utilizes this point not only
The specific recognition of object can be realized, it is also possible to the signal of target molecule is converted into the signal of DNA sequence thus carries out
Detection.
Differential Pulse Voltammetry, as one, electrochemical detection method has fast response time, instrument cost is low, detection is clever
Sensitivity high, is widely used analyzing detection field.Different metal ions under the same conditions, its differentiated pulse volt-ampere
The position at method gained scanning peak is different, and the content of the corresponding metal ion of peak height at gained scanning peak is directly proportional.Therefore,
By using the electrochemical properties difference of different metal ion, it is introduced in Electrochemical Detection system, it becomes possible to realization is many
Qualitative and quantitative analysis while component.The novel detection method that this patent is invented, swells with CEA and small peptide
Tumor markers MUC1 is simulated target thing, uses double aptamers technology to carry out the specific recognition of two kinds of objects, by biology
Good gold/bovine serum albumin the complex of the compatibility combines two kinds of different metal ions and carries out electrochemistry inspection as tracer
Surveying, it is achieved that detect while two kinds of tumor markerses, the method fast response time, sensitivity are good, and detection limit is low.
Summary of the invention
The technical problem to be solved in the present invention is to build a kind of electrochemical sensor to carry out two kinds of tumor marks of Sensitive Detection simultaneously
Will thing.
A kind of electrochemical method simultaneously detecting two kinds of tumor markerses based on aptamers technology, is characterized in that including following
Step:
The preparation of (1) two metal ion species tracer
The preparation of two metal ion species tracers comprises three steps；
First being the preparation of gold/bovine serum albumin complex, its step is as described below: 40 ~ 60 mg bovine serum albumin
Being dissolved under magnetic stirring in 10 mL ultra-pure waters, then 10 mL concentration are that the chlorauric acid solution of 10 ~ 30 mM joins
In above-mentioned solution and be stirred at room temperature 5 min, finally, rapidly join the ascorbic acid powder of 50 mg, react 5 min, gained
Solid product ethanol and water wash for several times, and 4 C preserve；
Next to that the preparation of gold/bovine serum albumin-metal ion, its step is as described below: be 40 mg by 0.5 mL concentration
mL-1Gold/bovine serum albumin complex solution be dispersed in respectively 10 mL concentration be respectively 10 mM lead nitrate solution and
In cadmium nitrate solution, it is centrifuged after stirring 24 h, washs for several times, products therefrom is scattered in respectively in 2 mL ultra-pure waters；
Being finally spike DNA connection procedure, its step is as described below: concentration is first spike DNA 1 and the spike of 10 M
DNA 2 solution heats 0.5 h respectively under 95 C, is then placing 1 h under 25 C, the spike DNA that will handle well afterwards
1 solution joins in gold/bovine serum albumin-lead ion solution, and spike DNA 2 solution joins gold/bovine serum albumin-cadmium
In solion；So far, two metal ion species tracer DNA-gold/bovine serum albumin-lead ion and DNA-gold/Sanguis Bovis seu Bubali are pure
Prepared by albumen-cadmium ion；
(2) DNA denaturation
Aptamers 1 and aptamers 2, and the capture probe 1 of sulfydryl modification and capture probe 2 heat 0.5 under 95 C respectively
h；These three solution is placed under 25 C 1 h；Wherein, the English name of aptamers is aptamer；
(3) electrode modification process
The each step of this step modified after all concentration be 0.01 M, pH 7.4 phosphate buffer solution clean, hatch temperature
Degree is 25 C；First, respectively capture probe 1 and capture probe 2 solution that 10 L concentration are 2 M are added drop-wise to pretreatment
On the gold electrode crossed, overnight incubation；Drip the sulfydryl hexanol that 5 L concentration are 1 mM, hatch 2 h；Drip 10 L respectively dense
Degree is aptamers 1 and aptamers 2 solution of 2 M, hatches 6 h；Respectively by two kinds of tumors that volume is the 5 a series of concentration of L
Mark solution is added drop-wise on electrode, hatches 1 h；Take DNA-gold/bovine serum albumin-lead ion that volume is 10 L respectively
It is added drop-wise on electrode with DNA-gold/bovine serum albumin-cadmium-ion solution, hatches 1 h；By the Ru that 10 L concentration are 1 mM
(NH3)6 3+It is added drop-wise on electrode；
(4) Electrochemical Detection
Electrochemical Detection uses Differential Pulse Voltammetry, and its scanning voltage scope is 0.9 ~ 0.3 V, and electrolyte used is pH
It it is the acetic acid/sodium acetate buffer of 4.5.
Beneficial effects of the present invention
(1) feature that aptamers is combined is utilized with object, it is possible to realize the specific recognition of two kinds of objects；
(2) the metal ion tracer that two kinds different is incorporated in detection system, it is achieved two kinds of object signal difference outputs；
(3) present invention used electrochemical detection method fast response time, highly sensitive.
Accompanying drawing explanation
Fig. 1 is the experimental principle figure of methods described herein.
Detailed description of the invention
In order to be more fully understood that the present invention, it is further elucidated with present disclosure below in conjunction with embodiment and accompanying drawing, but this
The content of invention is not limited solely to following enforcement.
Embodiment 1
A kind of electrochemical method simultaneously detecting two kinds of tumor markerses based on aptamers technology, is characterized in that including following step
Rapid:
The preparation of (1) two metal ion species tracer
The preparation of two metal ion species tracers comprises three steps；
First being the preparation of gold/bovine serum albumin complex, its step is as described below: 50 mg bovine serum albumin are at magnetic force
Being dissolved under stirring in 10 mL ultra-pure waters, then 10 mL concentration are that the chlorauric acid solution of 10 mM joins in above-mentioned solution
And it is stirred at room temperature 5 min, and finally, rapidly joining the ascorbic acid powder of 50 mg, react 5 min, gained solid product is used
Ethanol and water wash for several times, and 4 C preserve；
Next to that the preparation of gold/bovine serum albumin-metal ion, its step is as described below: be 40 mg by 0.5 mL concentration
mL-1Gold/bovine serum albumin complex solution be dispersed in respectively 10 mL concentration be respectively 10 mM lead nitrate solution and
In cadmium nitrate solution, it is centrifuged after stirring 24 h, washs for several times, products therefrom is scattered in respectively in 2 mL ultra-pure waters；
Being finally spike DNA connection procedure, its step is as described below: concentration is first spike DNA 1 and the spike of 10 M
DNA 2 solution heats 0.5 h respectively under 95 C, is then placing 1 h under 25 C, the spike DNA that will handle well afterwards
1 solution joins in gold/bovine serum albumin-lead ion solution, and spike DNA 2 solution joins gold/bovine serum albumin-cadmium
In solion；So far, two metal ion species tracer DNA-gold/bovine serum albumin-lead ion and DNA-gold/Sanguis Bovis seu Bubali are pure
Prepared by albumen-cadmium ion；
(2) DNA denaturation
Aptamers 1 and aptamers 2, and the capture probe 1 of sulfydryl modification and capture probe 2 heat 0.5 under 95 C respectively
h；These three solution is placed under 25 C 1 h；Wherein, the English name of aptamers is aptamer；
(3) electrode modification process
The each step of this step modified after all concentration be 0.01 M, pH 7.4 phosphate buffer solution clean, hatch temperature
Degree is 25 C；First, respectively capture probe 1 and capture probe 2 solution that 10 L concentration are 2 M are added drop-wise to pretreatment
On the gold electrode crossed, overnight incubation；Drip the sulfydryl hexanol that 5 L concentration are 1 mM, hatch 2 h；Drip 10 L respectively dense
Degree is aptamers 1 and aptamers 2 solution of 2 M, hatches 6 h；Respectively by two kinds of tumors that volume is the 5 a series of concentration of L
Mark solution is added drop-wise on electrode, hatches 1 h；Take DNA-gold/bovine serum albumin-lead ion that volume is 10 L respectively
It is added drop-wise on electrode with DNA-gold/bovine serum albumin-cadmium-ion solution, hatches 1 h；By the Ru that 10 L concentration are 1 mM
(NH3)6 3+It is added drop-wise on electrode；
(4) Electrochemical Detection
Electrochemical Detection uses Differential Pulse Voltammetry, and its scanning voltage scope is 0.9 ~ 0.3 V, and electrolyte used is pH
It it is the acetic acid/sodium acetate buffer of 4.5.
Embodiment 2
Detecting step, with example 1, is a difference in that: in step (4), scanning voltage scope is 1.0 ~ 0.3 V, and electrolyte used is
PH is the phosphate buffer solution of 7.4.
SEQUENCE LISTING
<110>University Of Ji'nan
<120>a kind of electrochemical method simultaneously detecting two kinds of tumor markerses
<130> 2016
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> DNA
<213>synthetic
<400> 1
ttagcagttg atcctttgga taccctgg 28
<210> 2
<211> 31
<212> DNA
<213>synthetic
<400> 2
tacccacgat accagcttat tcaattcgtg g 31
<210> 3
<211> 25
<212> DNA
<213>synthetic
<400> 3
ccagggtatc caaaggatca actgc 25
<210> 4
<211> 28
<212> DNA
<213>synthetic
<400> 4
ccacgaattg aataagctgg tatcgtgg 28
<210> 5
<211> 25
<212> DNA
<213>synthetic
<400> 5
gcagttgatc ctttggatac cctgg 25
<210> 6
<211> 28
<212> DNA
<213>synthetic
<400> 6
ccacgatacc agcttattca attcgtgg 28
Claims (3)
1. detect an electrochemical method for two kinds of tumor markerses simultaneously, it is characterized in that comprising the following steps:
The preparation of (1) two metal ion species tracer
The preparation of two metal ion species tracers comprises three steps；
First being the preparation of gold/bovine serum albumin complex, its step is as described below: 40 ~ 60 mg bovine serum albumin
Being dissolved under magnetic stirring in 10 mL ultra-pure waters, then 10 mL concentration are that the chlorauric acid solution of 10 ~ 30 mM joins
In above-mentioned solution and be stirred at room temperature 5 min, finally, rapidly join the ascorbic acid powder of 50 mg, react 5 min, gained
Solid product ethanol and water wash for several times, and 4 C preserve；
Next to that the preparation of gold/bovine serum albumin-metal ion, its step is as described below: be 40 mg by 0.5 mL concentration
mL-1Gold/bovine serum albumin complex solution be dispersed in respectively 10 mL concentration be respectively 10 mM lead nitrate solution and
In cadmium nitrate solution, it is centrifuged after stirring 24 h, washs for several times, products therefrom is scattered in respectively in 2 mL ultra-pure waters；
Being finally spike DNA connection procedure, its step is as described below: concentration is first the sulfydryl modification spike DNA 1 of 10 M
Under 95 C, heat 0.5 h with spike DNA 2 solution respectively, then under 25 C, place 1 h, afterwards by showing of handling well
Track DNA 1 solution joins in gold/bovine serum albumin-lead ion solution, and it is pure that spike DNA 2 solution joins gold/Sanguis Bovis seu Bubali
In albumen-cadmium-ion solution；So far, two metal ion species tracer DNA-gold/bovine serum albumin-lead ion and DNA-gold/
Prepared by bovine serum albumin-cadmium ion；
(2) DNA denaturation
Aptamers 1 and aptamers 2, and the capture probe 1 of sulfydryl modification and capture probe 2 heat 0.5 under 95 C respectively
h；These three solution is placed under 25 C 1 h；Wherein, the English name of aptamers is aptamer；
(3) electrode modification process
The each step of this step modified after all concentration be 0.01 M, pH 7.4 phosphate buffer solution clean, hatch temperature
Degree is 25 C；First, respectively capture probe 1 and capture probe 2 solution that 10 L concentration are 2 M are added drop-wise to pretreatment
On the gold electrode crossed, overnight incubation；Drip the sulfydryl hexanol that 5 L concentration are 1 mM, hatch 2 h；Drip 10 L respectively dense
Degree is aptamers 1 and aptamers 2 solution of 2 M, hatches 6 h；Respectively by two kinds of tumors that volume is the 5 a series of concentration of L
Mark solution is added drop-wise on electrode, hatches 1 h；Take DNA-gold/bovine serum albumin-lead ion that volume is 10 L respectively
It is added drop-wise on electrode with DNA-gold/bovine serum albumin-cadmium-ion solution, hatches 1 h；By the Ru that 10 L concentration are 1 mM
(NH3)6 3+It is added drop-wise on electrode；
(4) Electrochemical Detection
Electrochemical Detection uses Differential Pulse Voltammetry, and its scanning voltage scope is 0.9 ~ 0.3 V, and electrolyte used is pH
It it is the acetic acid/sodium acetate buffer of 4.5.
A kind of electrochemical method simultaneously detecting two kinds of tumor markerses, is characterized in that step (1)
In the preparation method of two metal ion species tracers.
A kind of electrochemical method simultaneously detecting two kinds of tumor markerses, is characterized in that step (2)
Middle aptamers 1 and the capture probe 1 of aptamers 2 and sulfydryl modification and capture probe 2, their nucleotides sequence is classified as description
Sequence described in nucleotide and aminoacid sequence table.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100122223A (en) * | 2009-05-12 | 2010-11-22 | 한국화학연구원 | Single-stranded dna aptamers specifically binding to cea |
| KR20110070845A (en) * | 2011-06-14 | 2011-06-24 | 한국화학연구원 | Single-stranded dna aptamers specifically binding to cea |
| CN103257167A (en) * | 2012-12-14 | 2013-08-21 | 苏州大学 | Electrode modification method for adopting nucleic acid aptamers for high sensitivity detection of tumor cells |
| CN104655616A (en) * | 2015-01-23 | 2015-05-27 | 宁波大学 | Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 |
| CN105241943A (en) * | 2015-09-25 | 2016-01-13 | 济南大学 | Preparation and applications of electrochemical immunosensor used for detecting circulating tumor cells |
-
2016
- 2016-08-31 CN CN201610769134.9A patent/CN106198662B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100122223A (en) * | 2009-05-12 | 2010-11-22 | 한국화학연구원 | Single-stranded dna aptamers specifically binding to cea |
| KR20110070845A (en) * | 2011-06-14 | 2011-06-24 | 한국화학연구원 | Single-stranded dna aptamers specifically binding to cea |
| CN103257167A (en) * | 2012-12-14 | 2013-08-21 | 苏州大学 | Electrode modification method for adopting nucleic acid aptamers for high sensitivity detection of tumor cells |
| CN104655616A (en) * | 2015-01-23 | 2015-05-27 | 宁波大学 | Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 |
| CN105241943A (en) * | 2015-09-25 | 2016-01-13 | 济南大学 | Preparation and applications of electrochemical immunosensor used for detecting circulating tumor cells |
Non-Patent Citations (1)
| Title |
|---|
| CHUNYAN LIU ET AL.: "A simple regenerable electrochemical aptasensor for the parallel and continuous detection of biomarkers", 《RSC ADVANCES》 * |
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