CN107860765B - Probe for metal ion detection, kit, preparation method and application - Google Patents
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Abstract
The invention relates to the technical field of biosensor metal ion detection, and particularly relates to a probe for metal ion detection, a kit, a preparation method and application. The invention adopts a metal ion deoxynucleotide sequence and a substrate chain sequence to modify a probe, modifies a group molecule with a catalytic luminescence effect on a chemiluminescence system at the end part of a substrate chain, realizes the detection of metal ions by a chemiluminescence principle, further modifies a group molecule which is inhibited by metal ions and can catalyze the chemiluminescence system to emit light at the end part of the substrate chain, realizes the dual signal amplification of the catalytic chemiluminescence activity inhibition, and improves the detection sensitivity; meanwhile, the method can realize the adjustment of the interval range of the normal linear region by changing the ionic strength of the reaction buffer solution. The method combines the Cu-Sub cleavage by Cu-DNAzyme and Cu2+Inhibition of HRP-catalyzed chemiluminescent activity produces a dual signal amplification.
Description
Technical Field
The invention relates to the technical field of biosensor metal ion detection, and particularly relates to a probe for metal ion detection, a kit, a preparation method and application.
Background
Metals are characterized by losing electrons to positively charged ions, and thus present in biological fluids are various forms of metal ions that can produce various bonding actions with biomolecules, thereby performing various functions in the body, such as electron transfer, oxygen carrying, active centers of enzymes, and the like. Too much or too little metal ions in the body can cause the disorder of the normal physiological functions of the human body, thereby causing various diseases. Transition metal ions such as iron, copper, manganese, zinc, etc. are important neurochemical factors, and their metabolic abnormalities are closely related to diseases of the nervous system, such as early senile dementia, familial myogenic amyotrophic lateral sclerosis and mad cow disease or creutzfeldt-jakob disease.
For example, copper (Cu) is one of the essential trace elements of a living body, and may participate in life activities as a coenzyme, and may also participate in formation of erythrocytes, as Cu is widely used in the fields of manufacturing, chemical industry, and pharmaceutical synthesis, people have more and more chances to contact Cu through environmental exposure, professional exposure, and iatrogenic exposure, and a large amount of radicals generated after an organism takes in excessive amount of Cu may cause oxidative damage to the body, Cu is loosely bound to albumin after entering blood, wherein 90% to 98% of Cu is transported into the liver to be strongly bound to α 2 globulin to form ceruloplasmin and then released into the blood, ceruloplasmin may be classified into saturated ceruloplasmin and unsaturated ceruloplasmin according to the number of bound Cu atoms, when ceruloplasmin synthesis in the body is impaired, the content of free Cu in the blood increases, and a large amount of radicals is generated, and thus the organ dysfunction is caused when the free Cu is deposited in organs such as liver, kidney, brain, and the relevant diseases including liver fibrosis (Wilson), kidney, brain, kidney, brain, and other organs are difficult to be detected by a high sensitivity of serum protein binding protein, which is calculated by a relatively complicated method of serum protein binding to detect a high-induced by a high-induced plasma induced.
The biosensor detection method constructed based on the recognition molecules is gradually applied to various fields due to the advantages of strong specificity and the like, has related applications in the fields of metal ion detection and the like, can quickly and specifically detect target metal ions compared with the traditional method, but has the defects of low sensitivity and low detection accuracy in the detection of free low-concentration metal ions in a biological sample. Therefore, how to improve the sensitivity and accuracy of detecting metal ions in organisms based on the recognition molecule biosensor becomes a technical problem to be solved urgently at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a probe for detecting metal ions, which can quickly, sensitively and accurately detect the specificity of target metal ions.
Another object of the present invention is to provide a method for producing a probe for detecting metal ions.
The invention also aims to provide a kit for detecting metal ions, which can quickly, sensitively and accurately detect the specificity of target metal ions in human serum.
The fourth purpose of the invention is to provide an application of the kit for detecting metal ions.
In order to achieve the purpose, the invention adopts the following technical scheme:
a metal ion detection probe is modified and connected with a metal ion deoxynuclease sequence and a substrate chain sequence which is complementarily matched and connected with the metal ion deoxynuclease sequence, and the 3' end of the substrate chain sequence is modified with a group molecule which can catalyze a chemiluminescence system to generate chemiluminescence.
Optionally, the metal ion inhibits catalytic activity of the group molecule modified at the 3' end of the substrate strand sequence.
Optionally, the metal ions are copper ions;
the deoxynuclease sequence is as follows: 5'-GGTAAGCCTGGGCCTCTTTCTTTTTA AGAAAGAAC-3', respectively;
the 3' end modified group molecule of the substrate chain sequence is horseradish peroxidase (HRP);
the substrate chain sequence is:
5’-Biotin-TTTTTTTTTTTTTTTTTTTTAGCTTCTTTCTAATACGGCTTAC C-HRP-3’。
optionally, the probe takes streptavidin-modified nano magnetic beads as a solid phase carrier. Optionally, the nano magnetic bead is Fe3O4。
The DNA sequence and the substrate chain sequence are artificially synthesized sequences, purified by HP L C after synthesis, prepared into 100 mu mol/L by TE buffer solution before use, and stored for later use;
the preparation method of the probe for detecting the metal ions is characterized by comprising the following operation steps of:
1) washing streptavidin modified nano magnetic beads by using a buffer solution 1, adding the buffer solution 1 and a substrate chain, carrying out vortex reaction at room temperature in a dark place, carrying out magnetic separation after the reaction is finished, and washing by using the buffer solution 1 to obtain nano magnetic beads combined with the substrate chain;
2) adding the deoxynuclease sequence and a hybridization buffer solution into the substrate chain-bound nano magnetic beads prepared in the step 1), carrying out incubation reaction, and carrying out magnetic separation after the reaction is finished to obtain the deoxynuclease sequence and substrate chain-bound nano magnetic beads;
3) and (3) cleaning the nano magnetic beads prepared in the step 2) by using a buffer solution 2, and storing in the buffer solution 2 to obtain the probe.
Optionally, in the step 1), the molar mass usage of the substrate chain corresponding to 1mg of the nano magnetic beads is 0.2-0.8 nmol, and the molar mass usage of the corresponding deoxynuclease sequence is 1-4 nmol.
Optionally, the vortex reaction time in the step 1) is 20-40 min; the incubation reaction time in step 2) was 60 min.
Optionally, the buffer solution 1 is a TE buffer solution containing NaCl and 0.05%, v/v, Tween-20, the hybridization buffer solution is a 0.05 mol/L HEPES buffer solution containing 1.5 mol/L NaCl and has a pH of 7.0, and the buffer solution 2 is a 0.01 mol/L Tris-HCl buffer solution containing NaCl and has a pH of 8.0.
A kit for detecting metal ions, comprising the probe.
The kit also comprises a chemiluminescence plate, chemiluminescence substrate liquid, a standard sample, a dilution buffer solution, a washing buffer solution, an EDTA solution, a PBST buffer solution, a sample digestion treatment liquid and a pH regulator.
In serum, copper is bound to proteins or amino acids in a bound state and is not present in an ionic form, so that before detection, a serum sample needs to be digested to obtain Cu in an ionic state, and the sample digestion solution contains a mixture of concentrated sulfuric acid with a mass concentration of 98%, concentrated nitric acid with a mass concentration of 70%, and hydrogen peroxide with a mass concentration of 30% in a volume ratio of 2:1: 1.
Optionally, the pH regulator is a KOH solution.
Optionally, the dilution buffer solution is 0.05-1.5 mol/L HEPES buffer solution containing NaCl, the pH value is 7.0, and the washing buffer solution is 0.01 mol/L Tris-HCl buffer solution containing NaCl, the pH value is 8.0.
Optionally, the chemiluminescent substrate solution comprises a chemiluminescent substrate A and a chemiluminescent substrate B, wherein the chemiluminescent substrate A is a mixture of L um and BIP, and the chemiluminescent substrate B is H2O2。
The application of the kit for detecting the metal ions in the aspect of detecting the concentration of the copper ions in the serum is characterized in that the detection method comprises the following operation steps:
a, adding a probe into a chemiluminescent plate, carrying out magnetic separation, washing by using a washing buffer solution, adding ascorbic acid, carrying out a light-shielding reaction at room temperature, carrying out magnetic separation after the reaction is finished, washing by using PBST, adding a chemiluminescent substrate solution, and measuring the luminous intensity of the chemiluminescent plate, wherein the luminous intensity is recorded as R L U0；
B, adding ascorbic acid into the copper ion standard solution, adding the copper ion standard solution containing the ascorbic acid into the chemiluminescence plate according to the method in the step A, measuring the luminous intensity and recording as R L U, and corresponding to different copper ion concentrations (R L U)0-RLU)/RLU0Drawing a standard curve for detecting the concentration of the copper ions to obtain (R L U)0-RLU)/RLU0Corresponding relation with the concentration of copper ions;
c, determining the total copper content of the serum sample, namely adding the human serum sample into a sample digestion treatment solution for digestion treatment, diluting the human serum sample with a dilution buffer solution to obtain a total copper sample to be detected, adding ascorbic acid into the total copper sample to be detected according to the same method in the step A, detecting the luminous intensity R L U, and carrying the total copper sample to the (R L U) obtained in the step B0-RLU)/RLU0Calculating the total copper content of the serum sample in a corresponding relation between the concentration of the copper ions and the concentration of the copper ions;
and (2) measuring the content of free copper in the serum sample, namely taking the human serum sample, adding an EDTA solution into the human serum sample for reaction, filtering and collecting filtrate, adding the filtrate into a sample digestion treatment solution for digestion treatment, adding a dilution buffer solution into the filtrate digestion solution for dilution to obtain a free copper sample to be measured, adding ascorbic acid into the free copper sample to be measured according to the same method in the step A, detecting the luminous intensity R L U, and carrying the free copper sample to the (R L U) obtained in the step B0-RLU)/RLU0And calculating the free copper content of the serum sample in a corresponding relation between the concentration of the copper ions and the concentration of the copper ions.
The ascorbic acid is added in the copper ion detection process to ensure that Cu is added2+Reduction to Cu+And (3) accelerating the detection rate, wherein the concentration of the ascorbic acid is 20-200 mu mol/L.
Optionally, the digestion treatment method comprises adding 400 μ L sample digestion treatment solution into each 100 μ L sample to be digested, reacting at 70 deg.C for 1h, heating to 150 deg.C, evaporating to dryness, dissolving with 800 μ L ultrapure water, and adjusting pH to neutral with KOH solution.
The metal ion deoxynuclease sequence is a DNA single-stranded fragment with a specific catalytic function obtained by screening through an exponential enrichment ligand system evolution technology (SE L EX), and consists of a deoxynucleic acid sequence and a corresponding substrate chain sequence, when metal ions exist, the catalytic activity is exerted, the substrate chain is cut into two parts, the cutting activity is related to the concentration of the metal ions, according to the principle, the metal ion deoxynucleic acid sequence and the substrate chain sequence are adopted to modify a probe, the end part of the substrate chain is modified with a group molecule with a catalytic luminescence effect on a chemiluminescence system, and the detection of the metal ions is realized through a chemiluminescence principle.
The specific invention adopts a copper ion deoxynucleic acid sequence (Cu-DNAzyme) and a corresponding substrate chain (Cu-Sub) modification probe, and the Cu-DNAzyme and Cu-Sub sequences are modified to a certain extent on the basis of keeping the specific functional structure unchanged, and the sequences are as follows: Cu-DNAzyme: 5'-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3', Cu-Sub: 5' -Biotin-T20AGCTTCTTTCTAATACGGCTTA CC-HRP-3 ', elongation of 20T bases at the 5' end of Cu-Sub can be reduced at its Fe3O4Steric hindrance when the surface is hybridized with Cu-DNAzyme, and the 3' end labeled Horse Radish Peroxidase (HRP) can catalyze L nucleic-H2O2The system generates chemiluminescence and is in Cu2+Under the action of Cu, its activity is inhibited2+The method has a synergistic effect on the cutting of the Cu-DNAzyme, double amplification is generated on detection signals, the detection sensitivity is improved, and the detection of high specificity and high sensitivity of total copper and free copper in a serum sample can be realized.
The principle is as follows: in the absence of Cu2+In the case of (1), the Cu-DNAzyme complex on the surface of the probe keeps stable conformation, wherein the HRP modified at the 3' end of Cu-Sub keeps the natural conformation and catalyzes L uinol-H2O2The system generates strong chemiluminescence (C L), and Cu is added2+Thereafter, the Cu-DNAzyme binds Cu2+And generates catalytic activity to catalytically cut Cu-Sub to generate S1 (-T)20AGCTTCTTTCTAATACG) and S2(-GCTTACC-HRP), wherein the HRP-labeled S2 end is released into the solution due to the decrease of the base pair complementary to Cu-DNAzyme, the HRP content on the surface of the probe is decreased after magnetic separation and washing, and further, Cu is added2+Binding to HRP results in a conformational change that reduces its catalytic activity, and therefore, due to Fe3O4The reduction of the content and activity of the HRP on the surface leads to the reduction of the catalytic chemiluminescence capacity of the probe according to the chemiluminescence intensity (R L U) and the Cu content in the sample to be detected2+The content is inversely proportional to the quantitative analysis.
Drawings
FIG. 1 shows the synthesis of probe Fe in example 1 of the present invention3O4The principle schematic diagram of detecting copper ions with @ Cu-Sub @ Cu-DNAzyme;
FIG. 2 is a diagram showing the synthesis of a probe Fe in example 1 of the present invention3O4@ Cu-Sub @ Cu-DNAzyme particle size distribution; in the figure, 1 is Fe3O4Microparticles; 2 is Fe3O4@ Cu-Sub; 3 is Fe3O4@Cu-Sub@Cu-DNAzyme；
FIG. 3 is a diagram showing the synthesis of a probe Fe in example 1 of the present invention3O4@ Cu-Sub @ Cu-DNAzyme Zeta potential characterization; in the figure, 4 is Fe3O4Microparticles; 5 is Fe3O4@ Cu-Sub; 6 is Fe3O4@Cu-Sub@Cu-DNAzyme；
FIG. 4 is Cu2+For Fe in example 1 of the present invention3O4@ Cu-Sub and Fe3O4The response curve of @ Cu-Sub @ Cu-DNAzyme;
FIG. 5 shows the effect of NaCl concentration in the dilution buffer on the detection line and sensitivity during the detection of serum samples in example 4 of the present invention;
FIG. 6 is a standard curve corresponding to different NaCl concentrations in the dilution buffer during the serum sample testing process in example 4 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail by specific examples.
The following examples use instruments and reagents:
autoamtic sample injection chemiluminescence apparatus (Victor X L light, PerkinElmer), laser particle size analyzer (NanoZS90, Malvern), magnetic streptavidin beads (Fe)3O4SA, tin-free bmerg BioMag biotechnology ltd), Cu-DNAzyme (sequence: 5'-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3'), Cu-Sub (sequence: 5' -Biotin-T20AGCTTCTTTCTAATACGGCTTACC-HRP-3') was synthesized by Takara, a Dalibao organism and purified by HP L C, before use, stock solutions of 100. mu. mol/L were prepared with TE buffer solution 1.0 mol/L pH 8.0Tris-HCl buffer solution, 1.0 mol/L pH 8.8Tris-HCl buffer solution, TE buffer solution (Beijing Solibao Tech Co., Ltd.), HEPES buffer solution, p-hydroxybiphenyl (BIP), ascorbic acid, potassium hydroxide (electronic grade, 99.999%) (Shanghai Allantin Biotech Co., Ltd.), luminol (L uminol, Sigma), 30% hydrogen peroxide (H.H.H.2O2Chinese medicine), Cu2+,Mn2+,Te2+,Bi3+,Ni2+,Hg2+,Pb2+,Se2+,Cd2 +,In3+,Ca2+,Mg2+,Fe3+,Co2+All solutions were prepared with Milli-Q ultrapure water (resistivity greater than 18.2M Ω. cm), and glassware for the experiments was HNO3After soaking overnight, the solution was rinsed thoroughly with purified water.
The buffers used in the experiment were (1) Buffer A which was a TE Buffer containing NaCl and 0.05% (v/v) Tween-20, (2) Buffer B which was a 0.01 mol/L Tris-HCl Buffer containing NaCl at pH 8.0, (3) Buffer C which was a 0.05 mol/L HEPES Buffer containing 0.05 to 1.5 mol/L NaCl at pH7.0, (4) hybridization Buffer which was a 0.05 mol/L HEPES Buffer containing 1.5 mol/L NaCl at pH7.0, (5) PBST which was a mixture of 0.5% (v/v) Tween-20 PBS Buffer, pH 7.4. (6) chemiluminescent substrate A which was 1.60 mmol/L L uminol and 0.03 mmol/L BIP, (7) chemiluminescent substrate B which was 10 mmol/L H-202O2。
Example 1
This example provides a probe for copper ion detection, which uses streptavidin magnetic bead Fe3O4the-SA is a solid phase carrier, a copper ion deoxynucleic acid sequence Cu-DNAzyme and a substrate chain sequence Cu-Sub matched and connected with the copper ion deoxynucleic acid sequence Cu-DNAzyme are modified and connected on the surface of streptavidin magnetic beads, and the 3' end of the substrate chain sequence Cu-Sub is modified with Horse Radish Peroxidase (HRP), and the synthesis method specifically comprises the following steps:
(1) taking 1mg of Fe3O4-SA in centrifuge tube, washing 3 times with Buffer 1(Buffer a) and magnetic separation;
(2) adding 6 mu L100 mu mol/L Cu-Sub into the centrifuge tube, adding Buffer A to 500 mu L, and carrying out vortex reaction for 30min at room temperature in the dark;
(3) after the reaction was completed, unbound Cu-Sub was discarded by magnetic separation and washed 3 times with Buffer A to obtain Fe3O4@Cu-Sub；
(4) Then adding 30 mu L100 to the system, 100 mu mol/L Cu-DNAzyme and adding hybridization buffer to 500 mu L, and incubating for 60 min;
(5) after the reaction is finished, the obtained product is washed 3 times by using a Buffer solution 2(Buffer B), and is resuspended in the Buffer B, and is stored at 4 ℃, and the probe Fe is obtained3O4@ Cu-Sub @ Cu-DNAzyme, which is a probe for detecting copper ions.
The particle size distribution and Zeta potential changes during the probe synthesis were measured using a laser particle sizer, and the results are shown in FIGS. 2 and 3, which are in comparison with Fe3O4In contrast, Fe3O4@ Cu-Sub and Fe3O4The particle size of @ Cu-Sub @ Cu-DNAzyme was slightly increased, probably due to the increased water solubility of modified Cu-Sub and Cu-DNAzyme. The three are all negative, and the Zeta potential absolute value is increased along with the modification of Cu-Sub and Cu-DNAzyme, probably due to the negative charge of a DNA chain in water, thereby judging that the synthesis of the probe is successful, wherein Fe3O4The Zeta potential of @ Cu-Sub @ Cu-DNAzyme is-31.8 mV, which indicates that the enzyme has good stability in aqueous solution.
Example 2
This example provides a probe for copper ion detection, which uses streptavidin magnetic bead Fe3O4the-SA is a solid phase carrier, a copper ion deoxynucleic acid sequence Cu-DNAzyme and a substrate chain sequence Cu-Sub matched and connected with the copper ion deoxynucleic acid sequence Cu-DNAzyme are modified and connected on the surface of streptavidin magnetic beads, and the 3' end of the substrate chain sequence Cu-Sub is modified with Horse Radish Peroxidase (HRP), and the synthesis method specifically comprises the following steps:
(1) taking 1mg of Fe3O4-SA in centrifuge tube, washing 3 times with Buffer 1(Buffer a) and magnetic separation;
(2) adding 2 mu L100 mu mol/L Cu-Sub into the centrifuge tube, adding Buffer A to 500 mu L, and carrying out vortex reaction for 20min at room temperature in the dark;
(3) after the reaction was completed, unbound Cu-Sub was discarded by magnetic separation and washed 3 times with Buffer A to obtain Fe3O4@Cu-Sub；
(4) Then adding 10 mu L100 to the system, 100 mu mol/L Cu-DNAzyme and adding hybridization buffer to 500 mu L, and incubating for 60 min;
(5) after the reaction is finished, the obtained product is washed 3 times by using a Buffer solution 2(Buffer B), and is resuspended in the Buffer B, and is stored at 4 ℃, and then the probe Fe is obtained3O4@ Cu-Sub @ Cu-DNAzyme, which is a probe for detecting copper ions.
Example 3
This example provides a probe for copper ion detection, which uses streptavidin magnetic bead Fe3O4the-SA is a solid phase carrier, a copper ion deoxynucleic acid sequence Cu-DNAzyme and a substrate chain sequence Cu-Sub matched and connected with the copper ion deoxynucleic acid sequence Cu-DNAzyme are modified and connected on the surface of streptavidin magnetic beads, and the 3' end of the substrate chain sequence Cu-Sub is modified with Horse Radish Peroxidase (HRP), and the synthesis method specifically comprises the following steps:
(1) taking 1mg of Fe3O4-SA in centrifuge tube, washing 3 times with Buffer 1(Buffer a) and magnetic separation;
(2) adding 8 mu L100 mu mol/L Cu-Sub into the centrifuge tube, adding Buffer A to 500 mu L, and carrying out vortex reaction for 40min at room temperature in the dark;
(3) after the reaction was completed, unbound Cu-Sub was discarded by magnetic separation and washed 3 times with Buffer A to obtain Fe3O4@Cu-Sub；
(4) Then adding 40 mu L100 to the system, 100 mu mol/L Cu-DNAzyme and adding hybridization buffer to 500 mu L, and incubating for 60 min;
(5) after the reaction is finished, the obtained product is washed 3 times by using a Buffer solution 2(Buffer B), and is resuspended in the Buffer B, and is stored at 4 ℃, and then the probe Fe is obtained3O4@ Cu-Sub @ Cu-DNAzyme, which is a probe for detecting copper ions.
Example 4
This example provides a kit for detecting copper ions, comprising the probe prepared in example 1, a chemiluminescent plate, a chemiluminescent substrate a, a chemiluminescent substrate B, a copper ion standard solution, a dilution buffer, a washing buffer, a PBST buffer, a sample digestion treatment solution, and a pH adjusting agent; wherein the dilution Buffer solution is Buffer C in the reagent, and the washing Buffer solution is Buffer B in the reagent; the sample digestion treatment solution comprises a volume ratio of 2:1:1 of a mixture of concentrated sulfuric acid with a mass concentration of 98%, concentrated nitric acid with a mass concentration of 70% and hydrogen peroxide with a mass concentration of 30%; the pH regulator is KOH.
The kit of the embodiment is used for detecting the total copper content and the free copper content in a blood sample, and the specific detection method comprises the following steps:
a, adding 100 mu L800-time diluted probes into each hole of a chemiluminescent plate, carrying out magnetic separation, cleaning twice by using Buffer B, adding 100 mu L of ascorbic acid with the concentration of 100 mu mol/L into each hole, carrying out light-shielding reaction at room temperature, carrying out magnetic separation after the reaction is finished, cleaning 5 times by using PBST, adding 50 mu L of chemiluminescent substrate A and 50 mu L of chemiluminescent substrate B respectively, and measuring the luminous intensity of the chemiluminescent plate, wherein the luminous intensity is marked as R L U0；
Adding ascorbic acid into copper ion standard solution, adding 100 μ L copper ion standard solution containing ascorbic acid into chemiluminescence plate according to the method described in step A, wherein the concentration of ascorbic acid is equal to that of ascorbic acid added in step A, measuring the luminous intensity and recording as R L U, corresponding to different copper ion concentrations (R L U)0-RLU)/RLU0Drawing a standard curve for detecting the concentration of the copper ions to obtain (R L U)0-RLU)/RLU0Corresponding relation with the concentration of copper ions;
c, measuring the total copper content of the serum sample, namely taking 100 mu L of the human serum sample, adding 400 mu L of sample digestion treatment liquid, reacting at 70 ℃ for 1h, heating to 150 ℃ for drying, dissolving with 800 mu L of ultrapure water, adjusting the pH value to be neutral by using KOH solution, adding Buffer C for dilution to serve as a total copper sample to be measured, adding 100 mu L of the total copper sample to be measured containing ascorbic acid into a chemiluminescence plate according to the same method of the step A, wherein the concentration of the ascorbic acid is equal to that of the ascorbic acid added in the step A, measuring the luminous intensity to be recorded as R L U, and substituting the luminous intensity into the (R L U) obtained in the step B0-RLU)/RLU0And between the concentration of copper ionsCalculating the total copper content of the serum sample in the corresponding relation;
and (2) determining the content of free copper in the serum sample, namely taking the human serum sample, adding an EDTA solution into the human serum sample for reaction, performing ultrafiltration by using a 10KD ultrafiltration tube, collecting filtrate, digesting the filtrate by the same method, adding Buffer C into the filtrate digestive juice for dilution to obtain a free copper sample to be detected, adding 100 mu L of the free copper sample to be detected containing ascorbic acid into the chemiluminescence plate by the same method as the step A, wherein the concentration of the ascorbic acid is equal to that of the ascorbic acid added in the step A, determining the luminous intensity and recording the luminous intensity as R L U, and substituting the luminous intensity into the (R L U) obtained in the step B (R L U)0-RLU)/RLU0And calculating the free copper content of the serum sample in a corresponding relation between the concentration of the copper ions and the concentration of the copper ions.
In this embodiment, the copper ions are detected by using the chemiluminescence principle, which is shown in FIG. 1, and Fe3O4SA is a solid support, first on Fe by means of a biotin-streptavidin system3O4Surface modification of Cu-Sub, and subsequent formation of stable secondary structure, Fe, by base complementary pairing of Cu-DNAzyme and Cu-Sub3O4Fe with Cu-DNAzyme complex modified on the surface3O4@ Cu-Sub @ Cu-DNAzyme nanoprobe. In the absence of Cu2+In the case of (1), Fe3O4The Cu-DNAzyme complex on the surface keeps stable conformation, wherein the HRP modified at the 3' end of Cu-Sub keeps the natural conformation and catalyzes L ulinol-H2O2The system generates strong chemiluminescence (C L), and Cu is added2+Thereafter, the Cu-DNAzyme binds Cu2+And generates catalytic activity to catalytically cut Cu-Sub to generate S1 (-T)20AGCTTCTTTCTAATACG) and S2(-GCTTACC-HRP), wherein the HRP-labeled S2 end is released into the solution due to the decrease of the complementary base pair to Cu-DNAzyme, and after washing by magnetic separation, Fe3O4Reduced surface HRP content, and, in addition, Cu2+Binding to HRP results in a conformational change that reduces its catalytic activity, and therefore, due to Fe3O4The reduction of the content and activity of the HRP on the surface leads to the reduction of the catalytic chemiluminescence capacity of the probe according to the intensity of chemiluminescenceDegree (R L U) and Cu in the sample to be tested2+The content is inversely proportional to the quantitative analysis.
1. Effect test to verify whether the decrease of R L U in the copper ion detecting probe of this example is due to the Cu-Sub cleavage by Cu-DNAzyme or Cu2+Fe is selected respectively for inhibition of HRP activity3O4@ Cu-Sub and Fe3O4@ Cu-Sub @ Cu-DNAzyme is Cu2+Acting on the substrate and plotting different Cu's respectively2+The results of the response curves for both are shown in FIG. 4, from which it can be seen that Cu2+Both of them can reduce R L U and use Fe3O4@ Cu-Sub @ Cu-DNAzyme as substrate for low-concentration Cu2+Is more sensitive, while a higher concentration of Cu2+Will be to Fe3O4Production inhibition of R L U in the @ Cu-Sub group therefore, the present example probe binds Cu-DNAzyme to cleave Cu-Sub and Cu2+The dual action of HRP activity inhibition generates signal amplification, and the Cu-DNAzyme fluorescence sensing method and the HRP activity inhibition detection Cu are simply utilized2+Compared with the method, the method has higher sensitivity and is more suitable for trace Cu2+Detection of (3).
2. Condition optimization test: optimizing the influence of the NaCl concentration in the Buffer C of the dilution Buffer on the linear range of the concentration of the detected copper ions:
for different actual samples, the concentration of target analytes is greatly different, for example, the reference value of total serum copper is 10.9-21.8 mu mol/L, the free serum copper only accounts for 5% -15% of the total serum copper, and the urine copper content of patients with copper metabolic disorder is related to the copper-expelling treatment process2+As shown in FIG. 5, as the NaCl concentration in Buffer C decreased, IC50 gradually decreased and the sensitivity increased, and in the 5 NaCl concentrations selected, when the NaCl concentration was 0.05 mol/L, IC50 reached the lowestThe reason for this may be the following two aspects: firstly, the Cu-DNAzyme gradually inhibits the Cu-Sub cleavage activity along with the increase of NaCl concentration in buffer C; on the other hand, the ionic strength influences the suspension stability of the nano magnetic beads in the buffer C to a certain extent, and the high-concentration NaCl leads the nano magnetic beads to be aggregated to reduce the surface area of the nano magnetic beads, thereby reducing the Cu-DNAzyme and the Cu2+As shown in FIG. 6, in the case where other conditions are optimal, when the NaCl concentration in Buffer C is 1.5 mol/L, Cu is present2+The linear response interval is 50-1500 nmol/L, when the NaCl concentration in Buffer C is reduced to 0.05 mol/L, Cu2+The linear response interval of (a) is 0.01-200 nmol/L, therefore, the method can achieve Cu simply by adjusting the ionic strength of Buffer C of the reaction Buffer2+The detection linear range and the sensitivity can be adjusted, and the method is more convenient to be suitable for different Cu2+And (4) detecting the concentration of the sample.
In addition, for the same reason, since the activities of Cu-DNAzyme and HRP are also pH sensitive, it is reasonable to conclude that the linear range of the method can also be adjusted by adjusting the pH of Buffer C.
3. Method standard curve, detection limit and specificity
Under the condition that the NaCl concentration in Buffer C is reduced to 0.05 mol/L, Cu with different concentrations is detected according to the method described in example 42+Luminous intensity R L U of (1), with Cu2+Concentration is in the abscissa, (R L U)0-RLU)/RLU0Plotting a standard curve for the ordinate, in which Cu2+The concentration of (R L U) is in the range of 0.01 to 200 nmol/L0-RLU)/RLU0Has good linear relation, and the regression equation is that Y is 0.06125+0.00344X (wherein Y is (R L U)0-RLU)/RLU0X is Cu2+Concentration), R2Is 0.9923.
Method detection Limit (L OD) the results of limiting dilution are shown in Table 1, and Cu in the low concentration region is detected2+R L U corresponding to the standard product, and detecting a plurality of Cu2+Blank sample acquisition
Has a value of 4901996 to
Corresponding Cu2+The concentration is the detection limit of the process, and therefore, under optimal conditions, the detection limit of the process is 0.001 nmol/L.
TABLE 1 Low concentration of Cu2+The result of the detection
4. Detection of method specificity:
example 4 the detection of free Cu in a complex system by using the kit may be interfered by other metal ions, so that 13 common metal ions Mn are selected2+、Te2+、Bi3+、Ni2+、Hg2+、Pb2+、Se2+、Cd2+、In3+、Ca2+、Mg2+、Fe3+、Co2+Investigation of the specificity of the method, where Cu2+Was 0.1. mu. mol/L, and the remaining concentrations of each metal ion were 3 (10, 100 and 500. mu. mol/L), respectively, it was found that 0.1. mu. mol/L Cu was used2+Namely, the strong inhibition is generated on C L except 500 mu mol/L Hg2+、Cd2+And Fe3+Slightly inhibits C L, and the degree of inhibition is far less than 0.1 mu mol/L Cu2+And the probe has no response to other metal ions. Thus the probe pair Cu2+Has a selectivity of at least higher than Hg2+、Cd2+And Fe 3+1000 times higher than that of the rest metal ions, 5000 times higher than that of the rest metal ions.
5. Accuracy and precision of the method
Test kit in example 4 for Cu2+The accuracy and the precision of the method are examined, and Cu with high, medium and low (150, 80, 1.0 nmol/L) concentrations are prepared respectively2+Samples, each concentration was assayed in triplicate and the recovery and Relative Standard Deviation (RSD) calculated, with the results shown in tables 2 and 3. Wherein the recovery rate in the plates ranges from 91.9% to 110.0%, the recovery rate between the plates ranges from 94.0% to 103.9%, and the corresponding RSD ranges from 3.0% to 11.8% and from 4.2% to 12.8% respectively;the recovery rates in the day and the daytime are respectively 97.2-118.0% and 92.3-106.0%, and the corresponding RSD is respectively 3.3-13.5% and 8.7-10.6%. Indicating that the method is used for Cu2+The detection accuracy and precision are high.
Table 2 methods accuracy and precision in and between plates (n ═ 3)
TABLE 3 method day-to-day and day-to-day accuracy and precision (n ═ 3)
6. Effect of practical application
By adopting the detection method described in the embodiment 4, the NaCl concentration in Buffer C is reduced to 0.05 mol/L, the serum of 9 patients with hepatolenticular degeneration (WD) in drug treatment and the serum of 10 healthy human sera are taken as detection samples, the total Cu and free Cu of different serum samples are detected, the detection results are shown in a table, the total Cu range of the healthy human sera is 12.71-23.86 mu mol/L according to the detection results, the free Cu range of the serum is 0.59-2.61 mu mol/L, the detection results are consistent with the results reported in the literature, the detection results of the method and the Atomic Absorption (AAS) detection results are analyzed by using a matched sample t test, and the P value is P value>0.01, the difference of the detection results of the two detection methods has no statistical significance, and proves that the method has high accuracy and can effectively and effectively detect Cu in a complex system2+Detection of (3).
In addition, the detection results of total Cu, free Cu and relative free Cu of the serum of a WD patient and the serum of a healthy person are respectively subjected to statistical analysis by using an independent sample t test, and the result shows that the difference of the total serum Cu and the relative free Cu between the WD patient and the healthy person has statistical significance (P <0.01), but the difference of the free serum Cu has no statistical significance (P >0.01), and the result indicates that the free serum Cu of the WD patient can be reduced to a normal level through the copper-expelling drug treatment, but the relative free Cu is still higher than that of the normal person due to the reduction of the total serum Cu caused by the copper-cyanin synthesis disorder.
TABLE 4 serum sample test results
7. Conclusion
The invention takes Cu-DNAzyme as Cu2+And (3) identifying molecules, and synthesizing a nano probe capable of quickly identifying and separating copper ions in a sample by using nano magnetic beads as a solid phase carrier and a separation tool, so as to construct an ultra-sensitive chemiluminescence detection method for the copper ions in a complex system. Further methods of the invention combine Cu-Sub cleavage by Cu-DNAzyme with Cu2+The inhibition of HRP catalytic chemiluminescence activity generates double signal amplification, the ultra-sensitive rapid detection of copper ions in complex samples is realized, and the ion strength of reaction buffer solution can be adjusted to Cu2+The linear range of detection is adjusted, and the method is suitable for Cu in different samples2+And optimized to react with Cu when the concentration of NaCl in the dilution buffer is 0.05 mol/L2+In the range of 0.01 to 200 nmol. L-1Has good linear relation in the range, and the detection limit is as low as 0.001 nmol. L-1Meanwhile, the method has good selectivity and high accuracy and precision, and in addition, the statistical analysis shows that the method has no statistical significance on the difference between the detection result of the serum sample and the detection result of the atomic absorption, which indicates that the method can be effectively applied to the trace Cu in complex systems such as biological samples, environmental samples, food and the like2+The detection probe and the detection method of other metal ions, such as zinc, manganese and the like, can be designed according to the copper ion detection probe and the copper ion detection method provided in the embodiment.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. A probe for detecting metal ions is characterized in that a metal ion deoxynuclease sequence and a substrate chain sequence which is complementarily matched and connected with the metal ion deoxynuclease sequence are modified and connected on the probe, and a group molecule which can catalyze a chemiluminescence system to generate chemiluminescence is modified at the 3' end of the substrate chain sequence;
the metal ions are copper ions;
the deoxynuclease sequence is as follows: 5'-GGTAAGCCTGGGCCTCTTTCTTTTTA AGAAAGAAC-3', respectively;
the 3' end modified group molecule of the substrate chain sequence is horseradish peroxidase (HRP);
the substrate chain sequence is:
5’-Biotin-TTTTTTTTTTTTTTTTTTTTAGCTTCTTTCTAATACGGCTTACC-HRP-3’。
2. the probe for detecting a metal ion according to claim 1, wherein the metal ion inhibits a catalytic activity of a group molecule modified at the 3' -end of the substrate strand sequence.
3. The probe for detecting metal ions according to claim 1, wherein the probe is a solid phase carrier comprising streptavidin-modified nanobead.
4. A method for preparing the probe for detecting metal ions according to claim 3, comprising the steps of:
1) washing streptavidin modified nano magnetic beads by using a buffer solution 1, adding the buffer solution 1 and a substrate chain, carrying out vortex reaction at room temperature in a dark place, carrying out magnetic separation after the reaction is finished, and washing by using the buffer solution 1 to obtain nano magnetic beads combined with the substrate chain;
2) adding the deoxynuclease sequence and a hybridization buffer solution into the substrate chain-bound nano magnetic beads prepared in the step 1), carrying out incubation reaction, and obtaining the deoxynuclease sequence and substrate chain-bound nano magnetic beads after the reaction is finished;
3) and (3) cleaning the nano magnetic beads prepared in the step 2) by using a buffer solution 2, and storing in the buffer solution 2 to obtain the probe.
5. A kit for detecting a metal ion, comprising the probe according to any one of claims 1 to 3.
6. The kit for detecting metal ions according to claim 5, wherein the kit is for detecting copper ions and comprises the probe according to claim 3.
7. The kit for detecting metal ions according to claim 6, further comprising a chemiluminescent plate, a chemiluminescent substrate solution, a standard sample, a dilution buffer, a washing buffer, an EDTA solution, a PBST buffer, a sample digestion treatment solution, and a pH adjusting agent.
8. The use of the kit for metal ion detection according to claim 7 for detecting the concentration of copper ions in serum, wherein the detection method comprises the following steps:
a, adding a probe into a chemiluminescence plate, carrying out magnetic separation, washing by using a washing buffer solution, adding ascorbic acid, carrying out a reaction at room temperature in a dark place, carrying out magnetic separation after the reaction is finished, washing by using PBST, adding a chemiluminescence substrate solution, and measuring the luminous intensity of the chemiluminescence plate, wherein the luminous intensity is recorded as R L U0；
B, adding ascorbic acid into the copper ion standard solution, adding the copper ion standard solution containing the ascorbic acid into the chemiluminescence plate according to the method in the step A, measuring the luminous intensity and recording as R L U, and corresponding to different copper ion concentrations (R L U)0-RLU）/RLU0Drawing a standard curve for detecting the concentration of the copper ions to obtain (R L U)0-RLU）/RLU0Corresponding relation with the concentration of copper ions;
c: determining the total copper content of the serum sample: taking human serumAdding sample digestion solution into the sample for digestion treatment, diluting the serum digestion solution with dilution buffer solution to obtain total copper sample to be detected, adding ascorbic acid into the total copper sample to be detected according to the same method in the step A, detecting the luminous intensity R L U, and substituting into the sample (R L U) obtained in the step B0-RLU）/RLU0Calculating the total copper content of the serum sample in a corresponding relation between the concentration of the copper ions and the concentration of the copper ions;
and (2) determining the content of free copper in the serum sample, namely taking the human serum sample, adding an EDTA solution into the human serum sample for reaction, performing ultrafiltration by using an ultrafiltration tube to collect filtrate, adding the filtrate into a sample digestion treatment solution for digestion treatment, adding a dilution buffer solution into the filtrate digestion solution for dilution to obtain a free copper sample to be detected, adding ascorbic acid into the free copper sample to be detected according to the same method in the step A, detecting the luminous intensity R L U, and carrying the free copper sample to the (R L U) obtained in the step B0-RLU）/RLU0And calculating the free copper content of the serum sample in a corresponding relation between the concentration of the copper ions and the concentration of the copper ions.
9. The use of claim 8, wherein the dilution buffer is 0.05 mol/L HEPES buffer with NaCl, pH = 7.0.
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