CN110806484A - Sarcosine detection method based on single-walled carbon nanotube and aptamer - Google Patents
Sarcosine detection method based on single-walled carbon nanotube and aptamer Download PDFInfo
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- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 title claims abstract description 172
- 108010077895 Sarcosine Proteins 0.000 title claims abstract description 86
- 229940043230 sarcosine Drugs 0.000 title claims abstract description 86
- 239000002109 single walled nanotube Substances 0.000 title claims abstract description 70
- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 108091023037 Aptamer Proteins 0.000 title claims description 30
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- 238000000034 method Methods 0.000 claims abstract description 26
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- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 14
- 230000008859 change Effects 0.000 claims description 14
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
Abstract
The invention relates to a detection method for determining specific target sarcosine of prostate cancer, in particular to a method for forming a monodisperse solution based on the adsorption of a fluorescent carbon nanotube by a nucleic acid aptamer; and then the nucleic acid aptamer recognizes sarcosine, dissociates from the surface of the carbon nano tube, the carbon nano tube is agglomerated, and fluorescence is quenched, so that the sarcosine content is measured. The invention has the advantages that: (1) the detection system constructed by the invention does not need to carry out functional modification on SWNTs and does not need to carry out any marking on ss-DNA, and the detection method is a label-free detection method and is rapid, simple and convenient to detect. (2) The invention utilizes the near infrared fluorescence of the monodisperse SWNTs for detection, avoids the error caused by the autofluorescence of organism tissues and body fluid samples, and is favorable for directly applying to the detection of the content of sarcosine in blood or urine.
Description
Technical Field
The invention belongs to the field of chemical detection. Relates to a detection method for determining specific target sarcosine of prostate cancer, in particular to a sarcosine detection method based on a single-walled carbon nanotube and a nucleic acid aptamer.
Background
Prostate cancer is an epithelial malignancy that occurs in the prostate gland, with morbidity and mortality increasing with age in men. The generation of the prostate cancer is deeply influenced by the environment and the life style of a patient, and the time from the development of tumor cells to the appearance of clinical symptoms is usually 10-20 years, so that the survival rate of the patient can be remarkably improved and improved by early screening of the prostate cancer patient. Prostate Specific Antigen (PSA) which is generally adopted clinically is used for early screening, but detection sensitivity and specificity are greatly controversial, false positive rate and false negative rate are high, and unnecessary biopsy causes great pain to patients. Therefore, a new method for detecting prostate cancer markers is urgently needed.
Sarcosine is an intermediate product of glycine metabolism, and alterations in the levels of sarcosine and sarcosine-related enzymes are closely associated with a variety of diseases. Recent studies have shown that sarcosine content increases significantly during cancer metastasis and can be used as a biomarker for monitoring the development and progression of prostate cancer. The traditional sarcosine determination methods are: an electrochemical method (Chinese patent: sarcosine detection device and a preparation method and application thereof, publication No. CN 106770588A.); electrophoresis method: (Chinese patent: a method for detecting sarcosine, publication No. CN 101718746A.); fluorescence spectroscopy: (Chinese patent: method for quantitative determination of sarcosine content and reaction kit, publication No. CN 101587076A.); chromatographic mass spectrometry: (literature: Partial enzymationalization and quantification of sarcosine from alkane using liquid chromatography. anal. biological. chem. 2013, 405, 3153-3158.). The method has the advantages that the sample preparation process is complex, professional personnel are required to operate instruments skillfully, and the method is not suitable for daily operation and analysis, so that the novel detection method with good specificity and stability is established, the rapid and sensitive determination of the sarcosine content in the complex sample environment is realized, and the method has important practical significance and value.
Single-walled carbon nanotubes (SWNTs) have a wide application prospect in many fields such as biomedicine due to their unique structures and excellent optical, electrical, physical and magnetic properties. Especially, the single-walled carbon nanotube can emit near-continuous photoluminescence and electroluminescence in the near-infrared wavelength range, has high quantum yield and good photobleaching tolerance and fluorescence stability, and is an ideal near-infrared fluorescent material.
The single-walled carbon nanotube can be combined with periodic single-stranded DNA, and has great prospect in the field of biological nondestructive testing. Aptamers (aptamers) are a class of synthetic, aperiodic, single-stranded DNA sequences that can fold into a particular conformation and specifically recognize a target through noncovalent interactions. The aptamer has excellent selectivity, specificity and affinity, and is extremely suitable for the field of biosensing. The method for detecting sarcosine without the mark is designed by combining the single-walled carbon nanotube with the aptamer, and has higher research significance and application value.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for measuring sarcosine based on a nucleic acid aptamer and a single-walled carbon nanotube.
The purpose of the invention is realized by the following scheme: a sarcosine detection method based on single-walled carbon nanotubes and aptamers is characterized in that fluorescent carbon nanotubes are adsorbed based on the aptamers to form a monodisperse solution, wherein the 5 'end of a designed recognition sequence is a aptamer sequence, and the 3' end of the designed recognition sequence contains (TAT)3The recognition sequence is wound on the surface of the carbon nano tube through pi-pi acting force; the nucleic acid aptamer recognizes sarcosine, dissociates from the surface of the carbon nano tubes (SWNTs), the carbon nano tubes are agglomerated, and fluorescence is quenched, so that the sarcosine content is measured, and the method comprises the following steps:
(1) aptame binds to fluorescent carbon nanotubes (CoMoCAT SWNTs):
adding 1mL of Sodium Dodecyl Sulfate (SDS) phosphate buffer solution (2 wt%) into 1 mg of CoMoCAT SWNTs, performing ultrasonic homogenization, adding 3-10 mg of DNA (deoxyribonucleic acid) in 1-5 of the identification sequence seq. No.1-5 into the mixed solution to enable the mass ratio of the SWNTs to the DNA to be 1: 3-1: 10, performing ultrasonic treatment in an ice bath for 2 hr, centrifuging the mixed solution for 1.5 hr at 12000 rpm/min to remove impurities and agglomerated carbon nanotubes in the solution, taking the centrifuged supernatant, and centrifuging for 15 min at 6000 rpm/min by using an ultrafiltration tube (MWCO 100 KD) to remove free ss-DNA in the solution. Re-dissolving the precipitate with 1mL phosphate buffer solution (PBS, pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing sarcosine (0-50 mu M) solution, storing in ice bath, opening a fluorescence spectrophotometer, setting parameters, correcting a base line, sequentially and uniformly mixing 10 mu L sarcosine solution with 89 mu L PBS solution (pH 7.4), transferring the mixture to a cuvette, then adding 1 mu L aptamer-SWNTs compound, uniformly mixing, reacting for 10 min, exciting with exciting light 580 nm, analyzing sarcosine content with the change of fluorescence intensity of a peak close to 1000nm, drawing a standard curve with the change of sarcosine concentration corresponding to quenching efficiency (F) =1-F1/F0Wherein, the fluorescence intensities of the single-walled carbon nanotubes before and after addition of sarcosine are respectively shown.
A preferred method for synthesizing the CoMoCAT SWNTs is the method disclosed in 2019, 9, 16, volume 91, 20, 12704, 12712, Design of folding DNA aptamer on single-walled carbon nanotubes for enhanced optical detection of targetprotein, S2.
The preferred mass ratio of SWNTs to DNA is 1: 6.
The principle of the invention is as follows: designing the recognition sequence with aptamer sequence at 5 'end and (TAT) at 3' end3And the recognition sequence is wound on the surface of the CoMoCAT SWNTs through pi-pi acting force to form a monodisperse DNA-SWNTs compound. Further, after being combined with sarcosine, the aptamer is folded into a specific three-dimensional structure, and then is dissociated from the surface of the single-walled carbon nanotube, so that the carbon nanotube is changed from a monodisperse state to an accumulation state, and fluorescence quenching is performed, and therefore output of a detection signal is achieved.
The method of the invention forms a monodisperse nanotube by adsorbing the aptamer to the single-walled carbon nanotube, and after the aptamer recognizes sarcosine, the sarcosine is dissociated from the single-walled carbon nanotube and the fluorescence of the nanotube is quenched, thereby quantifying the concentration of the sarcosine. The method adopts near-infrared fluorescence signals, is slightly interfered by autofluorescence, is a label-free detection method, has high sensitivity, and is suitable for quantitative detection of sarcosine content in complex sample environment.
The invention has the advantages that:
(1) the detection system constructed by the invention does not need to carry out functional modification on SWNTs and does not need to carry out any marking on ss-DNA, and the detection method is a label-free detection method and is quick, simple and convenient to detect;
(2) the invention utilizes the near infrared fluorescence of the monodisperse SWNTs for detection, avoids the error caused by the autofluorescence of organism tissues and body fluid samples, and is favorable for directly applying to the detection of the content of sarcosine in blood or urine.
Detailed Description
The technical solution of the present invention is further described below by specific examples. The following examples are further illustrative of the present invention and do not limit the scope of the present invention.
Example 1
A sarcosine detection method based on single-walled carbon nanotubes and aptamers is characterized in that fluorescent carbon nanotubes are adsorbed based on the aptamers to form a monodisperse solution, wherein the 5 'end of a designed recognition sequence is a aptamer sequence, and the 3' end of the designed recognition sequence contains (TAT)3The recognition sequence is wound on the surface of the carbon nano tube through pi-pi acting force; and then the nucleic acid aptamer recognizes sarcosine, dissociates from the surface of the carbon nano tube (SWNTs), agglomerates the carbon nano tube, and quenches fluorescence, so that the sarcosine content is measured according to the following steps:
(1) aptamer binding to CoMoCAT SWNTs:
adding 1mL of Sodium Dodecyl Sulfate (SDS) phosphate buffer (2 wt%) into 1 mg of CoMoCAT SWNTs, ultrasonically mixing uniformly, adding 6 mg of identification sequence seq.No.1 DNA1 into the mixed solution, and ultrasonically treating in ice bath for 2 hr to obtain a mixed solution; centrifuging the mixed solution at 12000 rpm/min for 1.5 hr to remove impurities and aggregated carbon nanotube, collecting supernatant, centrifuging at 6000 rpm/min with ultrafiltration tube (MWCO 100 KD) for 15 min to remove free ss-DNA in the solution; re-dissolving the precipitate with 1mL phosphate buffer solution (PBS, pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing a 0-50 mu M sarcosine solution, and storing in ice bath; opening a fluorescence spectrophotometer, setting parameters and correcting a base line; sequentially and uniformly mixing 10 mu L sarcosine solution and 89 mu L PBS solution (pH 7.4), and transferring the mixture to a cuvette; subsequently, adding 1 mu L aptamer-SWNTs compound, uniformly mixing, reacting for 10 min, exciting by exciting light 580 nm, and analyzing the content of sarcosine by peak fluorescence intensity change at 995 nm; a standard curve was plotted for the quenching efficiency (F) against the change in the concentration of sarcosine, (F)% =1-F1/F0Wherein, the fluorescence intensities of the single-walled carbon nanotubes before and after addition of sarcosine are respectively shown.
Example 2
A sarcosine detection method based on single-walled carbon nanotubes and aptamer is similar to that in example 1, and comprises the following steps:
(1) aptamer binding to CoMoCAT SWNTs:
adding 1mL of phosphate buffer (2 wt%) of Sodium Dodecyl Sulfate (SDS) into 1 mg of CoMoCAT SWNTs, uniformly performing ultrasonic treatment, adding 6 mg of DNA 2 of an identification sequence seq.No.2 into the mixed solution, and performing ultrasonic treatment in ice bath for 2 hr; centrifuging the mixed solution at 12000 rpm/min for 1.5 hr to remove impurities and agglomerated carbon nanotube; taking the centrifugal supernatant, centrifuging for 15 min at 6000 rpm/min by adopting an ultrafiltration tube (MWCO 100 KD), and removing free ss-DNA in the solution; re-dissolving the precipitate with 1mL phosphate buffer solution (PBS, pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing a 0-50 mu M sarcosine solution, and storing in ice bath; opening a fluorescence spectrophotometer, setting parameters and correcting a base line; sequentially taking 10 mu L sarcosine solution with each concentration, uniformly mixing the sarcosine solution with 89 mu L PBS solution (pH 7.4), and transferring the mixture to a cuvette; subsequently, adding 1 mu L aptamer-SWNTs compound, uniformly mixing, reacting for 10 min, exciting by exciting light 580 nm, and analyzing the content of sarcosine by peak fluorescence intensity change at 995 nm; a standard curve is drawn for the quenching efficiency (f) versus the change in sarcosine concentration (f)%=1-F1/F0Wherein, the fluorescence intensities of the single-walled carbon nanotubes before and after addition of sarcosine are respectively shown.
Example 3
A sarcosine detection method based on single-walled carbon nanotubes and aptamer is similar to that in example 1, and comprises the following steps:
(1) aptamer binding to CoMoCAT SWNTs:
adding 1mL of phosphate buffer (2 wt%) of Sodium Dodecyl Sulfate (SDS) into 1 mg of CoMoCAT SWNTs, uniformly performing ultrasonic treatment, adding 6 mg of DNA 3 with an identification sequence seq.No.3 into the mixed solution, and performing ultrasonic treatment in ice bath for 2 hr; centrifuging the mixed solution at 12000 rpm/min for 1.5 hr to remove impurities and agglomerated carbon nanotube; taking the centrifugal supernatant, centrifuging for 15 min at 6000 rpm/min by adopting an ultrafiltration tube (MWCO 100 KD), and removing free ss-DNA in the solution; re-dissolving the precipitate with 1mL phosphate buffer solution (PBS, pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing a 0-50 mu M sarcosine solution, and storing in ice bath; opening a fluorescence spectrophotometer, setting parameters and correcting a base line; sequentially and uniformly mixing 10 mu L sarcosine solution and 89 mu L PBS solution (pH 7.4), and transferring the mixture to a cuvette; subsequently, adding 1 mu L aptamer-SWNTs compound, uniformly mixing, reacting for 10 min, exciting by exciting light 580 nm, and analyzing the content of sarcosine by peak fluorescence intensity change at 995 nm; a standard curve was plotted for the quenching efficiency (F) against the change in the concentration of sarcosine, (F)% =1-F1/F0Wherein, the fluorescence intensities of the single-walled carbon nanotubes before and after addition of sarcosine are respectively shown.
Example 4
A sarcosine detection method based on single-walled carbon nanotubes and aptamer is similar to that in example 1, and comprises the following steps:
(1) aptamer binding to CoMoCAT SWNTs:
adding 1mL of Sodium Dodecyl Sulfate (SDS) phosphate buffer (2 wt%) into 1 mg of CoMoCAT SWNTs, uniformly performing ultrasonic treatment, adding 8 mg of DNA 4 with an identification sequence seq.No.4 into the mixed solution, and performing ultrasonic treatment in ice bath for 2 hr; centrifuging the mixed solution at 12000 rpm/min for 1.5 hr to remove impurities and agglomerated carbon nanotube; taking the centrifugal supernatant, centrifuging for 15 min at 6000 rpm/min by adopting an ultrafiltration tube (MWCO 100 KD), and removing free ss-DNA in the solution; re-dissolving the precipitate with 1mL phosphate buffer solution (PBS, pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing a 0-50 mu M sarcosine solution, and storing in ice bath; and opening the fluorescence spectrophotometer, setting parameters and correcting the base line. Sequentially and uniformly mixing 10 mu L sarcosine solution and 89 mu L PBS solution (pH 7.4), and transferring the mixture to a cuvette; subsequently, adding 1 mu L aptamer-SWNTs compound, uniformly mixing, reacting for 10 min, exciting by exciting light 580 nm, and analyzing the content of sarcosine by peak fluorescence intensity change at 995 nm; a standard curve was plotted for the quenching efficiency (F) against the change in the concentration of sarcosine, (F)% =1-F1/F0Wherein, the fluorescence intensities of the single-walled carbon nanotubes before and after addition of sarcosine are respectively shown.
Example 5
A sarcosine detection method based on single-walled carbon nanotubes and aptamer is similar to that in example 1, and comprises the following steps:
(1) aptamer binding to CoMoCAT SWNTs:
adding 1mL of Sodium Dodecyl Sulfate (SDS) phosphate buffer (2 wt%) into 1 mg of CoMoCAT SWNTs, uniformly performing ultrasonic treatment, adding 8 mg of identification sequence DNA 5 of identification sequence seq.No.5 into the mixed solution, and performing ultrasonic treatment in ice bath for 2 hr; centrifuging the mixed solution at 12000 rpm/min for 1.5 hr to remove impurities and agglomerated carbon nanotube; taking the centrifugal supernatant, centrifuging for 15 min at 6000 rpm/min by adopting an ultrafiltration tube (MWCO 100 KD), and removing free ss-DNA in the solution; re-dissolving the precipitate with 1mL phosphate buffer solution (PBS, pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing a 0-50 mu M sarcosine solution, and storing in ice bath; turn on the spectrofluorometer, set the parameters and correctA positive baseline; sequentially taking 10 mu L sarcosine solution with each concentration, uniformly mixing the sarcosine solution with 89 mu L PBS solution (pH 7.4), and transferring the mixture to a cuvette; subsequently, 1 mu L aptamer-SWNTs compound is added, after uniform mixing, reaction is carried out for 10 min, excitation is carried out at excitation light 580 nm, and sarcosine content is analyzed according to peak fluorescence intensity change at 995 nm. A standard curve was plotted for the quenching efficiency (F) against the change in the concentration of sarcosine, (F)% =1-F1/F0Wherein, the fluorescence intensities of the single-walled carbon nanotubes before and after addition of sarcosine are respectively shown.
<110> Shanghai nanotechnology and applied national center for engineering research Ltd
<120> sarcosine detection method based on single-arm carbon nanotube and aptamer
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Claims (3)
1. A sarcosine detection method based on single-walled carbon nanotubes and aptamers is characterized in that fluorescent carbon nanotubes are adsorbed based on the aptamers to form a monodisperse solution, wherein the 5 'end of a designed recognition sequence is a aptamer sequence, and the 3' end of the designed recognition sequence contains (TAT)3The recognition sequence is wound on the surface of the carbon nano tube through pi-pi acting force; the nucleic acid aptamer recognizes sarcosine, dissociates from the surface of the carbon nano tubes (SWNTs), the carbon nano tubes are agglomerated, and fluorescence is quenched, so that the sarcosine content is measured, and the method comprises the following steps:
(1) aptamer binding to CoMoCAT SWNTs:
adding 1mL of CoMoCAT SWNTs into 1mL of phosphate buffer solution (PBS buffer solution) with the concentration of 2 wt% of Sodium Dodecyl Sulfate (SDS), ultrasonically mixing uniformly, adding 3-10 mg of one of DNA 1-5 of an identification sequence seq.No.1-5 into the mixed solution, ultrasonically treating the mixed solution in ice bath for 2 hr, centrifuging the mixed solution for 1.5 hr at 12000 rpm/min, removing impurities and agglomerated carbon nanotubes in the solution, taking centrifugal supernatant, centrifuging the supernatant for 15 min at 6000 rpm/min by using an ultrafiltration tube (MWCO 100 KD), and removing free ss-DNA in the solution; re-dissolving the precipitate with 1mL PBS buffer (pH7.4) to obtain aptamer-SWNTs complex;
(2) determination of sarcosine content:
preparing 0-50 mu M sarcosine solution, storing in ice bath, opening a fluorescence spectrophotometer, setting parameters, correcting a base line, sequentially taking 10 mu L sarcosine solution, uniformly mixing with 89 mu L PBS buffer solution (pH 7.4), transferring to a cuvette, then adding 1 mu L aptamer-SWNTs compound, reacting for 10 min after uniform mixing, exciting with laser 580 nm, and analyzing sarcosine content by the change of fluorescence intensity of a peak near 1000nmAmount, standard curve was plotted as the quenching efficiency (F) against the change in sarcosine concentration, (F)% =1-F1/F0Wherein F is0、F1Respectively shows the fluorescence intensity of the single-walled carbon nanotube before and after the addition of sarcosine.
2. The method for detecting sarcosine based on single-walled carbon nanotubes and nucleic acid aptamers according to claim 1, wherein the method comprises the following steps: a preferred method for synthesizing the CoMoCAT SWNTs is the method disclosed in 2019, 9, 16, volume 91, 20, 12704, 12712, Design of folding DNA aptamer on single-walled carbon nanotubes for enhanced optical detection of target protein, S2.
3. The method for detecting sarcosine based on single-walled carbon nanotubes and nucleic acid aptamers according to claim 1, wherein the method comprises the following steps: the mass ratio of SWNTs to DNA was 1: 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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