CN109799197A - Purposes, lead ion detection kit and method of the cyanine dyes in detection lead ion - Google Patents
Purposes, lead ion detection kit and method of the cyanine dyes in detection lead ion Download PDFInfo
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Abstract
The invention proposes purposes, lead ion detection kit and method of the cyanine dyes in detection plumbum ion concentration.This method comprises: (1) mixes sample with cyanine dyes, aptamer；(2) mixture obtained in step (1) is subjected to wave spectrum detection；(3) lead ion and plumbum ion concentration whether there is as a result, determining based on wave spectrum detection in the sample.The detection specificity is strong and high sensitivity, is limited to 200nM to the detection of lead ion.
Description
Technical field
The present invention relates to detection fields, specifically, the present invention relates to cyanine dyes to detect the purposes in lead ion, lead ion
Detection kit and method.
Background technique
Lead is a kind of very strong metal pollutant of toxicity, and harm to the human body is serious, is almost all had an impact to each system of body,
It is especially bigger to the influence of children.Its major toxicity effect is to lead to anaemia, nervous function imbalance and injury of kidney, reproductive system
Damage etc., lead content reaches 100mg L in blood-1It can cause nervous system, reproduction, cardiovascular and developmental disorder etc. is a series of
Problem.Environmental Protection Agency provides that the maximum magnitude of Pb in Drinking Water ion is 72nM.Environmental Trace lead ion at present
Routine inspection mainly have atomic absorption method, fluorescent spectrometry, inductive coupling etc.；Ionomer emission spectrum method, electrochemical method
Deng.These methods are cumbersome, need the instrument of troublesome pre-treatment, special analytical technology personnel and valuableness, are unfavorable for
The quick analysis detection in scene.
Thus, still have to be developed for the detection of lead ion and improves.
Summary of the invention
The application is to be made based on inventor to the discovery of following facts and problem and understanding:
Inventors have found that aptamer (TBA) under the conditions of potassium/sodium ion buffer system, exists in the form of tetra- serobila of G-,
Tetra- serobila of G- can induce cyanine dyes and form J- aggressiveness, generate J- aggressiveness signal, and lead ion can be formed with aptamer (TBA)
More stable tetra- serobila of TBA G-, so that J- aggressiveness depolymerization, J- aggressiveness signal is lost, and is become according to the signal of detection J- aggressiveness
Change the concentration that can detecte out lead ion.Discovery based on the above issues, inventors herein propose cyanine dyes quantitative detection lead from
Purposes in son, and the kit that cyanine dyes is used to prepare detection plumbum ion concentration is proposed, and propose in detection sample
The method of plumbum ion concentration.Above-mentioned detection sensitivity height, high-specificity, reagent are easy to get, and detection is quick, is not necessarily to specific apparatus, inspection
Survey it is low in cost, convenient for promoting and applying in industry.
In the first aspect of the present invention, the invention proposes purposes of the cyanine dyes in detection plumbum ion concentration.According to this
The embodiment of invention, the purposes are purposes of the cyanine dyes in detection plumbum ion concentration.Inventors have found that passing through lead ion energy
So that the J- aggregation depolymerization that cyanine dyes is formed, reacts the concentration of lead ion by detecting the signal intensity of J- aggregation.Root
According to the embodiment of the present invention, 200nmol/L is reached to the detection limit of plumbum ion concentration, and reagent is easy to get, detection is quick, is not necessarily to
Specific apparatus, testing cost is cheap, convenient for promoting and applying in industry.
According to an embodiment of the invention, purposes of the above-mentioned cyanine dyes in detection plumbum ion concentration can further include
At least one following additional technical feature:
According to an embodiment of the invention, the purposes further comprises aptamer.Inventors have found that aptamer
(TBA) under the conditions of potassium/sodium ion buffer system, exist in the form of tetra- serobila of G-, can further induce cyanine dyes to form J- poly-
Collective generates J- aggregation signal, and when lead ion is added, lead ion and aptamer (TBA) are combined and formed more stable TBA
Tetra- serobila of G-, while making the gradually depolymerization of J- aggregation, and then can be more highly sensitive and more by detection J- aggregation signal intensity
Detect to high specificity the lead ion in sample.
According to an embodiment of the invention, the cyanine dyes is Formulas I compound represented,
Formulas I
Wherein: R1For C1~6Alkyl, optionally by alkyl-substituted phenyl, R2、R3、R4And R5Separately it is selected from H or C1
~6Alkyl or R2And R3Carbon atom connected to them is formed together 5 yuan to 7 yuan of ring structure or R4And R5With it
The carbon atom that is connected be formed together 5 yuan to 7 yuan of ring structure, R6And R7It is separately optionally to be replaced by sulfonic group
C1~6Alkyl, X1And X2Separately it is selected from C, O, S, Se or Te；Y is counter ion, and the counter ion is based on R6And R7Institute's band
Charge and select.According to an embodiment of the invention, above-mentioned cyanine dyes can more effectively be used to detect plumbum ion concentration, to lead ion
The detection of concentration has more specificity, further increases to the detection sensitivity of plumbum ion concentration.
According to an embodiment of the invention, the C1-C6Alkyl be selected from methyl, ethyl, n-propyl, isopropyl, normal-butyl,
Isobutyl group, tert-butyl, amyl, isopentyl, n-hexyl or isohesyl.
According to an embodiment of the invention, working as R6And R7When for alkyl, Y is halide anion；Work as R6And R7At least one carry
Sulfonate radical is free of Y；Work as R6And R7When carrying sulfonate radical, Y is triethylamine cation.
According to an embodiment of the invention, the cyanine dyes be selected from compound shown in following Formula II, formula III at least it
One:
Formula II,
Formula III.
Inventors have found that the cyanine dyes of compound shown in Formula II, formula III more effectively can be used to detect plumbum ion concentration, it is right
The detection of plumbum ion concentration has more specificity, also further increases to the detection sensitivity of plumbum ion concentration.
According to an embodiment of the invention, the molar ratio of the cyanine dyes and the aptamer is 1:(0.05~0.1).Hair
Bright people is had found by a large amount of research experiment, when the molar ratio of cyanine dyes and aptamer is not in 1:(0.05~0.1) range
Interior, no J- aggregation signal generates.
According to an embodiment of the invention, the aptamer has nucleotide sequence shown in SEQ ID NO:1.
GGTTGGTGTGGTTGG (SEQ ID NO:1).
Inventors have found that above-mentioned aptamer (TBA) is under the conditions of potassium/sodium ion buffer system, in the form of tetra- serobila of G-
In the presence of, can further induce cyanine dyes formed J- aggregation, generate J- aggregation signal, when lead ion is added, lead ion and
Aptamer (TBA) combines and forms more stable tetra- serobila of TBA G-, while making the gradually depolymerization of J- aggregation, and then pass through detection
J- aggregation signal intensity can detect to more highly sensitive and higher specificity the lead ion in sample.
In the second aspect of the present invention, the invention proposes a kind of purposes of cyanine dyes in reagent preparation box, the examinations
Agent box is for detecting plumbum ion concentration.Kit according to an embodiment of the present invention is high for the detection sensitivity of lead ion, exclusive
Property it is strong, and reagent is easy to get, and detection is quick, is not necessarily to specific apparatus, and testing cost is cheap, convenient for promoting and applying in industry.
According to an embodiment of the invention, such use can further include at least one following additional technical feature:
According to an embodiment of the invention, the kit further comprises aptamer.Inventors have found that aptamer
(TBA) under the conditions of potassium/sodium ion buffer system, exist in the form of tetra- serobila of G-, can further induce cyanine dyes to form J- poly-
Collective generates J- aggregation signal, and when lead ion is added, lead ion and aptamer (TBA) are combined and formed more stable TBA
Tetra- serobila of G-, while making the gradually depolymerization of J- aggregation, and then can be more highly sensitive and more by detection J- aggregation signal intensity
Detect to high specificity the lead ion in sample.
According to an embodiment of the invention, the aptamer has nucleotide sequence shown in SEQ ID NO:1.
According to an embodiment of the invention, the cyanine dyes is Formulas I compound represented,
Formulas I
Wherein: R1For C1~6Alkyl, optionally by alkyl-substituted phenyl, R2、R3、R4And R5Separately it is selected from H or C1
~6Alkyl or R2And R3Carbon atom connected to them is formed together 5 yuan to 7 yuan of ring structure or R4And R5With it
The carbon atom that is connected be formed together 5 yuan to 7 yuan of ring structure, R6And R7It is separately optionally to be replaced by sulfonic group
C1~6Alkyl, X1And X2Separately it is selected from C, O, S, Se or Te；Y is counter ion, and the counter ion is based on R6And R7Institute's band
Charge and select.According to an embodiment of the invention, above-mentioned cyanine dyes can more effectively be used to detect plumbum ion concentration, to lead ion
The detection of concentration has more specificity, further increases to the detection sensitivity of plumbum ion concentration.
According to an embodiment of the invention, the C1-C6Alkyl be selected from methyl, ethyl, n-propyl, isopropyl, normal-butyl,
Isobutyl group, tert-butyl, amyl, isopentyl, n-hexyl or isohesyl.
According to an embodiment of the invention, working as R6And R7When for alkyl, Y is halide anion；Work as R6And R7At least one carry
Sulfonate radical is free of Y；Work as R6And R7When carrying sulfonate radical, Y is triethylamine cation.
According to an embodiment of the invention, the cyanine dyes be selected from compound shown in following Formula II, formula III at least it
One:
Formula II,
Formula III.
Inventors have found that the cyanine dyes in kit is compound shown in Formula II or formula III, kit can be more effectively used for
The detection of plumbum ion concentration, detection sensitivity is higher, specificity further enhances.
According to an embodiment of the invention, the kit further comprises buffer.
According to an embodiment of the invention, the buffer is Tris-HCl-145mM Na+-5mM K+.On inventors have found that
The bioactivity of aptamer can be kept to the maximum extent by stating buffer, thus, lead ion can effectively induce cyanine dyes J- to assemble
Body depolymerization causes J- aggregation signal intensity.Therefore, the buffer makes the kit for detecting plumbum ion concentration,
Its specificity and sensitivity further increase.
According to an embodiment of the invention, the pH value of the buffer is 5.0~8.2.Inventors have found that above-mentioned pH can be maximum
The bioactivity of aptamer is kept to limit, thus, lead ion can effectively induce cyanine dyes J- aggregation depolymerization, cause J- poly-
Collective signal variation.Therefore, the buffer of the pH makes the kit for detecting plumbum ion concentration, specificity and spirit
Sensitivity further increases.
In the third aspect of the present invention, the invention proposes a kind of methods of lead ion in detection sample, which is characterized in that
It include: that (1) mixes sample with cyanine dyes, aptamer；(2) mixture obtained in step (1) is subjected to wave spectrum detection；
(3) lead ion and plumbum ion concentration whether there is as a result, determining based on wave spectrum detection in the sample.According to the present invention
The method of embodiment have the characteristics that specificity by force and high sensitivity for the detection of lead ion, and reagent is easy to get, and detection is fastly
Victory is not necessarily to specific apparatus, and testing cost is cheap, convenient for promoting and applying in industry.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, the wave spectrum detection includes being selected from circular dichroism detector, ultraviolet spectroscopy, fluorescence spectrum
At least one of method.Inventors have found that detecting the signal of J- aggregation by the above method, high sensitivity can be examined specifically
Measure lead ion.
According to an embodiment of the invention, the wave spectrum is detected as circular dichroism spectra detection.Inventors have found that circular dichroism spectra for
The signal of J- aggregation is detected, sensitivity is higher, can specifically detect lead ion.
According to an embodiment of the invention, the wave-length coverage of the circular dichroism detector is 200nm~700nm.Inventors have found that
Carry out circular dichroism detector detection when, using the wavelength of 200nm~700nm, may be implemented in sample lead ion it is higher exclusive
The detection of property and more high sensitivity.
According to an embodiment of the invention, there are the peaks 650nm in the circular dichroism detector testing result is in step (3)
There are the instructions of lead ion in the sample.It is surprisingly found by the inventors that after lead ion is mixed with cyanine dyes, aptamer,
Circular dichroism detector detection is carried out to the mixture, can occur specific peak at 650nm, therefore, sample to be tested is in circular dichroism
There are the instructions that the peak 650nm is in sample with the presence or absence of lead ion in spectrometry testing result.
According to an embodiment of the invention, the area based on the peak 650nm, determines the concentration of lead ion in the sample.
Inventors have found that the area at the peak specific peak 650nm is corresponding with the content of lead ion in sample.
According to an embodiment of the invention, by the way that the area at the peak 650nm is compared with standard curve, described in determination
The amount of lead ion in sample.Inventors have found that by the way that the area at the peak 650nm to be compared with standard curve, it can be more
Accurately determine the content of lead ion in the sample.
According to an embodiment of the invention, the standard curve be carried out using multiple samples containing known quantity lead ion it is flat
Row experiment determination.It should be noted that parallel laboratory test refers to the experiment carried out in the case where testing conditions are completely the same.Hair
Bright people's discovery carries out parallel laboratory test using multiple samples containing known plumbum ion concentration, and experiment condition is mentioned with of the present invention
The method condition of detection lead ion out is completely the same, to draw peak area-plumbum ion concentration standard curve, and then utilizing will
The area at the peak 650nm is compared with standard curve, can more accurately determine lead ion content in the sample.
According to an embodiment of the invention, the pH of the sample is 5.0~8.2.Inventors have found that pH is 5.0~8.2
Tris-HCl-145mM Na+-5mM K+Buffer system further increases the sensitivity for the method for detecting lead ion and specificity.
According to an embodiment of the invention, the content of lead ion is at least 200nM in the sample.Inventors have found that working as sample
When content of lead ion is at least 200nM in this, detect more accurate, sensitive.The method of embodiment according to the present invention is minimum
Detection is limited to 200nM.
According to an embodiment of the invention, the cyanine dyes is Formulas I compound represented,
Formulas I
Wherein: R1For C1~6Alkyl, optionally by alkyl-substituted phenyl, R2、R3、R4And R5Separately it is selected from H or C1
~6Alkyl or R2And R3Carbon atom connected to them is formed together 5 yuan to 7 yuan of ring structure or R4And R5With it
The carbon atom that is connected be formed together 5 yuan to 7 yuan of ring structure, R6And R7It is separately optionally to be replaced by sulfonic group
C1~6Alkyl, X1And X2Separately it is selected from C, O, S, Se or Te；Y is counter ion, and the counter ion is based on R6And R7Institute's band
Charge and select.According to an embodiment of the invention, above-mentioned cyanine dyes can more effectively be used to detect plumbum ion concentration, to lead ion
The detection of concentration has more specificity, further increases to the detection sensitivity of plumbum ion concentration.
According to an embodiment of the invention, the C1-C6Alkyl be selected from methyl, ethyl, n-propyl, isopropyl, normal-butyl,
Isobutyl group, tert-butyl, amyl, isopentyl, n-hexyl or isohesyl.
According to an embodiment of the invention, the cyanine dyes be selected from compound shown in following Formula II, formula III at least it
One:
Formula II,
Formula III.
Inventors have found that the cyanine dyes used is compound shown in Formula II or formula III, inspection of this method to plumbum ion concentration
Survey sensitivity is higher, specificity further enhances.
According to an embodiment of the invention, the molar ratio of the cyanine dyes and the aptamer is 1:(0.05~0.1).Hair
Bright people's discovery, when the molar ratio of cyanine dyes and aptamer is 1:(0.05~0.1), gained circular dichroism spectra detection peak figure exists
Occur characteristic peak at 650nm, if the molar ratio of cyanine dyes and aptamer is not in 1:(0.05~0.1) in range, justify two
Chromatography detection peak figure does not occur the characteristic peak of 650nm.It according to the method for the embodiment of the present invention being capable of higher specificity and more Gao Ling
Lead ion of the sensitivity ground in test sample.
According to an embodiment of the invention, the aptamer has nucleotide sequence shown in SEQ ID NO:1.Inventor
It was found that the sample containing lead ion is mixed with cyanine dyes, the aptamer with nucleotide sequence shown in SEQ ID NO:1
Afterwards, lead ion makes aptamer form tetra- stranded structure of G-, and tetra- serobila of G- further induces cyanine dyes to form J- aggregation, generates
J- aggregation signal, and then the lead ion content in sample can be determined by detecting J- aggregation signal.It is real according to the present invention
The detection method specificity for applying example is strong, high sensitivity further increases.
According to an embodiment of the invention, the concentration of the cyanine dyes is 4~16 micromoles per liters.Inventors have found that when cyanines contaminate
The concentration of material is 4~16 micromoles per liters, and characteristic peak occurs at 650nm in gained circular dichroism spectra detection peak figure.It is real according to the present invention
The method for applying example can be realized to the high specificity of lead ion in sample and the detection of high sensitivity.
According to an embodiment of the invention, the concentration of the cyanine dyes is 12 micromoles per liters.Inventors have found that working as cyanine dyes
Concentration when being 12 micromoles per liter, circular dichroism spectra detects peak value highest of the peak figure at 650nm.It is according to an embodiment of the present invention
Method can realize the detection of higher specificity and more high sensitivity to lead ion in sample.
According to an embodiment of the invention, the concentration of the aptamer is 0.6~2 micromoles per liter.Inventors have found that working as
The concentration of aptamer is 0.6~2 micromoles per liter, and characteristic peak occurs at 650nm in gained circular dichroism spectra detection peak figure.According to
The method of the embodiment of the present invention has high specificity and high sensitivity for the detection of lead ion in sample.
According to an embodiment of the invention, the concentration of the aptamer is 1 micromoles per liter.Inventors have found that when nucleic acid is suitable
When the concentration of body is 1 micromoles per liter, circular dichroism spectra detects peak value highest of the peak figure at 650nm.It is according to an embodiment of the present invention
Method has higher specificity and higher sensitivity for the detection of lead ion in sample.
Detailed description of the invention
Fig. 1 is specificity experimental result statistical chart according to an embodiment of the present invention；
Fig. 2 is detection limit experiment circular dichroism spectra test map according to an embodiment of the present invention；
Fig. 3 is the circular dichroism spectra test map of aptamer concentration screening experiment according to an embodiment of the present invention；
Fig. 4 is the circular dichroism spectra test map of cyanine dyes concentration screening experiment according to an embodiment of the present invention；
Fig. 5 is the circular dichroism of the concentration ratio range screening experiment of cyanine dyes and aptamer according to an embodiment of the present invention
Compose test map；
Fig. 6 a is lead ion detection range experiment circular dichroism spectra test map according to an embodiment of the present invention；And
Fig. 6 b is lead ion linear relationship experiment circular dichroism spectra test map according to an embodiment of the present invention.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.Implement
Particular technique or condition are not specified in example, described technology or conditions or is said according to the literature in the art according to product
Bright book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
It in the examples below, is the stability and consistency that guarantee testing result, sample storage and detection temperature are 4
Degree Celsius.
The experiment of 1 specificity of embodiment
Used cyanine dyes has structure shown in Formula II in the present embodiment, and aptamer has SEQ ID NO:1
Shown in nucleotide sequence, concentration is 1 micromole.
The present embodiment is to 8 kinds of different metal ions (Li+、NH4 +、Ca2+、Cu2+、Zn2+、Mg2+、Cd2+、Fe3+) carry out it is exclusive
Property investigate,
The sample that every metal ion species prepare 3 various concentrations is verified, and the concentration of each sample aptamer is concentration
It is 1 micromoles per liter, the concentration of methanol solution of cyanine dyes is 12 micromoles per liters.
Experimental procedure is as follows:
1) the Tris-HCl-5mM K of 10mM+-145mM Na+Buffer solution dissolves inorganic salts (LiCl, NH4Cl、CaCl2、
CuCl2、ZnCl2、MgCl2、CdCl2、FeCl3), the mother liquor that corresponding concentration of metal ions is 10 micromoles per liters is obtained, it is quasi- respectively
50,100,200 microlitres of each metal ion mother liquor are really measured as sample to be tested；
2) the aptamer solution 50 that 20 micromoles per liters are separately added into 3 parts of Xiang Shangshu each metal ion samples to be tested is micro-
It rises；State 60 microlitres of methanol solution that the cyanine dyes of 200 micromoles per liters is added in 3 parts of samples upwards in turn again；
3) Tris-HCl-5mM K is added respectively+-145mM Na+840 microlitres of buffer solution, 790 microlitres, 690 microlitres, system
Total volume is identical, aptamer is identical with cyanine dyes concentration, metal ion (Li+、NH4 +、Ca2+、Cu2+、Zn2+、Mg2+、Cd2+、
Fe3+) different 3 parts of sample to be tested of concentration；
4) circular dichroism detector detects solution to be measured, wavelength selective 2 00-700nm, circular dichroism spectrometer to above-mentioned sample to be tested into
Row analysis, sample storage and all operations all carry out at 4 deg. celsius；
5) interpretation of result: being absorbed as ordinate with circular dichroism spectra, and using the wavelength of circular dichroism spectra as abscissa, Fig. 1 is made.
As a result as shown in Figure 1, metal ion (Li+、NH4 +、Ca2+、Cu2+、Zn2+、Mg2+、Cd2+、Fe3+) concentration be 500,
1000,2000 nanomoles/liter, do not occur or occur lower cyanine dyes J- aggregation feature peak-to-peak signal in detection architecture
(650nm)。
As seen from Figure 1, method proposed by the present invention can specific detection lead ion, have very strong specificity.
The detection limit experiment of embodiment 2
In the present embodiment, cyanine dyes used in inventor has structure shown in Formula II.
Aptamer has nucleotide sequence shown in SEQ ID NO:1.
The present embodiment verifies the lead ion sample of 6 various concentrations, each sample plumbum ion concentration is respectively as follows: 0,
10,100,200,500,1000 nanomole.
Experimental procedure is as follows:
1)Tris-HCl-5mM K+-145mM Na+Buffer solution dissolves PbCl2, obtain the PbCl that concentration is 10 μM2Solution
For use, 10 μM of PbCl is separately added into 6 sample bottles20,1,10,20,50,100 microlitre of solution for later use；
2) 6 parts of PbCl of Xiang Shangshu210 microlitres of the aptamer, 200 micro- of 100 micromoles per liters is separately added into sample to be tested
60 microlitres of the methanol solution of the cyanine dyes of mol/L；
3) Tris-HCl-5mM K is added respectively+-145mM Na+Molten 930,929,920,910,880,830 microlitres are buffered,
Be made total volume is identical, aptamer with cyanine dyes concentration is identical, plumbum ion concentration is different 6 parts of sample to be tested；
4) circular dichroism spectrometer analyzes above-mentioned sample to be tested, and every operation all carries out at 4 deg. celsius.Circular dichroism spectra
Wave-length coverage is 200~700nm.
5) ordinate is absorbed as with circular dichroism spectra, using the wavelength of circular dichroism spectra as abscissa, Fig. 2 is made.
By Fig. 2, it can be concluded that, the detection of the method for lead ion is limited to 200 sodium mole in test sample proposed by the present invention.
3 aptamer concentration screening of embodiment
Used cyanine dyes has structure shown in Formula II in the present embodiment, and aptamer has SEQ ID NO:1
Shown in nucleotide sequence, plumbum ion concentration is 500nM；
The present embodiment verifies the aptamer sample of 10 various concentrations, the concentration of each sample aptamer are as follows:
0,0.4,0.6,0.8,1.0,1.2,1.4,1.6,1.8,2.0 micromoles per liter.
Experimental procedure is as follows:
1)Tris-HCl-5mM K+-145mM Na+Buffer solution dilutes Pb (NO3)2Mother liquor obtains the Pb that concentration is 5 μM2+
Solution for standby to be measured is separately added into Pb in 10 sample bottles2+100 microlitres of solution to be measured；
2) 10 parts of Pb (NO of Xiang Shangshu3)2It is molten that the aptamer that concentration is 100 micromoles per liters is separately added into sample to be tested
Liquid, volume are respectively 0,4,6,8,10,12,14,16,18,20 microlitre；Stating addition concentration in 10 parts of samples upwards in turn again is
60 microlitres of the cyanine dyes methanol solution of 200 micromoles per liters；
3) Tris-HCl-5mM K is added respectively+-145mM Na+Buffer solution 840,836,834,832,830,828,
826,824,822,820 microlitres, obtained total volume is identical, lead ion is identical with cyanine dyes concentration, aptamer concentration is different
10 parts of sample to be tested；
4) circular dichroism detector detects solution to be measured, wavelength selective 2 00-700nm, circular dichroism spectrometer to above-mentioned sample to be tested into
Row analysis, every operation all carry out at 4 deg. celsius；
5) interpretation of result: being absorbed as ordinate with circular dichroism spectra, and using the wavelength of circular dichroism spectra as abscissa, Fig. 3 is made.
As seen from Figure 3, there is J- aggregation in 0.6~2 micromoles per liter, in detection architecture in aptamer concentration
Feature peak-to-peak signal (650nm), when aptamer concentration is 1 micromoles per liter, circular dichroism spectra signal (CD) intensity is most strong, really with this
Fixed optimal aptamer concentration is 1 micromoles per liter.
4 cyanine dyes concentration screening of embodiment
Used cyanine dyes has structure shown in Formula II in the present embodiment, and aptamer has SEQ ID NO:1
Shown in nucleotide sequence, plumbum ion concentration is 500nM；
The present embodiment verifies cyanine dyes (methanol solution) sample of 9 various concentrations, each sample cyanine dyes it is dense
Degree are as follows: 0,1,2,4,8,10,12,16,20 micromoles per liters.
Experimental procedure is as follows:
1)Tris-HCl-5mM K+-145mM Na+Buffer solution dilutes Pb (NO3)2Mother liquor obtains the Pb that concentration is 5 μM2+
Solution for standby to be measured is separately added into Pb in 10 sample bottles2+100 microlitres of solution to be measured；
2) 9 parts of Pb of Xiang Shangshu2+10 microlitres of aptamer solution of 100 micromoles per liters are separately added into sample to be tested；Again according to
The methanol solution 0,5,10,20,40,50,60,80,100 of the secondary cyanine dyes that 200 micromoles per liters are added into above-mentioned 9 parts of samples
Microlitre；
3) 890,885,880,870,850,840,830,810,790 microlitres of Tris-HCl buffer solution are added respectively, system
Obtain total volume is identical, aptamer is identical with plumbum ion concentration, cyanine dyes concentration is different 9 parts of sample to be tested；
4) circular dichroism detector detects solution to be measured, wavelength selective 2 00-700nm, C. D. spectrum instrument to above-mentioned sample to be tested into
Row analysis, every operation all carry out at 4 deg. celsius；
5) interpretation of result: being absorbed as ordinate with circular dichroism spectra, and using the wavelength of circular dichroism spectra as abscissa, Fig. 4 is made.
As seen from Figure 4, there are J- aggregation body characteristics in 4~16 micromoles per liter, in detection architecture in cyanine dyes concentration
Peak-to-peak signal (650nm), when cyanine dyes concentration is 12 micromoles per liter, circular dichroism spectra (JASCO J-815) signal (CD) intensity is most
By force, determine optimal cyanine dyes concentration for 12 micromoles per liters with this.
The screening of the concentration ratio range of 5 cyanine dyes of embodiment and aptamer
Used cyanine dyes has structure shown in Formula II in the present embodiment, and aptamer has SEQ ID NO:1
Shown in nucleotide sequence, plumbum ion concentration is 500nM, and the concentration of cyanine dyes (methanol solution) is 12 micromoles per liters.
The present embodiment verifies (cyanine dyes/aptamer) sample of 10 various concentration ratios, each sample nucleic acid
The concentration of aptamer are as follows: 0,0.4,0.6,0.8,1,1.2,1.4,1.6,1.8,2.0 micromoles per liters.
Experimental procedure is as follows:
1)Tris-HCl-5mM K+-145mM Na+Buffer solution dilutes Pb (NO3)2Mother liquor obtains the Pb that concentration is 5 μM2+
Solution for standby to be measured is separately added into Pb in 10 sample bottles2+100 microlitres of solution to be measured；
2) 10 parts of Pb of Xiang Shangshu2+The methanol solution 60 that the cyanine dyes of 200 micromoles per liters is separately added into sample to be tested is micro-
It rises；State upwards in turn again the aptamer solution 0 that 100 micromoles per liters are added in 10 parts of samples, 4,6,8,10,12,14,16,
18,20 microlitres；
3) Tris-HCl-5mM K is added respectively+-145mM Na+Buffer solution 840,836,834,832,830,828,
826,824,822,820 microlitres, obtained total volume is identical, cyanine dyes is identical with plumbum ion concentration, aptamer concentration is different
10 parts of sample to be tested；
4) circular dichroism detector detects solution to be measured, wavelength selective 2 00-700nm, C. D. spectrum instrument to above-mentioned sample to be tested into
Row analysis, every operation all carry out at 4 deg. celsius；
5) interpretation of result: being absorbed as ordinate with circular dichroism spectra, and using the wavelength of circular dichroism spectra as abscissa, Fig. 5 is made.
As seen from Figure 5, the concentration ratio range of cyanine dyes and aptamer is 12 μM: (0.6-2.0) μM (1:
(0.05~0.1)), ratio is not in this range, no J- aggregation signal.
6 detection range of embodiment and linear relationship experiment
In the present embodiment, cyanine dyes used in inventor has structure shown in Formula II.
Aptamer has nucleotide sequence shown in SEQ ID NO:1.
The present embodiment verifies the lead ion sample of 10 various concentrations, each sample plumbum ion concentration is respectively as follows: 0,
50、100、200、500、1000、1500、2000、3000、5000nM。
Experimental procedure is as follows:
1)Tris-HCl-5mM K+-145mM Na+Buffer solution dilutes Pb (NO3)2Mother liquor obtains the Pb that concentration is 10 μM2 +Solution for standby to be measured is separately added into Pb in 10 sample bottles2+Solution 0,5,10,20,50,100,150,200,300,500 to be measured
Microlitre；
5) 9 parts of Pb of Xiang Shangshu2+It is separately added into 10 microlitres of the aptamer of 100 micromoles per liters in sample to be tested, 200 micro- rubs
You/liter 60 microlitres of methanol solution of cyanine dyes；
6) molten 930,925,920,910,880,830,780,730,630,430 microlitres of Tris-HCl buffering is added respectively,
Be made total volume is identical, aptamer with cyanine dyes concentration is identical, plumbum ion concentration is different 9 parts of sample to be tested；
7) circular dichroism spectrometer analyzes above-mentioned sample to be tested, and every operation all carries out at 4 deg. celsius.Circular dichroism spectra
Wave-length coverage is 200~700nm.
5) ordinate is absorbed as with circular dichroism spectra, using the wavelength of circular dichroism spectra as abscissa, Fig. 6 a and Fig. 6 b is made.
By Fig. 6 a and Fig. 6 b, it can be concluded that, the range of linearity of the method for detection lead ion proposed by the present invention is 200-2000
Nanomole.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
SEQUENCE LISTING
<110>Institute of Chemistry, Academia Sinica
<120>purposes, lead ion detection kit and method of the cyanine dyes in detection lead ion
<130> PIDC3175169
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> DNA
<213> Artificial
<220>
<223>nucleotide sequence of aptamer
<400> 1
ggttggtgtg gttgg 15
Claims (10)
1. purposes of the cyanine dyes in detection plumbum ion concentration.
2. purposes according to claim 1, which is characterized in that further comprise aptamer.
3. purposes according to claim 1, which is characterized in that the cyanine dyes is the chemical combination with structure shown in Formulas I
Object,
Wherein, R1For C1~6Alkyl or optionally by alkyl-substituted phenyl；
R2、R3、R4And R5Separately it is selected from H or C1~6Alkyl or R2And R3Carbon atom connected to them is together
Form 5 yuan to 7 yuan of ring structure or R4And R5Carbon atom connected to them is formed together 5 yuan to 7 yuan of ring structure；
R6And R7The separately C optionally to be replaced by sulfonic group1~6Alkyl；
X1And X2Separately it is selected from C, O, S, Se or Te；
Y is counter ion, and the counter ion is based on R6And R7It is electrically charged and select,
Optionally, work as R6And R7When for alkyl, Y is halide anion；
Work as R6And R7At least one of carry sulfonate radical, be free of Y；
Work as R6And R7When carrying sulfonate radical, Y is triethylamine cation；
Optionally, the cyanine dyes is selected from least one of compound shown in following Formula II, formula III:
4. purposes according to claim 2, which is characterized in that the molar ratio of the cyanine dyes and the aptamer is 1:
(0.05~0.1),
Optionally, the aptamer has nucleotide sequence shown in SEQ ID NO:1.
5. purposes of the cyanine dyes in reagent preparation box, the kit is for detecting plumbum ion concentration.
6. purposes according to claim 5, which is characterized in that the kit further comprises aptamer,
Optionally, the aptamer has nucleotide sequence shown in SEQ ID NO:1.
7. purposes according to claim 5, which is characterized in that the cyanine dyes is Formulas I compound represented,
Wherein, R1For C1~6Alkyl or optionally by alkyl-substituted phenyl；
R2、R3、R4And R5Separately it is selected from H or C1~6Alkyl or R2And R3Carbon atom connected to them is together
Form 5 yuan to 7 yuan of ring structure or R4And R5Carbon atom connected to them is formed together 5 yuan to 7 yuan of ring structure；
R6And R7The separately C optionally to be replaced by sulfonic group1~6Alkyl；
X1And X2Separately it is selected from C, O, S, Se or Te；
Y is counter ion, and the counter ion is based on R6And R7It is electrically charged and select,
Optionally, work as R6And R7When for alkyl, Y is halide anion；
Work as R6And R7One of carry sulfonate radical, be free of Y；
Work as R6And R7When carrying sulfonate radical, Y is triethylamine cation；
Optionally, the cyanine dyes is selected from least one of compound shown in following Formula II, formula III:
Optionally, the kit further comprises buffer,
Optionally, the buffer is Tris-HCl,
Optionally, the pH value of the buffer is 5.0~8.2.
8. a kind of method of plumbum ion concentration in detection sample characterized by comprising
(1) sample is mixed with cyanine dyes, aptamer；
(2) mixture obtained in step (1) is subjected to wave spectrum detection；
(3) lead ion and plumbum ion concentration whether there is as a result, determining based on wave spectrum detection in the sample.
9. according to the method described in claim 8, it is characterized in that, wave spectrum detection includes selected from circular dichroism detector, ultraviolet
At least one of spectroscopic methodology, fluorescent spectrometry,
Preferably, the wave spectrum is detected as circular dichroism spectra detection,
Optionally, the wave-length coverage of the circular dichroism detector is 200nm~700nm.
It optionally, there are the peak 650nm is that there are lead in the sample in the circular dichroism detector testing result in step (3)
The instruction of ion,
It is preferably based on the area at the peak 650nm, determines the concentration of lead ion in the sample,
Optionally, by the way that the area at the peak 650nm to be compared with standard curve, lead ion in the sample is determined
Amount,
Optionally, the standard curve is that the sample using multiple containing known quantity lead ion carries out what parallel laboratory test determined.
Optionally, the pH of the sample is 5.0~8.2,
Preferably, the concentration of lead ion is at least 200nM in the sample.
10. according to the method described in claim 8, it is characterized in that, the cyanine dyes be Formulas I compound represented,
Wherein, R1For C1~6Alkyl or optionally by alkyl-substituted phenyl；
R2、R3、R4And R5Separately it is selected from H or C1~6Alkyl or R2And R3Carbon atom connected to them is together
Form 5 yuan to 7 yuan of ring structure or R4And R5Carbon atom connected to them is formed together 5 yuan to 7 yuan of ring structure；
R6And R7The separately C optionally to be replaced by sulfonic group1~6Alkyl；
X1And X2Separately it is selected from C, O, S, Se or Te；
Y is counter ion, and the counter ion is based on R6And R7It is electrically charged and select,
Optionally, work as R6And R7When for alkyl, Y is halide anion；
Work as R6And R7One of carry sulfonate radical, be free of Y；
Work as R6And R7When carrying sulfonate radical, Y is triethylamine cation；
Optionally, the cyanine dyes is selected from least one of compound shown in following Formula II, formula III:
Optionally, the molar ratio of the cyanine dyes and the aptamer is 1:(0.05~0.1),
Optionally, the aptamer has nucleotide sequence shown in SEQ ID NO:1,
Optionally, the concentration of the cyanine dyes is 4~16 micromoles per liters,
Preferably, the concentration of the cyanine dyes is 12 micromoles per liters,
Optionally, the concentration of the aptamer is 0.6~2 micromoles per liter,
Preferably, the concentration of the aptamer is 1 micromoles per liter.
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| CN110714011A (en) * | 2019-11-11 | 2020-01-21 | 暨南大学 | Lead ion aptamer, electrochemical sensor and preparation method |
| CN112345749A (en) * | 2020-10-16 | 2021-02-09 | 吉林烟草工业有限责任公司 | Nucleic acid molecule embedded organic semiconductor small molecule aggregate, preparation method and application thereof in heavy metal ion detection |
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| CN110714011A (en) * | 2019-11-11 | 2020-01-21 | 暨南大学 | Lead ion aptamer, electrochemical sensor and preparation method |
| CN110714011B (en) * | 2019-11-11 | 2023-12-08 | 暨南大学 | Lead ion nucleic acid aptamer, electrochemical sensor and preparation method |
| CN112345749A (en) * | 2020-10-16 | 2021-02-09 | 吉林烟草工业有限责任公司 | Nucleic acid molecule embedded organic semiconductor small molecule aggregate, preparation method and application thereof in heavy metal ion detection |
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